US 20100028334A1

(19) United States

(12) Patent Application Publication (10) Pub. No.: US 2010/0028334 A1
Cottarel et al. (43) Pub. Date: Feb. 4, 2010
(54) COMPOSITIONS AND METHODS TO Publication Classification
POTENTIATE COLISTIN ACTIVITY
(51) Int. Cl.
Guillaume Cottarel, Mountain A638/12 (2006.01)
(75) Inventors: AOIN 37/00 (2006.01)
View, CA (US); Jamey A 6LX 39/395 (2006.01)
Wierzbowski, Stoneham, MA (US) A6IP3L/04 (2006.01)
Correspondence Address: CI2O I/68 (2006.01)
RONALDI. EISENSTEIN (52) U.S. Cl. ................... 424/130.1: 514/2: 514/9: 435/6
100 SUMMER STREET, NIXON PEABODY LLP
BOSTON, MA 02110 (US) (57) ABSTRACT

(73) Assignee: TRUSTEES OF BOSTON

A pharmaceutical composition comprising an antimicrobial
agent and an enhancer of an antimicrobial agent, wherein the
UNIVERSITY, Boston, MA (US) enhancer of an antimicrobial agent is an inhibitor of gene, that
by inactivating the gene product potentiates the effectiveness
(21) Appl. No.: 12/519,336 of the antimicrobial agent. In some embodiments, the phar
maceutical composition further comprises a pharmaceuti
(22) PCT Filed: Dec. 13, 2007 cally acceptable carrier. In some embodiments, the antimi
(86). PCT No.: PCT/US07/87397 crobial agent is an antimicrobial peptide Such as a polymyxin,
for example but not limited to colistin. In some embodiments
S371 (c)(1), of the present invention provides methods to treat and/or
(2), (4) Date: Jun. 15, 2009
prevent infection of a Subject with a microorganism by
administering a pharmaceutical composition comprising an
Related U.S. Application Data antimicrobial agent and an enhancer of an antimicrobial
agent. In some embodiments, the present invention provides
(60) Provisional application No. 60/875,003, filed on Dec. methods to inhibit growth of a microorganism by administer
15, 2006, provisional application No. 60/963,265, ing a pharmaceutical composition comprising an antimicro
filed on Aug. 3, 2007. bial agent and an enhancer of an antimicrobial agent.

Overnight culture in LB from a previous

onvernight incubation in presence of
Kanamycin

Transfer 30 into 30 us Transfer 30 ul into 30 ul LB

Containing Colistin

Stamp onto LB agar plate

e-plates
analysis

List of mutants
with overlapping score
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COMPOSITIONS AND METHODS TO
POTENTIATE COLISTIN ACTIVITY
FIELD OF THE INVENTION
0001. The present invention relates generally to methods
and compositions to treat drug resistant bacteria, and more
particularly to methods and compositions to potentate the
activity of lipopeptides.
BACKGROUND OF THE INVENTION
0002 Increasing multidrug resistance in Gram-negative
bacteria, in particular Pseudomonas aeruginosa, Acineto
bacter baumannii, and Klebsiella pneumoniae, presents a
critical problem. Limited therapeutic options have forced
infectious disease clinicians and microbiologists to reap
praise the clinical application of colistin, a polymyxin anti
biotic discovered more than 50 years ago. Colistin is now one
of the last drug resorts to fight Gram-negative bacteria.
0003. The world is facing an enormous and growing threat
from the emergence of microorganisms that are resistant to a
wide range of currently available antibiotics. For example,
infections caused by utility drug resistant gram-negative bac
teria, particularly Pseudomonas aeruginosa and Acineto
bacter baumannii, are becoming a critical challenge in com
promised hospital patients (e.g. patients in intensive care
unite, patients with cystic fibrosis or diffuse panbroncholitis),
and with seemingly trivial infections in sites Such as middle
and central ear and eyes. The level of resistance to front line
anti-pseudomonal agents is alarmingly high. Of particular
concern are reports on antibiograms of P aeruginosa and A.
baumannii in hospital outbreaks, where colistin, a member of
the polymyxin class of antibiotics, is the only effective anti
biotic. Unfortunately, polymyxin-resistant P aeruginosa has
been isolated from patients with eye infections, ear infec
tions, and particularly in the sputum of patients with cystic
fibrosis. The appearance of polymyxin-resistant gram-nega
tive pathogens is of great concern.
0004 Colistin is a polymyxin antibiotic produced by cer
tain strains of Bacillus polymyxa. Colistin is effective against
Gram negative bacilli, except Proteus and Burkholderia
cepacia and is particularly effective against multi-drug resis
tantisolates of Pseudomonas aeruginosa, Acinetobacter bau
mannii and Klebsiella pneumoniae. Colistin interacts with
the bacterial cytoplasmic membrane, changing its permeabil
ity therefore causing leakage of the cell contents.
0005. However, despite the ability of colistin to kill multi
drug resistant bacteria, its use in treating patients is highly
limited by the fact that it is highly neurotoxic and nephrotoxic
and results in adverse renal effects. Colistin must be admin
istered parenterally as it is not absorbed by the gastrointesti
nal tract, mucous membranes or intacted or denuded skin.
Colistin administration, particularly intravenously, was aban
doned due to a high incidence of nephrotoxicity and neuro
toxicity. In regard of the emergence of bacteria resistant to
major classes of antibiotics and of the lack of new classes of
antibiotics, colistin is re-appearing as a valuable antibacterial
therapeutic agent. In fact it is being reintroduced in clinical
practice due to the emergence of multidrug-resistant gram
negative bacteria therefore reflecting the critical situation to
cure Some bacterial infection.
0006 Colistin, also called Colimycin or polymycin E, a
cyclic lipopeptide, penetrates the cell wall of gram negative
bacteria by a self induced mechanism by chelating divalent
Feb. 4, 2010
ions, it destabilizes the wall and can insinuate into it. It per
forates the cell wall, causing distortion of this structure and
the release of intracellular constituents in the outside. Colistin
appears to re-emerge as recent clinical findings have been
published, focusing on evaluation of efficacy, emerging resis
tance and potential toxicities. The use of colistin at current
therapeutic effective doses is often associated with neurotox
icity and nephrotoxicity. Therefore it would be desirable to
reduce the toxicity of colistin.
0007. Therefore, there is great need in the art to for an
effective therapy against multi-drug resistant microorgan
isms, in particular gram-negative bacteria and multi-drug
resistant gram-negative bacteria, for example, polymyxin
resistant gram-negative bacteria Such as P. aeruginosa and A.
baumannii, or to increase the sensitivity of microorganisms to
treatment.
SUMMARY OF THE INVENTION
0008. The present invention relates to methods and com
positions to potentate the efficacy of antimicrobial agents, for
example antimicrobial peptides. The inventors have discov
ered that by inactivating certain genes, the effectiveness of
antimicrobial agents, such as antimicrobial peptides is
increased. Accordingly, the present invention relates to
inhibitors of such genes as enhancers of antimicrobial agents.
In alternative embodiments, the present invention relates to a
composition comprising one or more antimicrobial agents
with one or more enhancers of antimicrobial agents.
0009. As disclosed herein, the inventors have discovered a
method of reducing the toxicity of antimicrobial agents. Such
as for example, Colistin by co-administering an enhancer of
the antimicrobial agent, for example an inhibitor of the gene
products as disclosed herein in Table 4 or Table 2.
0010. Using a functional genomics approach, the inven
tors discovered that inactivation of specific gene products
render the bacteria more susceptible to colistin, a potent but
toxic antibiotic. For example, the inventors demonstrate that
deletion of the ubiFH and iscS loci render the bacterial cells
more sensitive to colistin. The inventors also demonstrate that
that potassium tellurite, an inhibitor of IscS, potentiates colis
tin efficacy. Accordingly, the inventors have discovered dif
ferent combination therapies with colistin, and their use with
a wide variety of major classes of antibiotics.
0011. Another aspect of the present invention relates to
methods to screen for gene products, which when inactivated,
potentiate the effect of antimicrobial agents, such as antimi
crobial peptides. Another aspect of the invention relates to
identification of inhibitors of such genes, and thus identifica
tion of enhancers of antimicrobial agents, such as antimicro
bial peptides. Another aspect of the present invention relates
to a pharmaceutical formulation comprising a composition of
at least one antimicrobial agent, such as an antimicrobial
peptide, and at least one enhancer to the antimicrobial agent.
In such an embodiment, the enhancer of the antimicrobial
agent is an inhibitor of a gene product, which by inactivating
the gene product potentiates the effectiveness of the antimi
crobial agent. For example, the enhancer of the antimicrobial
agent, such as an inhibitor of one of the genes listed in Table
1 or 4 as disclosed herein potentiates the efficacy of the
antimicrobial agent, e.g. peptides such as colistin, to a greater
extent than if either was used alone (synergy), and vice versa,
the antimicrobial agent potentiates the effect of the enhancer
of antimicrobial peptide, referred to as bi-directional synergy.
In some embodiments, the enhancer of antimicrobial agent
US 2010/0028334 A1
may not have any antimicrobial effect when used on its own,
whereas when Such enhancer of an antimicrobial agent is
used concurrently with an antimicrobial agent, it may have
antimicrobial activity.
0012. In a further embodiment, the present invention
relates to the use of a composition comprising an antimicro
bial agent, Such as an antimicrobial peptide, and an enhancer
to the antimicrobial agent for the treatmentofa Subject. In one
Such embodiment, the composition comprising an antimicro
bial agent and an enhancer thereof is used to prevent and/or
inhibit the growth of a microorganism. In alternative embodi
ments, the pharmaceutical formulations of the present inven
tion are used for the treatment and/or prophylaxis of an infec
tion in a subject.
0013. In some embodiments, the antimicrobial agent is a
peptide. Such as a lipopeptide, and in another embodiment,
the lipopeptide is a cyclic lipopeptide. In some embodiments,
the lipopeptide or cyclic lipopeptide is a polymyxin or
belongs to the polymyxin class of antibiotics orderivatives or
analogues thereof, which are defined in more detail below. In
Some embodiments, the polymyxin is polymyxin A, B1, B2,
D1, D2, E1, E2, F, G, M, P S and/or T or any derivative,
analogue or variant thereof. In some embodiments the poly
myxin is a colistin, for example, but not limited to colistin A
and/or colistin B. In some embodiments, the polymyxin is in
the form of a salt, for example a methoane sulfonate or sulfate
salt or a colistin salt.
0014. Otherantimicrobial agents can be used, for example
but not limited to, Small molecules, peptides, peptidomimet
ics, chemicals, compounds, and any entity that inhibits the
grown and/or kills a microorganism.
0015. In some embodiments, the gene products, which
when inactivated potentiate antimicrobial agents effective
ness are, for example but not limited to the genes as shown in
Table 1, for example, agaA, atp A, atpC, atpB, atp), atpE.
atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK,
lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA,
sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.
yiaY, yidK, yihV, yfhC), yibn and/or homologues, variants
and fragments thereof. In some embodiments, the gene prod
ucts are atpA, atpH, betB, guaA, guaB, lip A, lySA, rpiA,
and/or trXA or homologues, variants and fragments thereof.
0016. In some embodiments, the enhancers of antimicro
bial agents, such as antimicrobial peptides are for example,
compounds that inhibit the gene products which when inac
tivated potentiate the antimicrobial agent, for example anti
microbial peptides. Examples of such inhibitors are for
example but not limited to, mefloquine, Venturicidin A, anti
mycin, myxothiazol, Stigmatellin, diuron, iodoacetamide,
potassium tellurite hydrate, all L-vinylglycine, N-ethylmale
imide, L-allyglycine, diaryduinoline, betaine aldehyde chlo
ride, acivein, psicofuraine, buthionine Sulfoximine, diami
nopemelic acid, 4-phospho-D-erythronhydroxamic acid,
motexafin gadolinium and/or Xycitrin or modified versions or
analogues thereof. In particular embodiments, the enhancer
of antimicrobial agents is mefloquine or antimycin,
myxothiazol, Stigmatellin, diuron, iodoacetamide, potassium
tellurite hydrate, all L-vinylglycine, N-ethylmaleimide, L-al
lyglycine. The present invention also provides methods for
the modification of the enhancers of antimicrobial peptides
described herein, using structure-based design methods. Also
encompassed in the present invention is the use of structure
based design methods to design a single molecule that com
Feb. 4, 2010
prises both an antimicrobial agent, for example an antimicro
bial peptide and an enhancer of antimicrobial agents
described herein.
0017. In further embodiments, the enhancers of antimicro
bial agents are any molecule, compound and/or drug which
inhibits and/or inactivates a gene that results in the potentia
tion of an antimicrobial agent, such as, for example an anti
microbial peptide. In some embodiments, such inhibitors to
the gene products include antibodies (polyclonal or mono
clonal), neutralizing antibodies, antibody fragments, chi
meric antibodies, humanized antibodies, recombinant anti
bodies, peptides, proteins, peptide-mimetics, aptamers,
oligonucleotides, hormones, Small molecules, nucleic acids,
nucleic acid analogues, carbohydrates or variants thereofthat
function to inactivate the nucleic acid and/or protein of the
gene products identified herein, and those as yet unidentified.
Nucleic acids include, for example but not limited to, DNA,
RNA, oligonucleotides, peptide nucleic acid (PNA), pseudo
complementary-PNA (pcPNA), locked nucleic acid (LNA),
RNAi, microRNAi, siRNA, shRNA etc. The inhibitors can be
selected from a group of a chemical, Small molecule, chemi
cal entity, nucleic acid sequences, nucleic acid analogues or
protein or polypeptide or analogue or fragment thereof.
0018. In some embodiments, the enhancer of the antimi
crobial agent as disclosed herein does not have any effect on
decreasing cell viability when it is used by itself. In some
embodiments, an enhancer of the antimicrobial agent is
selected based on its ability to enhance the effect or activity of
the antimicrobial agent. In some embodiments therefore, an
enhancer of the antimicrobial agent may not have any anti
pathogenic effects or ability to decrease cell viability when
used itself, and thus will have no antibiotic or no antimicro
bial activity when used on its own, but when such enhancer of
the antimicrobial agent is used concurrently with an antimi
crobial agent, such as, for example with colistin, the enhancer
of the antimicrobial agent functions to enhance the activity of
the antimicrobial agent.
0019. In some embodiments, the microorganisms of the
invention are bacterium. In some embodiments, the bacteria
are gram positive and gram negative bacteria. In some
embodiments, the bacteria are multi-drug resistant bacterium.
In further embodiments, the bacteria are polymyxin-resistant
bacterium. Examples of gram-negative bacteria are for
example, but not limited to P. aeruginosa, A. baumannii,
Salmonella spp., Klebsiella pneumonia, Shigella spp. and/or
Stenotrophomonas maltophilia.
0020. In some embodiments, the pharmaceutical compo
sitions of the invention are administered in coformulations
with one or more other antibiotics or therapeutic agents, for
example but not limited to aminoglycosides, carbapenemes,
cephalosporins, cephems, glycoproteins fluroquinolones/
quinolones, oxazolidinones, penicillins, Streptogramins, Sul
fonamides and/or tetracyclines.
0021. In a further embodiment, the invention provides
methods of administration of the compositions and/or phar
maceutical formulations of the invention and include any
means commonly known by persons skilled in the art. In some
embodiments, the Subject is any organism, including for
example a mammalian, avian or plant. In some embodiments,
the mammalian is a human, a domesticated animal and/or a
commercial animal.
0022. One aspect of the present invention relates to a com
position comprising an antimicrobial agent and an enhancer
to the antimicrobial agent, wherein the enhancer to the anti
US 2010/0028334 A1
microbial agent is an inhibitor of a gene product that by
inactivating the gene product potentiates the effectiveness of
the antimicrobial agent. In some embodiments, the antimi
crobial agent is a peptide, for example but not limited to a
lipopeptide Such as a cyclic lipopeptide. In some embodi
ments, a cyclic lipopeptide is a polymyxin class of antibiotic
or derivative thereof, for example but not limited to a poly
myxin is selected from the group of polymyxin A, B1, B2, D1,
D2, E1 and/or E2, F, G, M, P S and/or T. In some embodi
ments, the polymyxin is selected from polymyZin B1 and/or
polymyxin B2 or from colistin A and/or colistin B.
0023. In some embodiments, the composition as disclosed
herein comprises colistin in the form of a colistin salt, for
example methane Sulphonate and/or sulfate salt.
0024. In some embodiments, the compositions comprises
an enhancer of an antimicrobial agent which is an agent which
inactivates a gene or gene product, for example agene or gene
product is selected from a group consisting of agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or
ynjD or homologues, variants or fragments thereof. In some
embodiments the compositions comprises an enhancer of an
antimicrobial agent, which is an agent that inactivates a gene
or gene product which is encoded by any of the genes listed in
Table 1 or Table 4. In some embodiments, an enhancer of an
antimicrobial agent is an inhibitor of a gene listed in Table 1
or 4, for example suitable inhibitors useful as enhancers of
antimicrobial agents as disclosed herein include, but are not
limited to mefloquine, Venturicidin A, diarycuinoline, betaine
aldehyde chloride, acivein, psicofuraine, buthionine Sulfox
imine, diaminopemelic acid, 4-phospho-D-erythronhydrox
amic acid, motexafin gadolinium and/or Xycitrin or modified
versions or analogues thereof.
0025. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is atpA, atpF or atpH or
homologues, variants or fragments thereof, and the enhancer
of the antimicrobial agent or inhibitor is mefloquine and/or
Venturicidin A and/or diarycuinoline or modified versions or
analogues thereof.
0026. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product betB or homologues or vari
ants thereof, and the inhibitor is betaine aldehyde chloride or
modified versions or analogues thereof.
0027. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is guaA or guaB or homo
logues or variants thereof, and the inhibitor or enhancer of the
antimicrobial agent is acivin and/or psicofluranine or modi
fied versions or analogues thereof.
0028. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is LipA or homologues or
variants thereof, and the inhibitor or enhancer of antimicro
bial agent is buthionine sulfoximine or modified versions or
analogues thereof.
0029. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is LySA or homologues or
Feb. 4, 2010
variants thereof, and the inhibitor or enhancer of antimicro
bial agent is diaminopimelic acid or modified versions or
analogues thereof.
0030. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is rpiA or homologues or
variants thereof, and the enhancer is inhibitor or enhancer of
antimicrobial agent is 4-phospho-D-erythronhydrixamic acid
or modified versions or analogues thereof.
0031. In some embodiments, the composition as disclosed
herein comprises an enhancer of an antimicrobial agent
which inhibits the gene product is trx A or homologues or
variants thereof, and the inhibitor or enhancer of antimicro
bial agent is motexafin gadolinium and/or Xycitrin acid or
modified versions or analogues thereof.
0032. In some embodiments, the enhancer of antimicro
bial agent inhibitor comprises a small molecule, nucleic acid,
nucleic acid analogue, peptide, ribosome, antibody, and vari
ants and fragments thereof. In some embodiments, the
nucleic acid is DNA, RNA, DNA/RNA hybrids, a triple helix
nucleic acid, an antisense nucleic acid, ribozyme oran RNAi
or a homologue or fragment thereof. In some embodiments,
the nucleic acid analogues comprises peptide nucleic acid
(PNA), pseudo-complementary PNA (pcPNA), locked
nucleic acid (LNA) and variants thereof.
0033. In some embodiments, the compositions as dis
closed herein are useful in the inhibition of growth and/or
decrease in viability of a microorganism, for example a bac
terium. In some embodiments, the bacterium is a gram-posi
tive bacterium, and in alternative embodiments, the bacte
rium is a gram-negative bacterium. In some embodiments, the
microorganism is a multi-drug resistant microorganism, for
example but not limited to a multi-drug resistant microorgan
ism is resistant to at least one member of the polymyxin class
of antibiotics or derivatives or analogues thereof. In some
embodiments, a multi-drug resistant microorganism is poly
myxin resistant Pseudomonas aeruginosa, Acinetobacter
baumannii, Stenotrophomonas maltophilia, Salmonella spp.
and/or Klebsiella pneumonia and/or Shigella spp. In some
embodiments, the multi-drug resistant microorganism is
resistant to colistin and/or a colistin salt.
0034. In some embodiments, the composition as disclosed
herein, can be administered at different times, for example a
antimicrobial agent can be administered prior to, or simulta
neously with, the administration of the enhancer of the anti
microbial agent or an inhibitor of the gene product.
0035 Another aspect of the present invention relates to a
pharmaceutical formulation comprising the composition as
disclosed herein and a pharmaceutically acceptable carrier. In
Some embodiments, the composition as disclosed herein us
useful to treat an infection caused by a microorganism. In
Some embodiments, the use of the composition as disclosed
herein comprises the amount of the antimicrobial agent is at
least 25% less than the same antimicrobial agent in an
isogenic cell except for the addition of the enhancer of anti
microbial agent without a reduction of antimicrobial effect.
0036) Another aspect of the present invention relates to a
method of killing and/or preventing or inhibiting growth of a
microorganism comprising administering to a subject in need
thereof an effective amount of a pharmaceutical formulation
of the composition as disclosed herein. In some embodi
ments, the present invention provides a method of treatment
and/or prophylaxis of an infection caused by a microorganism
comprising steps of administering to a subject in need thereof
US 2010/0028334 A1
an effective amount of a pharmaceutical formulation of the
composition as disclosed herein.
0037. In some embodiments, the compositions and meth
ods as disclosed herein are administered to a Subject, for
example, where a Subject is mammalian, avian or a plant. In
Some embodiments the Subject is a mammalian, for example
a human. In further embodiments, the mammalian is a domes
ticated animal.
0038. In some embodiments, the compositions and meth
ods as disclosed herein are administered to a subject to
decrease the viability of a microorganism, for example a
bacterium, Such as a gram-positive bacterium or a gram
negative bacterium.
0039. In some embodiments, the compositions and meth
ods as disclosed herein are administered to a Subject to treat
an infection, for example infections such as, but not limited
to, infection is selected from the group consisting of bacterial
wound infections, mucosal infections, enteric infections, sep
tic conditions, infectious in airways, cerebrospinal fluid,
blood, eyes and skin. In some embodiments, an infection is
caused by gram-negative bacteria. In some embodiments an
infection is caused by multi-drug resistant gram negative
bacteria, Such as for example multi-drug resistant microor
ganism is resistant to at least one member of the polymyxin
class of antibiotics and synthetic derivatives thereof. In some
embodiments, the compositions and methods as disclosed
herein are administered to a subject that is infected with a
microorganism, such as, for example, a microorganisms are
selected from a group comprising: Pseudomonas aeruginosa,
Acinetobacter baumannii, Stenotrophomonas maltophilia,
Salmonella spp and/or Klebsiella pneumonia and/or Shigella
spp.
0040 Another aspect of the present invention relates to the
use of an antimicrobial agent and an enhancer to the antimi
crobial agent, wherein the enhancer inhibits a gene product of
the treatment or prophylaxis of an infection caused by a
microorganism, wherein the antimicrobial agent and the
inhibitor interact synergistically against the microorganism.
In some embodiments, the infection is selected from the
group consisting of bacterial wound infections, mucosal
infections, enteric infections, septic conditions, infectious in
airways, cerebrospinal fluid, blood, eyes and skin.
0041 Another aspect of the present invention relates to the
use of an antimicrobial agent and an enhancer of an antimi
crobial agent, or an inhibitor to a gene product in the manu
facture of a medicament for inhibiting or preventing produc
tion of at least one pathogenic factor by a microorganism. In
Some embodiments, the use of an antimicrobial agent and an
inhibitor to a gene product in the manufacture of a medica
ment for inhibiting or preventing production of at least one
pathogenic factor by a microorganism in a Subject being
treated with a medicament comprising a antimicrobial agent
and the inhibitor.
0042 Another aspect of the present invention relates to a
pharmaceutical formulation comprising the composition as
disclosed herein, wherein the enhancer of the antimicrobial
agent enhances the anti-pathogenic activity of the antimicro
bial agent.
0043. Another aspect of the present invention relates to a
method for identifying gene products, wherein inactivation of
the gene products potentiates antimicrobial agent activity, the
method comprising the steps of: (a) mutating one or more
genes in a cell, (b) contacting the cell with the antimicrobial
agent, incubating the cell for a sufficient amount of time to
Feb. 4, 2010
allow for growth, and (c) assessing the number of cells,
wherein the number of cells is compared to steps (a)-(c)
performed on a non-mutated cell, wherein the decrease in
numbers of cells identifies a gene product that when inacti
vated potentiates antimicrobial peptide activity. In some
embodiments, the cell used in the method to identify antimi
crobial agents is a bacterium, for example E. Coli. In alterna
tive embodiments, the cell is selected from a group consisting
of Bacillus cereus, Bacillus anthracis, Bacillus cereus,
Bacillus anthracis, Clostridium botulinum, Clostridium dif
ficle, Clostridium tetani, Clostridium perfingens, Coryne
bacteria diptheriae, Enterococcus (Streptococcus D), List
eria monocytogenes, Pneumococcal infections
(Streptococcus pneumoniae), Staphylococcal infections and
Streptococcal infections; Gram-negative bacteria including
Bacteroides, Bordetella pertussis, Brucella, Campylobacter
infections, enterohaemorrhagic Escherichia coli (EHEC/E.
coli O157:17), enteroinvasive Escherichia coli (EIEC),
enterotoxigenic Escherichia coli (ETEC), Haemophilus
influenzae, Helicobacter pylori, Klebsiella pneumoniae,
Legionella spp., Moraxella catarrhalis, Neisseria gonor
rhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas
aeruginosa, Salmonella spp., Shigella spp., Vibrio cholera
and Yersinia, acid fast bacteria including Mycobacterium
tuberculosis, Mycobacterium avium-intracellulars, Myco
bacterium johnei, Mycobacterium leprae, atypical bacteria,
Chlamydia, Myoplasma, Rickettsia, Spirochetes, Treponema
pallidum, Borrelia recurrentis, Borrelia burgdorfii and Lep
to spira icterohemorrhagiae, Actinomyces, Nocardia, P
aeruginosa, A. baumannii, Salmonella spp., Klebsiella pneu
monia, Shigella spp. and/or Stenotrophomonas maltophilia
and other miscellaneous bacteria. In some embodiments, the
antimicrobial agent used in the methods to identify a gene
product that when inactivated potentiates antimicrobial pep
tide activity is any antimicrobial agent commonly known by
persons of ordinary skill in the art or are disclosed herein. In
Some embodiments, in the methods to identify gene product
that when inactivated potentiates antimicrobial peptide activ
ity, mutation results in inactivation of the gene or gene prod
uct.
BRIEF DESCRIPTION OF FIGURES
0044 FIG. 1 shows a schematic drawing of the screen
shown in a 96 well plate format for simplicity, the screen was
done in a 384 well plate format. The arrow points to the
expected result. Example of digital image analysis with the
iscS and ubiH mutants as example.
0045 FIG. 2 shows a flow chart of the genetic and chemi
cal screen leading to a combination therapy between colistin
and potassium tellurite. Mutants are listed in table 1 in the
Supplement section.
0046 FIG. 3 shows a heat map of gene homology of the
identified geneloci in different bacteria. The conserved genes
loci are dark, and less conserved gene loci are light in color.
0047 FIG. 4 shows an example of colistin and mefloquine
synergy in a minimum inhibitory concentration (MIC) assay
in E. coli. As the concentration of mefloquine increases, the
minimum inhibitory concentration of colistin decreases.
0048 FIG. 5 shows a phenotypic response to colistin. FIG.
5A shows the log change in colony forming units per ml
(CFU/mil) of wild-type, BW25113 and ubiHandiscS mutant
E. coli cells (meants.d.) in presence or absence of colistin.
The cells were incubated with colistinata final concentration
of 1.25 ug/ml. At regular time points (3 and 6 hours), aliquots
US 2010/0028334 A1 Feb. 4, 2010

were taken and serially diluted into PBS. 10 ul of cells were
plated onto LBagar no colistin plates. Number of colonies
were counted after overnight incubation at 37 C. Colony
count was normalized to get the same starting amount of cells
for all strains. FIG. 5B shows the phenotypic response to
colistin and potassium tellurite. Log change in colony form
ing units per ml (CFU/ml) of wild-type, BW25113 E. coli
cells (meants.d.) in presence or absence of colistin and potas
sium tellurite. Cell were grown in the absence or presence of
colistin (1.25 ug/ml), potassium tellurite (1.6 ug/ml) alone
and combined. At regular time points (3 and 6 hours)) aliquots
were taken and serially diluted into PBS. 10 ul of cells were
plated onto LBagar no colistin plates. Number of colonies
were counted after overnight incubation at 37 C. Colony
count was normalized to get the same starting amount of cells
for all strains.
DETAILED DESCRIPTION OF THE INVENTION
0049. The present invention relates to methods and com
positions to potentate the efficacy of antimicrobial agents
such as antimicrobial peptides. The inventors have discovered
that by inactivating certain genes, the effectiveness of an
antimicrobial agent, Such as an antimicrobial peptide is
increased. Accordingly, the present invention relates to
inhibitors of such genes as enhancers of antimicrobial agents,
for example antimicrobial peptides. In alternative embodi
ments, the present invention relates to compositions compris
ing one or more antimicrobial agents, such as antimicrobial
peptides, with one or more enhancers thereof.
0050. The present invention relates to methods and com
positions comprising antimicrobial agents, such as peptides
and enhancers thereof. Such as enhancers of antimicrobial
peptides. In particular embodiments, the antimicrobial agents
are antimicrobial peptides, and in Some embodiments, the
antimicrobial peptides are lipopeptides, in particular cyclic
lipopeptides. Enhancers to antimicrobial agents, for example
enhancers of antimicrobial peptides can include nucleic
acids, peptide, nucleic acid analogues, phage, phagemids,
polypeptides, peptidomimetics, antibodies, Small or large
organic or inorganic molecules or any combination of the
above. The enhancers to the antimicrobial agents can be natu
rally occurring or non-naturally occurring (e.g., recombinant)
and are sometimes isolated and/or purified.
0051. In some embodiments, the enhancers of antimicro
bial agents such as antimicrobial peptides are for example,
compounds that inhibit the gene products which when inac
tivated potentiate the antimicrobial agents. Examples of Such
inhibitors are for example but not limited to, mefloquine,
Venturicidin A, diaryduinoline, betaine aldehyde chloride,
acivein, psicofuraine, buthionine Sulfoximine, diaminope
melic acid, 4-phospho-D-erythronhydroxamic acid, motexa
fin gadolinium and/or xycitrin or modified versions or ana
logues thereof. In particular embodiments, an enhancer of
antimicrobial agents is mefloquine
0052. In some embodiments the enhancers to antimicro
bial agents, for example antimicrobial peptides inhibit a gene
or gene product. Examples of Such gene products are shown
in Table 1, and include for example, but are not limited to
agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA,
cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA,
nuvC, papa, pdxH, phnL, potE. rpiA, such3, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV.yfhO, yibn and/or homologues, variants and fragments
thereof.

TABLE 1.
List of example genes that can be inhibited.
SEQ GeneBank
ID NO: Gene BLAT ID ID Accession ID DESCRIPTION

1 agaA b313S 23671.98 AAC76169 putative N-acetylgalactosamine-6-phosphate deacetylase

2 atp A b3734 790172 AAC76757 membrane-bound ATP synthase, F1 sector, alpha-subunit
3 atpB b3738 7901.76 AAC76761 membrane-bound ATP synthase, FO sector, subunita
4 atpC b3731 790169 AAC76754 membrane-bound ATP synthase, F1 sector, epsilon-subunit
5 aptl) b3732 7901.70 AAC767SS Membrane-bound ATP synthase, F1 sector, beta-subunit
6 atpE b3737 7901.75 AAC76760 membrane-bound ATP synthase, FO sector, subunit c
7 atpC b3733 790171 AAC76756 membrane-bound ATP synthase, F1 sector, gamma-Subunit
8 atpH b3735 790173 AAC76758 membrane-bound ATP synthase, F1 sector, delta-subunit
9 betB bO312 786SO4 AAC7341S NAD+-dependent betaine aldehyde dehydrogenase
10 cscA b2810 789175 AAC75852 orf, hypothetical protein
11 cscA b3162 7895.53 AAC761.96 inducible ATP-independent RNA helicase
12 cscB b1680 787970 AAC747SO orf, hypothetical protein
13 epC bOS88 786803 AAC73689 ATP-binding component of ferric enterobactin transport
14 guaA b2507 788.854 AAC75560 GMP synthetase (glutamine-hydrolyzing)
15 guaB b2SO8 788.855 AAC7SS61 MP dehydrogenase
16 iscS b2S30 788879 AAC75583 putative aminotransferase
17 kdgK b3526 7899.45 AAC76SS1 ketodeoxgluconokinase
18 lip A bO628 786846 AAC73729 ipoate synthesis, Sulfur insertion?
19 lysA b2838 7892O3 AAC75877 diaminopimelate decarboxylase
20 minima 87081837 AAC74217.2 RNA (5-methylaminomethyl-2-thiouridylate)-
methyltransferase
21 nuvC b2S30 48994898 AAT48142 cysteine desulfurase monomer
22 papA b3734 790172 AAC76757 membrane-bound ATP synthase, F1 sector, alpha-subunit
23 pdhik 787926 AAC74710 pyridoxine 5'-phosphate oxidase,
24 phnL b4096 790534 AACTTO57 ATP-binding component of phosphonate transport
25 pot bO692 786908 AAC73786 putrescine transport protein
26 rpiA b2914 78928O AAC75951 ribosephosphate isomerase, constitutive
27 SucB bO727 786946 AAC73821 2-oxoglutarate dehydrogenase (dihydrolipoyltransSuccinase
E2 component)
US 2010/0028334 A1 Feb. 4, 2010

TABLE 1-continued
List of example genes that can be inhibited.
SEQ GeneBank
ID NO: Gene BLAT ID ID Accession ID DESCRIPTION
28 trix A b3781 790215 AACT6786 thioredoxin 1
29 tuSB b3343 789741 ACC76368 tRNA 2-thiouridine synthesizing protein
30 tuSE bO969 87081811 ACC74OSS tRNA 2-thiouridine synthesizing protein
31 ubiF b3833 2367307 AACT6836 2-octaprenyl-6-methoxy-1,4-benzoquinone --> 2-octaprenyl
3-methyl-6-methoxy-1,4-benzoquinone
32 ubi b2907 7892.74 AACTS945 2-octaprenyl-6-methoxyphenol--> 2-octaprenyl-6-methoxy-1,
4-benzoquinone
33 uncA b3734 710172 AACT6757 membrane-bound ATP synthase, F1 sector, alpha-subunit
34 wisB b2907 7892.74 AACTS945 2-octaprenyl-6-methoxyphenol--> 2-octaprenyl-6-methoxy-1,
4-benzoquinone
35 yeeY b2O15 788326 AACTSO76 putative transcriptional regulator LYSR-type
36 yiaY b3589 79001S AACT6613 putative oxidoreductase
37 yid K b3679 7901 13 AACT6702 putative cotransporter
38 yihV b3883 790316 AAD13445 putative kinase
39 yfhO b2S30 788879 AACTSS83 putative aminotransferase
40 ybN b4049 790483 AACF7019 orf, hypothetical protein
41 ynjD b1756 788053 AACT4826 YnjD is an ATP-binding component of a predicted metabolite
uptake ABC transporter

0053 Definitions
0054 For the purpose of this specification it will be clearly
understood that the word “comprising means “including but
not limited to’, and that the word “comprises have a corre
sponding meaning.
0055 As used in this specification the singular forms “a”,
“an and the include the plural references unless the context
clearly dictate otherwise, for example, reference to “an anti
microbial peptide' or “an antimicrobial agent” includes mix
tures of antimicrobial peptides or agents respectively, refer
ence to “a antimicrobial agent' includes mixtures of two or
more such components, and the like.
0056. The term “microorganism’ includes any micro
scopic organism or taxonomically related macroscopic
organism within the categories algae, bacteria, fungi, yeast
and protozoa or the like. It includes Susceptible and resistant
microorganisms, as well as recombinant microorganisms.
Examples of infections produced by Such microorganisms are
provided herein. In one aspect of the invention, the antimi
crobial agents and enhancers thereofare used to target micro
organisms in order to prevent and/or inhibit their growth,
and/or for their use in the treatment and/or prophylaxis of an
infection caused by the microorganism, for example multi
drug resistant microorganisms. In some embodiments, gram
negative microorganisms are also targeted.
0057 The anti-pathogenic aspects of the invention target
the broader class of “microorganism' as defined herein. How
ever, given that a multi-drug resistant microorganism is so
difficult to treat, the antimicrobial agent and an enhancer of
antimicrobial agent in the context of the anti-pathogenic
aspect of the invention is Suited to treating all microorgan
isms, including for example multi-drug resistant microorgan
isms.
0058. Unless stated otherwise, in the context of this speci
fication, the use of the term “microorganism” alone is not
limited to “multi-drug resistant organism’, and encompasses
both drug-susceptible and drug-resistant microorganisms.
The term “multi-drug resistant microorganism” refers to
those organisms that are, at the very least, resistant to more
than two antibiotics in different antibiotic classes. This
includes those microorganisms that have more resistance than
those that are resistant to three or more antibiotics in a single
antibiotic class. This also includes microorganisms that are
resistant to a wider range of antibiotics, i.e. microorganisms
that are resistant to one or more classes of antibiotics.
0059. The term “microorganism’ includes any micro
Scopic organism or taxonomically related macroscopic
organism within the categories algae, bacteria, fungi, yeast
and protozoa or the like. The microorganisms targeted in the
first aspect of the present invention are multi-drug resistant
microorganisms. Preferably, gram-negative microorganisms
are targeted
0060. The term “antimicrobial agent” as used herein refers
to any entity with antimicrobial activity, i.e. the ability to
inhibit the growth and/or kill bacterium, for example gram
positive- and gram negative bacteria. An antimicrobial agent
includes any chemical, peptide, peptidomimetic, entity or
moiety, or analogues of hybrids thereof, including without
limitation synthetic and naturally occurring non-proteina
ceous entities. In some embodiments, the antimicrobial agent
is a small molecule having a chemical moiety. For example,
chemical moieties include unsubstituted or substituted alkyl,
aromatic or heterocyclyl moieties including macrollides, lep
tomycins and related natural products or analogues thereof.
Antimicrobial agents can be any entity known to have a
desired activity and/or property, or can be selected from a
library of diverse compounds. The term "agent” as used
herein and throughout the application is intended to refer to
any means such as an organic or inorganic molecule, includ
ing modified and unmodified nucleic acids such as antisense
nucleic acids, RNAi, such as siRNA or shRNA, peptides,
peptidomimetics, receptors, ligands, and antibodies, aptam
ers, polypeptides, nucleic acid analogues or variants thereof.
0061 The term “antimicrobial peptide' as used herein
refers to any peptides with antimicrobial activity, i.e. the
ability to inhibit the growth and/or kill bacterium, for example
gram positive- and gram negative bacteria. The term antimi
crobial peptides encompasses all peptides that have antimi
crobial activity, and are typically, for example but not limited
to, short proteins, generally between 12 and 50 amino acids
long, however larger proteins with Such as, for example
lysozymes are also encompassed as antimicrobial peptides in
US 2010/0028334 A1
the present invention. Also includes in the term antimicrobial
peptide are antimicrobial peptidomimetics, and analogues or
fragments thereof. The term “antimicrobial peptide' also
includes all cyclic and non-cyclic antimicrobial peptides, or
derivatives or variants thereof, including tautomers, see Liet
al. JACS, 2006, 128: 5776-85 and http://aps.unmc.edu/AP/
main.php for examples, which are incorporated herein in their
entirety by reference. In some embodiments, the antimicro
bial peptide is a lipopeptide, and in some embodiments the
lipopeptide is a cyclic lipopeptide. The lipopeptides include,
for example but not limited to, the polymyxin class of anti
microbial peptides.
0062. The term “polymyxin' is used in its broadest sense
to encompass all members of the well known polymyxin class
of antibiotics and synthetic derivatives thereof. Derivatives
within this class are the non-cyclic derivatives of cyclic poly
myxin, derivatives containing amino acid variations, deriva
tives containing Substitutes of the fatty acid components with
other fatty acids or substituents, derivatives with D- and
L-amino acid conversions, and derivatives Substituted with
any one or more optional substituents identified below. Clas
sic polymyxins include, but are not limited to, polymyxin A,
B1, B2, C, D1, D2, E1 and/or E2, FC, M, P S and T. The
polymyxins are cationic detergents and are relatively simple
basic peptides with molecular masses of about 1000-1200
daltons.
0063. In this embodiment, the term “polymyxin resistant”
refers to those microorganisms that are resistant to the mem
ber of the polymyxin class of lipopeptides being used in one
embodiment.
0064. The term “prevent or inhibit growth of a microor
ganism, for example a multi-drug-resistant microorganism
refers to the interference with growth or replication of the
microorganism, which can include but does not necessarily
extend to killing of the microorganism.
0065. The term “treatment and/prophylaxis' refers gener
ally to afflicting a subject, tissue or cell to obtain a desired
pharmacologic arid/or physiologic effect. The effect may be
prophylactic in terms of completely or partially preventing a
disease or sign or symptom thereof, and/or may be therapeu
tic in terms of a partial or complete cure of a disease.
0066. The term “synergy' or “synergistically are used
interchangeably herein refers to the total increase in activity
of the antimicrobial agent and the enhancer of antimicrobial
agent components of the invention, over their single and/or
additive antimicrobial activity. It includes the increase in
activity of only one of the antimicrobial components. In the
present invention, the antimicrobial agent and enhancer of
antimicrobial agent component show at least synergistic anti
pathogenic activity.
0067. The term “bidirectional synergy” refers to the
increase in activity of each antimicrobial component (i.e. the
antimicrobial agent and/or antimicrobial peptide and
enhancer of the agent or enhancer of antimicrobial peptide)
when used in conjunction with the other antimicrobial com
ponent, and not merely an increase in activity of one of the
antimicrobial components. In the present invention, the anti
microbial agent and enhancer of antimicrobial agent compo
nent show at least synergistic antimicrobial activity. Advan
tageously, for example, the antimicrobial agent (for example
antimicrobial peptide) and enhancer of the antimicrobial
agent (for example antimicrobial peptide) show bidirectional
synergistic antimicrobial activity. Stated in other words, for
example, if an antimicrobial agent is the antimicrobial pep
Feb. 4, 2010
tide colistin, and the enhancer of such antimicrobial peptide is
mefloquin, then bidirectional synergy means mefloquin
enhances the activity of the antimicrobial peptide colistin and
Vice versa, the antimicrobial peptide colistin can be used to
enhance the activity of mefloquin.
0068. In the present invention, the antimicrobial agent and
an enhancer of antimicrobial agent show at least synergistic
anti-pathogenic activity. The term “bidirectional Synergy’
refers to the increase in activity of each antimicrobial com
ponent when used in conjunction with the otherantimicrobial
component, and not merely an increase in activity of one of
the antimicrobial components. In the present invention, the
antimicrobial agent and enhancer of the antimicrobial agent
component show at least synergistic, and in some instances
bidirectional Synergistic antimicrobial activity.
0069. The term “anti-pathogenic’ refers to activity inhib
iting or preventing production of a pathogenic factor released
by living microorganisms in a host which leads to destructive
effects of tissues at the site(s) of infection. This includes
inhibition of the expression of flagellin in P. aeruginosa,
perturbing of cytokine production and altering action of poly
morphonuclear cell functions in vivo and ex vivo, and/or the
inhibition of production of pyocyanin, which is produced by
microorganisms such as P. aeruginosa and is blue pigment
which disrupts human ciliary beating in vitro, inhibits epider
mal cell growth and also impedes lymphocyte proliferation.
0070 The term “analog as used herein refers to a com
position that retains the same structure or function (e.g., bind
ing to a receptor) as a polypeptide or nucleic acid herein.
Examples of analogs include peptidomimetics, peptide
nucleic acids, Small and large organic or inorganic com
pounds, as well as derivatives and variants of a polypeptide or
nucleic acid herein. The term “analog as used herein refers to
a composition that retains the same structure or function (e.g.,
binding to a receptor) as a polypeptide or nucleic acid herein.
(0071. The term “derivative' or “variant as used herein
refers to a peptide, chemical or nucleic acid that differs from
the naturally occurring polypeptide or nucleic acid by one or
more amino acid or nucleic acid deletions, additions, Substi
tutions or side-chain modifications. Amino acid substitutions
include alterations in which an amino acid is replaced with a
different naturally-occurring or a non-conventional amino
acid residue. Such substitutions may be classified as “conser
Vative', in which case an amino acid residue contained in a
polypeptide is replaced with another naturally occurring
amino acid of similar character either in relation to polarity,
side chain functionality or size.
0072 Substitutions encompassed by the present invention
may also be “non conservative', in which an amino acid
residue which is present in a peptide is substituted with an
amino acid having different properties. Such as naturally
occurring amino acid from a different group (e.g., Substitut
ing a charged or hydrophobic amino; acid with alanine), or
alternatively, in which a naturally-occurring amino acid is
Substituted with a non-conventional amino acid. In some
embodiments amino acid Substitutions are conservative.
(0073. The term “amino acid” within the scope of the
present invention is used in its broadest sense and is meant to
include naturally occurring L. C.-amino acids or residues, but
is not necessarily restricted to the naturally occurring amino
acids.
0074 By “pharmaceutically acceptable derivative' is
meant any pharmaceutically acceptable salt, hydrate or any
other compound which, upon administration to the Subject, is
US 2010/0028334 A1
capable of providing (directly or indirectly) an antimicrobial
peptide and/or enhancer of antimicrobial component or resi
due thereof.
0075. The term “pro-drug is used herein in its broadest
sense to include those compounds and entities which are
converted in vivo to active antimicrobial agents and/or active
enhancers of antimicrobial agents of the present invention.
0076. The term “tautomer' is used herein in its broadest
sense to include antimicrobial agents and/or or enhancers of
antimicrobial agents which are capable of existing in a state of
equilibrium between two isometric forms. Such compounds
may differ in the bond connecting two atoms or groups and
the position of these atoms or groups in the compound.
0077. The term “isomer' is used herein in its broadest
sense and includes structural, geometric and stereo isomers.
As the antimicrobial agents and/or or enhancers of antimicro
bial agents may have one or more chiral centers, they are
capable of existing in enantiomeric forms.
0078. The term “infection' or “microbial infection' which
are used interchangeably herein refers to in its broadest sense,
any infection caused by a microorganism and includes bac
terial infections, fungal infections, yeast infections and pro
toZomal infections.
0079 Suitable mammals include members of the orders
Primates, Rodentla, Lagomorpha, Cetacea, Homo sapiens,
Carnivora, Perissodactyla and Artiodactyla. Members of the
orders Perissodactyla and Artiodactyla are included in the
invention because of their similar biology and economic
importance, for example but not limited to many of the eco
nomically important and commercially important animals
Such as goats, sheep, cattle and pigs have very similar biology
and share high degrees of genomic homology.
0080. As used herein, the term “effective amount” is
meant an amount of antimicrobial agent and/or enhancers of
antimicrobial agent components of the present invention
effective to yield a desired antibiotic activity. The term “effec
tive amount as used herein refers to that amount of compo
sition necessary to achieve the indicated effect. The specific
“effective amount” will, obviously, vary with such factors as
the particular condition being treated, the physical condition
of the subject, the type of subject being treated, the duration of
the treatment, the route of administration, the type of antimi
crobial agent and/or enhancer of antimicrobial agent, the
nature of concurrent therapy (if any), and the specific formu
lations employed, the ratio of the antimicrobial agent and/or
enhancers antimicrobial agent components to each other, the
structure of each of these components or their derivatives.
0081. As used herein, a “pharmaceutical carrier' is a phar
maceutically acceptable solvent, Suspending agent or vehicle
for delivering the combination of antimicrobial agent and/or
enhancers of antimicrobial agent components to the Subject.
The carrier may be liquid or solid and is selected with the
planned manner of administration in mind. Each carrier must
be pharmaceutically “acceptable' in the sense of being com
patible with other ingredients of the composition and non
injurious to the Subject.
0082. The terms “gene(s) refers to a nucleic acid
sequence (DNA, RNA, or analogs and/or combinations
thereof) that encodes through its template or messenger RNA
a sequence of amino acids characteristic of a specific peptide.
The term “gene can includes intervening, non-coding
regions, as well as regulatory regions, and can include 5' and
3' ends. Examples of genes associated with, when inactivated,
potentiation the effect of antimicrobial agents are, for
Feb. 4, 2010
example but not limited to, agaA, atpA, atpC, atpB, atp).
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE,
rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV, yfhC), yibN and etc., and any
homologs, analogs or fragments thereof.
I0083. The term “gene product(s)' as used herein refers to
include RNA transcribed from a gene, or a polypeptide
encoded by a gene or translated from RNA.
I0084. The term “inhibition” or “inhibit” when referring to
the activity of an antimicrobial agent and/or enhancer of
antimicrobial agent refers to prevention of, or reduction in the
rate of growth. Inhibition and or inhibit when referring to the
activity of an enhancer of antimicrobial agent refers to the
prevention or reduction of activity of a gene or gene product,
that when inactivated potentiates the activity of an antimicro
bial agent.
I0085. The term “homolog’ or “homologous' as used
herein refers to homology with respect to structure and/or
function. With respect to sequence homology, sequences are
homologs if they are at least 50%, preferably at least 60%,
more preferably at least 70%, more preferably at least 80%,
more preferably at least 90%, more preferably at least 95%
identical, more preferably at least 97% identical, or more
preferably at least 99% identical. The term “substantially
homologous' refers to sequences that are at least 90%, more
preferably at least 95% identical, more preferably at least
97% identical, or more preferably at least 99% identical.
Homologous sequences can be the same functional gene in
different species.
I0086. The term “hybridize” refers to interaction of a
nucleotide sequence with a second nucleotide sequence. Such
interaction can be, e.g., in Solution or on a solid Support. Such
as cellulose or nitrocellulose. If a nucleic acid sequence binds
to a second nucleotide sequence with high affinity, it is said to
“hybridize' to the second nucleotide sequence. The strength
of the interaction between the two sequences can be assessed
by varying the stringency of the hybridization conditions.
Under highly stringent hybridization conditions only highly
complementary nucleotide sequences hybridize.
I0087. The term “organism' as used herein includes all
living cells including microorganisms (e.g., viruses, bacteria,
protozoa), plants, and animals (e.g., humans, birds, reptiles,
amphibians, fish, and domesticated animals, such as cows,
chicken, pigs, dogs, and goats).
I0088. The term “peptide' or “polypeptide' are used inter
changeably herein, refers to any composition that includes
two or more amino acids joined to each other by a peptide
bond or peptidomimetic thereof. The term includes both short
chains, which are also commonly referred to in the art as
peptides, oligopeptides and oligomers, for example, and to
longer chains, which generally are referred to in the art as
proteins. The term "peptide' includes all peptides as
described below. It will be appreciated that peptides often
contain amino acids other than the 20 amino acids commonly
referred to as the 20 naturally occurring amino acids, and that
many amino acids, including the terminal amino acids, can be
modified in a given polypeptide, either by natural processes
Such as glycosylation and other post-translational modifica
tions, or by chemical modification techniques which are well
known in the art. Known modifications which can be present
in peptides of the present invention include, but are not lim
ited to, acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme
US 2010/0028334 A1
moiety, covalent attachment of a polynucleotide or poly
nucleotide derivative, covalent attachment of a lipid or lipid
derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, dem
ethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formulation, gamma-c
arboxylation, glycation, glycosylation, GPI anchor forma
tion, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenyla
tion, racemization, selenoylation, Sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginy
lation, and ubiquitination.
0089. The term "peptidomimetic' as used herein refers to
molecules which mimic an aspect of a polypeptide structure.
The term “mimetic” as used herein is any entity, molecule,
chemical, Small or large molecule, organic or inorganic, Syn
thetic or natural, that mimics the mechanism of the molecule
of which it is a mimetic.
0090 The term “purified’ refers to a material (e.g., com
pound, molecule, or structure of interest) that is relatively free
of other materials that it normally is associated with and is
preferably at least 0.1%, 1%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% of total weight of the material.
0091. The term “recombinant as used herein refers with
reference to material (e.g., a cell, a nucleic acid, a protein, or
a vector) indicates that such material has been modified by the
introduction of a heterologous material (e.g., a cell, a nucleic
acid, a protein, or a vector). Thus, for example, recombinant
microorganisms or cells express genes that are not found
within the native (non-recombinant) form of the microorgan
ism or cell or express native genes that are otherwise abnor
mally expressed, under expressed or not expressed at all. For
example, a recombinant antibody is an antibody which is not
normally found in native (non-recombinant) antibodies form.
0092. The term “treatment” or “treating” as used herein
refers to reducing or alleviating symptoms in a Subject, pre
venting symptoms from worsening or progressing, or inhibi
tion, elimination, or prevention of the infection, disorder or
symptoms in a subject who is free therefrom. “Treating as
used herein covers any treatment of, or prevention of an
infection or disease in a vertebrate, a mammal, for example, a
human, and includes: (a) preventing the infection and/or dis
ease from occurring in a subject that may be predisposed to
the infection and/or disease, but has not yet been diagnosed as
having it; (b) inhibiting a infection and/or disease, i.e., arrest
ing its development; or (c) relieving or ameliorating the
effects of the infection and/or disease, i.e., cause regression of
the effects of the infection or disease.
0093. The term “unit dose when used in reference to a
therapeutic composition of the present invention refers to
physically discrete units Suitable as unitary dosage for the
Subject, each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect in association with the required physiologically accept
able diluents, i.e., carrier, or vehicle.
0094. The term “subject' as used herein refers to any
animal having a disease or condition which requires treat
ment with a pharmaceutically active agent. The Subject may
be a mammal, for example a human, or may be a domestic or
commercial or companion animal. While in one embodiment
of the invention it is contemplated that the antimicrobial
agents and/or enhancers of antimicrobial agents of the inven
tion are Suitable for use in medical treatment of humans, it is
Feb. 4, 2010
also applicable to veterinary treatment, including treatment of
companion animals such as dogs and cats, and domestic
animals such as horses, ponies, donkeys, mules, llama,
alpaca, pigs, cattle and sheep, or Zoo animals such as pri
mates, felids, canids, bovids, and ungulates
0.095 Antimicrobial Agents
0096. One aspect of the invention relates to antimicrobial
agents. In some embodiments, the antimicrobial agent is anti
microbial peptide. The term “antimicrobial peptide' refers to
peptides which have antimicrobial activity. In some embodi
ments, the antimicrobial agent may be any peptide with anti
microbial activity, for example cyclic and non-cyclic pep
tides, lipopeptides, and peptides (or proteins) with long
chains of amino acids, for example, lyoSymes etc. and modi
fied versions thereof. In some embodiments, the antimicro
bial peptide is a lipopeptide. The lipopeptides include, for
example, the polymyxin class of antimicrobial peptides. The
term “polymyxin' is used in its broadest sense to encompass
all members of the well known polymyxin class of antibiotics
and synthetic derivatives thereof. Derivatives within this class
are the non-cyclic derivatives of cyclic polymyxin, deriva
tives containing amino acid variations, derivatives containing
substitutes of the fatty acid components with other fatty acids
or substituents, derivatives with D- and L-amino acid conver
sions, and derivatives Substituted with any one or more
optional substituents identified below. Classic polymyxins
include polymyxin A, B1, B2, C, D1, D2, E1 and/or E2, FC,
M. P. S and T. The polymyxins are cationic detergents and are
relatively simple basic peptides with molecular masses of
about 1000-1200 daltons.
0097 Examples of members of the polymyxin class are
polymyxin B (Band B) and polymyxin E (colistin A and B).
Both these members include a cyclic heptapeptide ring with a
tripeptide side chain. It is envisaged that declyclisation of the
ring may resultina peptide with effective antimicrobial activ
ity. Accordingly, cyclic and non-cyclic derivatives and vari
ants of the polymyxins and similar peptides are encompassed
within the term “antimicrobial peptide'.
0098. Also included within the scope of “antimicrobial
peptide' are all the components of polymyxin B and poly
myxin B, as well as synthetic derivatives thereof.
0099. It is envisaged that one or more amino acid sequence
(s) of the peptides above can be varied without significant
effect on the structure or function of the peptide. Thus, the
invention further includes variations of the peptide which
show antimicrobial activity Such variations or mutants
include amino acid deletions, insertions, inversions, repeats
and type substitutions.
0100. The structural formula of polymyxin B (B1 and B2)
is as follows:
R -> (a) L-Dab -> L-Thr -> (a) L-Dab ->
US 2010/0028334 A1 Feb. 4, 2010

-continued
Y-NH2 -continued
(C) L-Dab -- D-Phe -- L-Leu Y-NH2

(C., Y) L-Dab | (C) L-Dab -- D-Leu -- L-Leu

L-Thr -- (C) p -- (C) p (C., Y) L-Dab

Y-NH2 Y-NH2 L-Thr -- (C) L-Dab -- (C) p

0101 Polymyxin B1: R-(+)-6-methyloctanyl
0102 Polymyxin B2: R-(+)-6-methylheptanyl
0103 Phe: Phenylalanine, Thr: Threonine, Leu: Leucine,
Dab=C.Y-diaminobutyric acid, wherein C. and Y indicate the
respective —NH involved in the peptide linkage.
Colistin
0104
0105 Colistin is a multicomponent polypeptide antibiotic,
comprised mainly of colistin A and B, that became available
for clinical use in the 1960s, but was replaced in the 1970s by
antibiotics considered less toxic. There are two forms of
colistin commercially available: colistin sulfate for oral and
topical use, and colistimethate Sodium (sodium colistin meth
ane Sulphonate, colistin Sulfomethate sodium) for parenteral
use (shown herein); both can be delivered by inhalation.
Although there have been a substantial number of clinical
reports on the Successful use of colistin or polymyxin B
(which differs by only one amino acid from colistin) against
infections caused by multidrug-resistant P aeruginosa, A
baumannii, and Kpneumoniae, there is a dearth of informa
tion on the clinical pharmacokinetics, pharmacodynamics,
and toxicodynamics of colistin. Such data are essential for
establishing optimal dosing regimens.
0106 Polymyxin B (colistin) has many different compo
nents. Its basic structure is a cyclic heptapeptide ring and a
tripeptide side chain covalently bound to a fatty acid at the
N-terminus via an acyl group. At least 30 components have
been isolated and thirteen identified. They differ from each
other by the composition of amino acids and fatty acids. The
two major components are colistin A and colistin B. The
structural formula is as follows:
Y-NH2 Y-NH2
0107 Colistin A: R-methyloctanoic acid
0.108 Colistin B: R=6-methylheptanoic acid
0109 Thr: Threonine, Leu: Leucifle, Dab=C.Y-diami
nobutyric acid, wherein C. and Y indicate the respective
—NH involved in the peptide linkage.
0110 Shown below in (A) are structures of Colistin A and
Colistin B. Shown in (B) are the structures of colistimethane
A and B, fatty acid: 6-methyloctanoic acid for colistin A and
6-methylheptanoic acid for colistin B: Thr-thereonine, Leu
leucine, Dab-C.Y-diaminobutyric acid, C. and Y indicate
respective amino groups involved in peptide linkage.
Fatty acid -- (a) L-Dab -- L-Thr --
Y-NH2
(a) L-Dab --
Y-NH2
Y-NH2
-1 (C) L-Dab -- D-Leu -- L-Leu
(C., Y) L-Dab
L-Thr -- (C) L-Dab -- (C) L-Dab
Fatty acid -- (a) L-Dab -- L-Thr --

Fatty acid -- (a) L-Dab -- L-Thr -> CH

SOH

(a) L-Dab --
(a) L-Dab --

CH
SOH
US 2010/0028334 A1 Feb. 4, 2010
11

vivo data. With respect to antibacterial activity against P

-continued aeruginosa, recent studies have indicated that colistimethate
SOH is a non-active prodrug of colistin. A recent review provides
more details on the considerable differences between colis
CH2 timethate and colistin in their chemistry, pharmacokinetics,
and pharmacodynamics.
0114. Another schematic showing the chemical structure
of colistin is shown below:
(C) L-Dab -- D-Leu -- L-Leu
-1
(C., Y) L-Dab Hosseo
L-Thr -- (C) L-Dab -- (C) L-Dab lose O
HN1)
r H H R2
hi, s
lot lot
0111 Amino acid substitutions are typically of single resi
dues, but may be of multiple residues, either clustered or
dispersed. Additions encompass the addition of one or more
naturally occurring or non-conventional amino acid residues. He

Deletion encompasses the deletion of one or more amino acid O NH N OtH

residues.
0112 Minor components of colistin include the poly OI lsO S=O
myxin E3 and E4, norvaline-polymyxin E1, Valine-poly -Sn NH CH
myxin E1, and valine-polymyxin E2, isoleucine-polymyxin HO O
E1, isoleucine-polymyxin E2, polymyxin E7 and isoleucine N
H
O OH
polymyxin E0. In some embodiments, the antimicrobial pep O NH
tide comprises any one or more components of colistin, and in
some embodiments, colistin A and/or colistin B. The propor
tion of colistin A and colistin B in commercial material varies
between pharmaceutical Suppliers and batches, but it is gen Ho1 S No1\N
NH
R
erally between 4.5:1 to 0.9:1. Colistin is available commer H
cially in two forms, colistin Sulphate and sodium colistin colimycin
methaneSulphonate. Sodium colistin methanesulphonate HN
hydrolyses in aqueous media and forms a complex mixture of
partially sulphomethylated derivatives plus colistin. One or
more of the above forms of colistin or its derivatives are
encompassed within the scope of the invention. In some H 2
embodiments, antimicrobial peptide comprises salts of colis HN N N
tin, for example salts of pharmaceutically acceptable cations NH
Such as sodium, potassium, lithium and the like, acid addition O
salts of pharmaceutically acceptable inorganic acids Such as HN O O CH3
hydrochloric orthophosphoric, sulphuric and the like, and/or O
salts of pharmaceutically acceptable organic acids such as H3C O
O NH CH3
acetic, propionic, methanesulfonic and the like. O
0113 Although widely used in the literature, the terms HO
colistin and colistimethate are not interchangeable. Colistin HN
(usually used as the Sulphate salt) is a polycation, whereas N
H
colistimethate (used as the Sodium salt) is a polyanion at
physiological pH. Colistimethate is prepared from colistin by NH2
reaction of the free y-amino groups of the five C.Y-diaminobu
tyric acid residues with formaldehyde followed by sodium
bisulphite. Colistimethate is not stable in vitro or in vivo, and
is hydrolysed to a series of methanesulphonated derivatives
plus colistin. Colistin is more stable than colistimethate in
human plasma. The differences in chemistry between colis
timethate and colistin also translate into differences in phar
macokinetics and pharmacodynamics. Whereas colistime
thate is eliminated mainly by the kidney and the urinary
excretion involves renal tubular secretion, colistin is elimi
nated predominantly by the non-renal route because, at least
in part, the compound undergoes very extensive renal tubular
reabsorption. After intravenous administration of colistime
thate (sodium), the plasma half-lives of colistimethate are colistin (free base)
approximately half of those of the colistin generated from in
US 2010/0028334 A1
12
0115. Where R1 and R2 are as follows:
component I
ca
CH3
aest
CH3
component III CH3
O2 CH3 as
CH3
component IV an
ca CH
CH3
0116 Methods to produce pure biologically active colis
tin, or a pharmaceutically acceptable salt thereof, and meth
ods for its use for treatment for Pseudmonomia aeruginosa,
Stenotrophomonas maltophilia and other are disclosed in
U.S. Pat. No. 5,767,068 and International Application WO
98/20839 which are incorporated herein in its entirety by
reference. In some embodiments, the colistin can be admin
istered with additional agents, as disclosed in International
Patent Number WO2006/045156, which is incorporated
herein by reference. In particular, International Patent Num
ber WO2006/045156 discloses using colistin with a mac
rolide component, such as erythromycin, clarithromycin,
azithromycin and other components with a lactone ring,
where the combination of the macrollide and colistinenhances
the anti-pathogenic effects of the macrollide component and
the colisitin as compared to their effects alone.
0117. In one embodiment, the antimicrobial peptide com
prises colistin methanesulphonate and/or colistin Sulphate. In
another embodiment, the antimicrobial peptide comprises
colistin Sulphate.
0118. It is envisaged that variation of these components,
for example, by Substituting a D-amino acid residue for the
same or different L-amino acid residue or vice versa, varying
the R substituents and/or conservative amino acid substitu
tions, while maintaining the synergistic is antimicrobial
activity with the peptide enhancer component of the inven
tion, is encompassed within the scope of the invention.
0119 Colisitin is associated with neurotoxicity and neph
rotoxicity. The inventors have discovered a dosage regimen
and combination of colistin with enhancers of antimicrobial
agents as disclosed herein, colistin can be used at reduced
doses for the same effect and thereby the inventors have
discovered a method whereby colistin can be administered to
reduce toxicity effects. Colistin, a cyclic lipopeptide, pen
etrates the cell wall of G-bacteria by a self induced mecha
nism by chelating divalent ions, it destabilizes the wall and
can insinuate into it. Without being bound by theory, colistin
Feb. 4, 2010
CH
CH
CH3
CH
CH
perforates the cell wall, causing distortion of this structure
and the release of intracellular constituents in the outside.
I0120 Enhancers of Antimicrobial Agents
I0121 One aspect of the invention relates to enhancers of
antimicrobial agents. In some embodiments, enhancers of
antimicrobial agents are inhibitors of the gene products listed
in Table 1 or Table 4 as disclosed herein. The inactivation of
a gene product by inhibition is considered to potentiate the
effectiveness of the antimicrobial agent if the amount of anti
microbial agent used after inactivation is reduced by at least
10% without adversely affecting the result, for example,
without adversely effecting the level of antimicrobial activity.
In another embodiment, the criteria used to select an enhanc
ers that potentiate the activity of an antimicrobial agent is a
reduction of at least ... 10%, ... 15%, ... 20%, ... 25%, ..
.35%, ... 50%, ... 60%, ...90% and all amounts in-between
of the antimicrobial agent without adversely effecting the
antimicrobial effect when compared to the similar cell with
out the addition of an enhancer of the antimicrobial agent.
I0122. In some embodiments, example of such enhancers
of antimicrobial agents can include nucleic acids, peptides,
nucleic acid analogues, phage, phagemids, polypeptides,
peptidomimetics, antibodies, Small or large organic or inor
ganic molecules or any combination of the above. The
enhancers of antimicrobial agents can also be naturally occur
ring or non-naturally occurring (e.g., recombinant) and are
Sometimes isolated and/or purified.
(0123. In some embodiments, where the antimicrobial
agent is an antimicrobial peptide, the enhancer is an enhancer
to an antimicrobial peptide. In Such embodiments, these
enhancers are, for example, but not limited to inhibitors of
gene products. In some embodiments, the gene products are,
for example but not limited to, agaA, atp A, atpC, atpB, atp).
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE,
rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV. y?hC), yibn and/or ynjD or homo
US 2010/0028334 A1
logues thereof. In some embodiments, the gene products are,
for example but not limited to, those genes listed in Table 4. In
Some embodiments, the gene products are homologues, vari
ants, fragments or Substantially homologous to the following
genes; agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB,
cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK.
yihV. y?hO, yibn and/orynjD or alternatively those listed in
Table 4. In other embodiments, the gene products are, for
example but not limited to, agaA, atp A, atpC, atpB, atp).
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE,
rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD. The identi
fication of gene products, which when inactivated enhance
antimicrobial peptides is discussed in more detail below (see
"Screening to Identify Gene Products that suppress the activ
ity of antimicrobial peptides’). Similarly, identification and
screening of inhibitors of such gene products is also described
in more detail below (see sections titled “Screening For Small
Molecules That Inhibit the Gene Product and “Structure
Based Design Methods to Create Small Molecule enhancers
of antimicrobial peptide').
0124. In some embodiments, enhancers of antimicrobial
agents are inhibitors to the gene products listed in table 1, and
such inhibitors can be the small molecules as disclosed in
Table 2. In some embodiments, an enhancer of the antimicro
bial agent as disclosed herein does not include macrollides,
Such as for example erthyromycin, clarithromycin,
azithromyxin, clindamycin, azithromycin and ketolides Such
as tellithromycin. In some embodiments, the enhancer of the
antimicrobial agent as disclosed herein does not include the
following list of antibiotics; rifampicin, meropenem, ampi
cillin-Sulbactam ciprofloZacin, poperacillin-clavulanic acid,
imipenem, amikacin, gentamicin and ciproflxicin. While
Some of these agents may have been used in combination with
colistin, their ability to decrease the amount of colistin to be
used without decreasing colistin antimicrobial effect as com
pared to the use of colistin alone cannot be determined
because of the limitations in the study design.
012.5 ATP synthases: In one embodiment, the gene prod
uct, which when inactivated potentiates the activity of anti
microbial agents, is for example an ATP synthase. In some
embodiments, the ATP synthase is for example atpA. In alter
native embodiments, the gene product is atp A, atpH and/or
atpH. ATP synthases is a general term for an enzyme that can
synthesize adenosine triphosphate (ATP) from adenosine
diphosphate (ADP) and inorganic phosphate by utilizing
Some form of energy.
0126 An ATP synthase (EC 3.6.3.14) is a general term for
an enzyme that can synthesize adenosine triphosphate (ATP)
from adenosine diphosphate (ADP) and inorganic phosphate
by utilizing some form of energy. ATP syntheses are impor
tant in energy metabolism, for example ATP-proton motive
force interconversion in the synthesis of energy for use by the
cell and/or organism. ATP is formed by proton-conducting,
membrane-bound ATP synthase. ATP synthase is a multi
component enzyme complex consisting of two main compo
nents: F1 and FO. F1 is on the inner surface of the membrane
and is the catalytic center; F1 consists of nine polypeptide
chain subunits of five different types. FO is embedded within
the membrane and forms the membrane proton channel; in E.
coli FO consists of three subunits (A=atpB, BatpF, C-atpE),
Feb. 4, 2010
F1 consists of five subunits (alpha=atpA, beta-atpl),
gamma atpG, delta atpH, epsilon atpC).
I0127 ATP synthesis is the fundamental process to provide
cell energy and is generated by oxidative phosphorylation.
The Source of energy is an electrochemical gradient of pro
tons issued from electron transfer across the membranes (Se
nior et al., 2002). Theatp operon of the E. coli ATP syntheses
consists of nine genes arranged in the order atp, atpB, atpE.
atpF, atpH, atpA, atpG, atp), and atpC; similar gene organi
Zation have been characterized in other bacteria. Theses gene
products assembled into two components: F1 and F0. F 1 is on
the inner Surface of the membrane and is the catalytic center;
F1 consists of nine polypeptide chain subunits of five differ
ent types. Both the atpA and atpF mutants were primarily
identified in the primary screen using the mutant library. The
atpF mutant was not retained for further studies. The atp A
Sub-unit is conserved among bacterial species and mamma
lians. The level of identity is over 80% for Salmonella sp.
Klebsiella pneumo, Yersina, Haemophilus influenza, Yersinia
and Vibrio cholera.
I0128. Inhibitors of ATP synthases: In some embodiments
where the gene product is an ATP synthase, for example atpA,
atpH and/or atpH, the inhibitor to the gene product, is an
inhibitor to ATP synthases. In some embodiments, the inhibi
tor of ATP synthases is mefloquine or analogues, mimetics or
derivatives thereof. Examples of Mefloquine are the orally
administered antimalarial drug used as a prophylaxis against
and treatment for malaria, known by the trade name Lariam R.
(manufactured by Roche Pharmaceuticals) and chemical
name mefloquine hydrochloride (formulated with HCI).
Mefloquine was developed in the 1970s at the Walter Reed
Army Institute of Research in the U.S. as a chemical synthetic
similar to quinine. In some embodiments, the ATP synthase
inhibitor is Mefloquine hydrochloride, and in some embodi
ments it is a 4-quinolinemethanol derivative. While Meflo
quine is known to have some side effects, its co-administra
tion with an antimicrobial agent, for example an
antimicrobial peptide may enable its use at lower therapeuti
cally effective doses, and Such, reduce the occurrence of side
effects.
0129. In another embodiment, the inhibitor of an ATP
synthase can be selected from; venturicidin A, diaryduino
line, Betaine, Acivin, Psicofuranine or derivatives, mimectics
or analogues thereof. In alternative embodiments, the inhibi
tor of ATP synthases is venturicidin or oligomycin or ossa
mycin or derivatives, mimetics or analogues thereof. The
antibiotics venturicidin, oligomycin and ossamycin were
investigated as potential inhibitors of the Escherichia coli
H+-ATP synthase. It was found that venturicidin strongly
inhibited ATP-driven proton transport and ATP hydrolysis,
while oligomycin weakly inhibited these functions. Inhibi
tion of the H+-ATP synthase by venturicidin and oligomycin
was correlated with inhibition of FO-mediate proton trans
port. Venturicidin had been shown to be an antifungal of
Potential Use in Agriculture.
0.130. Any inhibitor of ATP synthase is also encompassed
for use in the present invention, including those described
herein, as well as those yet unidentified.
I0131 Inhibitors of BetB. In another embodiment, the gene
product, which when inactivated potentiates the activity of
antimicrobial agents, is for example is a Betaine aldehyde
dehydrogenase. In some embodiments, the gene product is,
for example, a NAD-dependent Betaine aldehyde dehydro
genase. In some embodiments, the Betaine aldehyde dehy
US 2010/0028334 A1
drogenase is, for example betB. Bet genes confer protection
against osmotic stress by making the osmoprotectant glycine
betaine from choline. The bet genes are induced by choline,
oxygen, and osmotic stress.
0.132. In some embodiments, an inhibitor of an ATP syn
thases is Betaine. One such inhibitor is, for example but not
limited to Cystadane(R) (betaine anhydrous for oral solution),
which is currently used as an agent for the treatment of
homocystinuria. In another embodiment, an inhibitor of an
ATP synthase which can be used in the methods and compo
sitions as disclosed herein is Acivin, which is an irreversible
inhibitor of gamma-glutamyl transpeptidase (ID50-0.54
mM). Inhibits the enzymatic conversion of LTC4 to LTD4.
Acivin is currently used as a potent anti-tumor and anti
leishmania agent.
0133. In some embodiments where the gene product is a
Betaine aldehyde dehydrogenase, for example betB, the
inhibitor to the gene product, is an inhibitor to Betaine alde
hyde dehydrogenase. In some embodiments, the inhibitor of
Betaine aldehyde dehydrogenase is Betaine or anhydrous
betaine or betaine aldehyde chloride oran analogue orderiva
tive or mimetic thereof. Examples of Betaine are Cystadane(R)
(betaine anhydrous for oral Solution), which is typically used
in the treatment of homocystinuria. Any inhibitor of Betaine
aldehyde dehydrogenase is also encompassed for use in the
present invention, including those described herein, as well as
those yet unidentified.
0134. Inhibitors of GuaA and GuaB: In another embodi
ment, the gene product, which when inactivated potentiates
the activity of antimicrobial agents, is for example is a gua
nine-monophosphate (GMP) synthase or an inosine-5'-
monophosphate (IMP) dehydrogenase or homologues or
variants thereof. In some embodiments, the GMP synthase is,
for example, guaA and IMP dehydrogenase is, for example,
guaB or homologues or variants thereof. GMP synthases and
IMP dehydrogenases important in the conversion of IMP to
AMP and GMP. These are also used for de novo synthesis as
well as in the salvage of purine bases. In some embodiments,
the gene products are for example also purA and purB, which
are required for the reactions to of IMP to AMP, whereas in
other embodiments, the gene products are guaB and guaA,
which are required for synthesis of GMP.
0135) In another embodiment, the gene product, which
when inactivated potentiates the activity of antimicrobial
agents, is for example is a 2-octaprenyl-6-methylphonel
hydroxylase, producing 2-octaprenl-6-methyoxy-1,4-benzo
quinone and is involved in the electron transport pathway.
0136. In some embodiments where the gene product is a
guanine-monophosphate (GMP) synthase, for example guaA
or an inosine-5'-monophosphate (IMP) dehydrogenase, for
example guaB, the enhancer of the antimicrobial agent is for
example Acivin and/or Psicofurine. Acivin is an irreversible
inhibitor of gamma-glutamyl transpeptidase (IDso 0.54
mM) and inhibits the enzymatic conversion of LTC4 to LTD4.
Typically it is used as a potent anti-tumor and anti-leishmania
agent. Psicofuranine was shown to have some antibacterial
activities. (Hanka, 1959). Any inhibitor of a guanine-mono
phosphate (GMP) synthase, for example guaA or an inosine
5'-monophosphate (IMP) dehydrogenase, for example guaB
is also encompassed for use in the present invention, includ
ing those described herein, as well as those as yet unidenti
fied. Accordingly, in another embodiment, an inhibitor of an
ATP synthase which can be used in the methods and compo
Feb. 4, 2010
sitions as disclosed herein is Psicofuranine, which was shown
to have some antibacterial activities. (Hanka, 1959).
0.137 Inhibitors of LipA: In another embodiment, the
gene product, which when inactivated potentiates the activity
of antimicrobial agents, is for example a Lipoyl synthase, or
a homologue or variant thereof. An example of a lipoyl Syn
thase is LipA, which is an iron-sulfur protein with SAM
dependent chemistry. Lipoyl synthase is involved in lipoic
acid biosynthesis, where the pathway of lipoic acid biosyn
thesis consists solely of two steps in which two sulfur atoms
are introduced into the carbon skeletonat C-8 and C-6. White
showed that the sulfur atom in lipoic acid is derived from
cysteine. As with the incorporation of the sulfur atom into
biotin, the insertion of sulfur in lipoid acid occurs without the
formation of either unsaturated or hydroxylated intermedi
ates. Two different sulfur atoms are inserted in lipoic acid
biosynthesis, whereas a single Sulfur atom inserts into both
carbon atoms in biotin biosynthesis. There is evidence to
indicate that a in each case, a single enzyme is responsible for
the sulfur insertions in each molecule, for example the bioB
gene product in biotin biosynthesis and the lip A gene product
in lipoic acid biosynthesis. Accordingly, in one embodiment,
the enhancer of antimicrobial agentis, for example an inhibi
tor to Lipoyl synthase, or a homologue or variant thereof. In
one embodiment, the inhibitor of Lipoyl synthase, is for
example, buthionine sulfoximine or derivatives or modified
versions, mimetics or analogues thereof. Any inhibitor of
Lipoyl synthase, for example lip A is also encompassed for
use in the present invention, including those described herein,
as well as those as yet unidentified.
0.138. Inhibitors of Lys A: In another embodiment, the
gene product, which when inactivated potentiates the activity
of antimicrobial agents, is for example a diaminopimelate
decarboxylase or a homologue or variant thereof. An example
of a diaminopimelate decarboxylase is lySA. The expression
of diaminopimelate decarboxylase depends on the intracel
lular concentration of both diaminopimelate, which acts as an
inducer, and lysine, which acts as a corepressor. This double
regulation reflects the special situation of the diaminopime
late decarboxylase, as a catabolic enzyme for diaminopime
late and an anabolic enzyme for lysine. Moreover, ppGpp
must play a role inlySA expression, which is modified in relA
mutants. Accordingly, in one embodiment, the enhancer of
antimicrobial agent is an inhibitor to diaminopimelate decar
boxylase, or homologues or mimetics or variants thereof. In
one embodiment, the inhibitor is for example, diami
nopimelic acid and/or lysine or analogues, derivatives or
homologues thereof. Any inhibitor of a diaminopimelate
decarboxylase, for example lySA, is also encompassed foruse
in the present invention, including those described herein, as
well as those as yet unidentified.
0.139. Inhibitors of RpiA: In another embodiment, the
gene product, which when inactivated potentiates the activity
of antimicrobial agents, is for example a ribose-5-phosphate
isomerase or a homologue or variant thereof. In some
embodiments, the ribose-5-phosphate isomerase is ribose-5-
phosphate isomerase A. In some embodiments, the ribose-5-
phosphate isomerase A is alkali-inducible. An example of
ribose-5-phosphate isomerase A is rpiA. Ribose 5-phosphate
isomerases interconvert ribose 5-phosphate and ribulose
5-phosphate. This reaction allows the synthesis of ribose
from the pentose phosphate pathway and represents a means
for the salvage of carbohydrates after nucleotide breakdown.
However, such a dual role for a metabolic pathway is unusual
US 2010/0028334 A1
and presents problems for effective metabolic regulation.
Two unrelated types of enzymes can catalyze the reaction.
RpiA is highly conserved and present in almost all organisms.
In E. coli and Salmonella, the enzyme is constitutively
expressed. The second type of ribose isomerase, RpiB, is
Sometimes referred to as AIsB because it can also take part in
the metabolism of the rare Sugar allose. E. coli strains defec
tive in rpiA are ribose auxotrophs, despite the presence of
wild-typerpiB. Ribose prototrophs of an rpiA genetic back
ground arise spontaneously. RpiA exhibits a a/131(a13)/13/a
fold, some portions of which are similar to proteins of the
alcoholdehydrogenase family. The two subunits of the dimer
in the asymmetric unit have different conformations, repre
senting the opening/closing of a cleft. The enzyme presum
ably acts by an acid-base catalysis mechanism.
0140. Accordingly, in one embodiment, the enhancerofan
antimicrobial agent is an inhibitor to ribose-5-phosphate
isomerase, for example an inhibitor to ribose-5-phosphate
isomerase A, or a homologue or variant thereof. In one
embodiment, the inhibitor is for example, 4-phospho-D-er
thronhydrixamic acid or analogues, derivatives or homo
logues or mimetics thereof. Any inhibitor of ribose-5-phos
phate isomerases, for example rpiA are also encompassed for
use in the present invention, including those described herein,
as well as those as yet unidentified.
0141 Inhibitors of TrXA. In another embodiment, the gene
product, which when inactivated potentiates the activity of
antimicrobial agents, is for example is a Thioredoxin or a
homologue or variant thereof. An example of a Thioredoxin is
trXA or trxB, which has general chaperone activity, and func
tions as a processitiviy factor for phage T7 gene 5 DNA
polymerase. Accordingly, in one embodiment, an enhancer of
antimicrobial agent is an inhibitor to thioredoxin or a variant
or homologue thereof. In one embodiment, the inhibitor of
thioredoxin is, for example, motexafin gadolinium and/or
Xycistrin or derivatives, analogues or variants or mimetics
thereof. Any inhibitor of thioredoxin, for example an inhibitor
of trXA, is also encompassed for use in the present invention,
including those described herein, as well as those as yet
unidentified.
0142. In some embodiments, the gene product is involved
in RNA modifications. Biosynthesis of thionucleoside is a
complex process associated with Sulfur metabolism and
involving numerous proteins including, but not limited to,
iscS, tuSA, tusB and mnmA.
0143. In another embodiment, the gene product, which
when inactivated potentiates the activity of antimicrobial
agents, is for example is a ubi or a homologue or variant
thereof. An example of a ubigenes is ubiH, which is involved
in the ubiquinone synthesis pathway. Ubiquinone is also
referred to as coenzyme Q. ubiH is a 2-octaprenyl-6-methox
yphenol hydroxylase and produces 2-octaprenyl-6-methoxy
1,4-benzoquinone. Coenzyme Q is found in the membranes
Examples of enhancers of antimicrobial agents (referred to as “inhibitors') which inhibit the
genes (or gene products) which, when inactivated, potentiate the effect of antimicrobial agents.
SEQ ID GeneBank Accession
NO: Gene
44 ace.
2 atp A
9 betB
45 fo
Feb. 4, 2010
of endoplasmic reticulum, peroxisomes, lysosomes, Vesicles
and notably the inner membrane of the mitochondrion where
it is an important part of the electron transport chain; there it
passes reducing equivalents to acceptors such as Coenzyme
Q: cytochrome c-oxidoreductase. ubiH gene product is con
served among bacteria showing over 75% identity with the
ubiH homologs in Shigella, salmonella and Klebsiella. The
level of identity with Pseudomonas is lower at 40%.
0144. Inhibitors of ubiH: ubiH gene which is in the
ubiquinone synthesis pathway and the atpA which is a com
ponent of the ATP synthase indirectly or directly involved in
the electron transfer chain. Ubiquinone is part of a cascade of
electron transfer leading to the production of ATP by the ATP
synthase. The electron transport chain is the major consumer
of oxygen in the cell. The cascade of redox reactions allows
the phosphorylation of ADP, forming ATP utilizing the
energy derived from various Substrates through the central
metabolism (glycolysis, TCA cycle) while reducing NADU
or FADH2. The reasons for these genes once inactivated
genetically or chemically to potentiate colistin are not clear. It
is possible that the membrane potential chain is required to
maintain an intact plasma membrane, as the gene products
ubiH and atpA are necessary but not essential tough for this
process.
0145. It is also encompassed within the present invention
that an antimicrobial agent disclosed herein can be combined
with any of the inhibitors of the gene products mentioned
above, thereby enhancing the activity of antimicrobial agent.
AS Such, inhibitors to the gene products; agaA, atp A, atpC,
atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA,
guaB, iscS, kdgK, lip A, lySA, mnmA. nuvC, papa, pdxH.
phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH,
uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD
or homologues or variants thereofare referred to as "enhanc
ers of antimicrobial agents’ or "enhancers of antimicrobial
peptides' herein. In some embodiments, an inhibitor of a
gene product is any inhibitor or agent which inhibits the
function of a gene listed in Table 4. Such enhancers of anti
microbial agents are for example, but not limited to, meflo
quine, Venturicidin A, antimycin A, myxothiazol, Stigma
tellin, diuron, idoacetamide, potassium tellurite hydrate,
aDL-vinylglycine, N-Ethylmaleimide, L-Allyglycine, dia
ryduinoline, betaine aldehyde chloride, acivein, psicofuraine,
buthionine Sulfoximine, diaminopemelic acid, 4-phospho-D-
erythronhydroxamic acid, motexafin gadolinium and/or
Xycitrin and are Summarized in Table 2. In some embodi
ments, one or more enhancer of antimicrobial agent can be
used to enhance or potentiate the activity of antimicrobial
agents described herein. Any combination of antimicrobial
agent enhancers can be used, in any amount, in any form and
by any route of administration. In some embodiments, the
enhancers of the antimicrobial agent are administered at the
same time or sequentially in any order.
TABLE 2
ID ID Inhibitor
2-oxoglutarate or 3-hydroxybutyrate
1790172 AACT6757 mefloquine, venturicidin A
1786SO4 AACT341S betaine aldehyde chloride
Sulfamethoxazole, Phosphanilic acid
US 2010/0028334 A1
16
TABLE 2-continued
Examples of enhancers of antimicrobial agents (referred to as “inhibitors') which inhibit the
genes (or gene products) which, when inactivated, potentiate the effect of antimicrobial agents.
SEQID GeneBank Accession
NO: Gene ID ID Inhibitor
14 guaA 1788.854 AAC75560 acivicin, psicofuranine
15 guaB 1788.855 AAC75561 Tiazofurin, mycophenolic acid
19 lipA 1786846 AAC73729 buthionine Sulfxoximine
20 lySA 1789203 AAC75877 diaminopimelic acid
46 nuo menaquinone oxidoreductase
27 rpiA 1789280 AAC75951 4-phospho-D-erythronohydroxamic acid
47 shC iethylpyrocarbonate, Methylmalonic acid, Myeloperoxidase
29 trix A 1790215 AAC76786 motexafin gadolinuim, Xyctrin
0146 Inhibitors of Gene Products
0147 In some embodiments, the inhibitors to the gene
products which when inactivated, potentiate the effect of
antimicrobial agents, include for example antibodies (poly
clonal or monoclonal), neutralizing antibodies, antibody
fragments, peptides, proteins, peptide-mimetics, aptamers,
oligonucleotides, hormones, Small molecules, nucleic acids,
nucleic acid analogues, carbohydrates or variants thereofthat
function to inactivate the nucleic acid and/or protein of the
gene products identified herein, and those as yet unidentified.
Nucleic acids include, for example but not limited to, DNA,
RNA, oligonucleotides, peptide nucleic acid (PNA), pseudo
complementary-PNA (pcPNA), locked nucleic acid (LNA).
RNAi, microRNAi, siRNA, shRNA etc. The inhibitors can be
selected from a group of a chemical, Small molecule, chemi
cal entity, nucleic acid sequences, nucleic acid analogues or
protein or polypeptide or analogue or fragment thereof. In
some embodiments, the nucleic acid is DNA or RNA, and
nucleic acid analogues, for example can be PNA, pcPNA and
LNA. A nucleic acid may be single or double stranded, and
can be selected from a group comprising: nucleic acid encod
ing a protein of interest, oligonucleotides, PNA, etc. Such
nucleic acid sequences include, for example, but not limited
to, nucleic acid sequence encoding proteins that act as tran
Scriptional repressors, antisense molecules, ribozymes, Small
inhibitory nucleic acid sequences, for example but not limited
to RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense
oligonucleotides etc. A protein and/or peptide inhibitor or
fragment thereof, can be, for example, but not limited to
mutated proteins; therapeutic proteins and recombinant pro
teins. Proteins and peptides inhibitors can also include for
example; mutated proteins, genetically modified proteins,
peptides, synthetic peptides, recombinant proteins, chimeric
proteins, antibodies, humanized proteins, humanized anti
bodies, chimericantibodies, modified proteins and fragments
thereof.
0148. In some embodiments, an enhancer of an antimicro
bial agent, such as, but not limited to those listed under
inhibitors in Table 2 can be combined with the antimicrobial
agent to form one dual mode of action compound. Such
methods are well known by person of ordinary skill in the art
and include chemical conjugation of two molecules or con
jugation by any means known in art. Methods of conjugation
of two molecules or entities to form a binary conjugate is
described in PCT Patent Application No: WO99/66944,
which is specifically incorporated herein in its entirety by
reference. In some embodiments, where the enhancer is a
polypeptide. Such as for example an antibody or peptide
inhibitor as disclosed below, methods of conjugation can be,
Feb. 4, 2010
for example, by chemical means, linkers and the like. In some
embodiments, the conjugation may be by protein fusion, the
methods of which are well know in the art.
0149. In some embodiments, the pharmaceutical compo
sition can comprise a pharmaceutically acceptable carrier and
pharmaceutically effective amounts of a microbial agent and
an enhancer of an antimicrobial agent which are polypeptide
and proteins. In one embodiment, the antimicrobial agent is a
colistin, or a homologue or analogue thereof, and in another
embodiment, the enhancer of an antimicrobial agent is an
inhibitory antibody or polypeptide inhibitor of one of the
genes listed in Table 1 or Table 4. In one embodiment, the
antimicrobial agent and an enhancer of an antimicrobial agent
can be conjugated together, using such methods of protein or
polypeptide conjugation which are well known in the art. One
can use any method for conjugation of molecules known by
persons of ordinary skill in the art, for example, conjugation
by chemical means, covalent bonds, linkers and the like. In
Some embodiments, the conjugation may be protein fusion,
the methods of which are well known in the art. For example,
BioVertis of Vienna has a dual action compound called
Oxaquin, which combines the therapeutic moieties of two
different antibiotic compounds into one molecule.
0150. In some embodiments, multi-binding agents are
useful in the methods and compositions as disclosed herein,
for example multi-binding agents which comprise an antimi
crobial agents such as colistin and an enhancer of Such anti
microbial agent, such as those inhibiting at least one gene
listed in Table 2 or Table 4. Multivalent binding interactions
are characterized by the concurrent interaction of multiple
ligands with multiple ligand binding sites on one or more
cellular receptors. Multivalent interactions differ from collec
tions of individual monovalent interactions by imparting
enhanced biological and/or therapeutic effect. Just as multi
Valent binding can amplify binding affinities; it can also
amplify differences in binding affinities, resulting in
enhanced binding specificity as well as affinity. An example
of a multi-binding agent is an avimer, which relates to a
peptide agent which is capable of binding to one or more sites.
0151. Antibodies
0152. In some embodiments, inhibitors of genes and/or
gene products useful in the methods of the present invention
that function as enhancers to antimicrobial peptides include,
for example, antibodies, including monoclonal, chimeric
humanized, and recombinant antibodies and fragment
thereof. In some embodiments, neutralizing antibodies can be
used as inhibitors of the gene products identified herein.
Antibodies are readily raised in animals such as rabbits or
mice by immunization with the gene product, which when
US 2010/0028334 A1
inactivated, potentiate the effect of an antimicrobial agent.
Immunized mice are particularly useful for providing sources
of B cells for the manufacture of hybridomas, which in turn
are cultured to produce large quantities of monoclonal anti
bodies. Chimeric antibodies are immunoglobin molecules
characterized by two or more segments or portions derived
from different animal species. Generally, the variable region
of the chimeric antibody is derived from a non-human mam
malian antibody, Such as murine monoclonal antibody, and
the immunoglobin constant region is derived from a human
immunoglobin molecule. Preferably, both regions and the
combination have low immunogenicity as routinely deter
mined. Humanized antibodies are immunoglobin molecules
created by genetic engineering techniques in which the
murine constant regions are replaced with human counter
parts while retaining the murine antigenbinding regions. The
resulting mouse-human chimeric antibody should have
reduced immunogenicity and improved pharmacokinetics in
humans. Some examples of high affinity monoclonal antibod
ies and chimeric derivatives thereof, useful in the methods of
the present invention, are described in the European Patent
Application EP 186,833; PCT Patent Application WO
92/16553; and U.S. Pat. No. 6,090,923.
0153. In one embodiment of this invention, the inhibitor to
the gene products identified herein can be an antibody mol
ecule or the epitope-binding moiety of an antibody molecule
and the like. Antibodies provide high binding avidity and
unique specificity to a wide range of target antigens and
haptens. Monoclonal antibodies useful in the practice of the
present invention include whole antibody and fragments
thereof and are generated in accordance with conventional
techniques, such as hybridoma Synthesis, recombinant DNA
techniques and protein synthesis. Useful monoclonal anti
bodies and fragments may be derived from any species (in
cluding humans) or may be formed as chimeric proteins
which employ sequences from more than one species. Human
monoclonal antibodies or “humanized' murine antibody are
also used in accordance with the present invention. For
example, murine monoclonal antibody may be “humanized'
by genetically recombining the nucleotide sequence encod
ing the murine Fv region (i.e., containing the antigenbinding
sites) or the complementarily determining regions thereof
with the nucleotide sequence encoding a human constant
domain region and an Fc region. Humanized targeting moi
eties are recognized to decrease the immunoreactivity of the
antibody or polypeptide in the host recipient, permitting an
increase in the half-life and a reduction in the possibly of
adverse immune reactions in a manner similar to that dis
closed in European Patent Application No. 0.411,893 A2. The
murine monoclonal antibodies should preferably be
employed in humanized form. Antigen binding activity is
determined by the sequences and conformation of the amino
acids of the six complementarily determining regions (CDRS)
that are located (three each) on the light and heavy chains of
the variable portion (Fv) of the antibody. The 25-kDa single
chain FV (ScFV) molecule, composed of a variable region
(VL) of the light chain and a variable region (VH) of the
heavy chain joined via a short peptide spacer sequence, is the
Smallest antibody fragment developed to date. Techniques
have been developed to display schv molecules on the surface
of filamentous phage that contain the gene for the ScFv. Sclv
molecules with a broad range of antigenic-specificities can be
present in a single large pool of ScFV-phage library.
Feb. 4, 2010
0154) One limitation of scFv molecules is their monova
lent interaction with target antigen. One of the easiest meth
ods of improving the binding of a Schv to its target antigen is
to increase its functional affinity through the creation of a
multimer. Association of identical scFv molecules to form
diabodies, triabodies and tetrabodies can comprise a number
of identical Fv modules. These reagents are therefore multi
valent, but monospecific. The association of two different
ScFv molecules, each comprising a VH and VL domain
derived from different parent Ig will form a fully functional
bispecific diabody. A unique application of bispecific ScFVS is
to bind two sites simultaneously on the same target molecule
via two (adjacent) Surface epitopes. These reagents gain a
significant: avidity advantage over a single Sclv or Fab frag
ments. A number of multivalent scEv-based structures has
been engineered, including for example, miniantibodies,
dimeric miniantibodies, minibodies, (sclv)2, diabodies and
triabodies. These molecules span a range of Valence (two to
four binding sites), size (50 to 120 kDa), flexibility and ease
of production. Single chain Fv antibody fragments (scFvs)
are predominantly monomeric when the VHandVL domains
are joined by, polypeptide linkers of at least 12 residues. The
monomer schv is thermodynamically stable with: linkers of
12 and 25 amino acids length under all conditions. The non
covalent diabody and triabody molecules are easy to engineer
and are produced by shortening the peptide linker that con
nects the variable heavy and variable light chains of a single
schv molecule. The schv dimers are joined by amphipathic
helices that offer a high degree of flexibility and the minian
tibody structure can be modified to create a dimeric bispecific
(DiBi) miniantibody that contains two miniantibodies (four
schv molecules) connected via a double helix. Gene-fused or
disulfide bonded scFv dimers provide an intermediate degree
of flexibility and are generated by straightforward cloning
techniques adding a C-terminal Gly4CyS sequence. Scv
CH3 minibodies are comprised of two schv molecules joined
to an IgG CH3 domain either directly (LD minibody) or via a
very flexible hinge region (Flex minibody). With a molecular
weight of approximately 80kDa, these divalent constructs are
capable of significant binding to antigens. The Flex minibody
exhibits impressive tumor localization in mice. Bi- and tri
specific multimers can be formed by association of different
schv molecules. Increase in functional affinity can be reached
when Fab or single chain Fv antibody fragments (scFv) frag
ments are complexed into dimers, trimers or larger aggre
gates. The most important advantage of multivalent scvs
over monovalent scFv and Fab fragments is the gain in func
tional binding affinity (avidity) to target antigens. High avid
ity requires that Schv multimers are capable of binding simul
taneously to separate target antigens. The gain in functional
affinity for schv diabodies compared to schv monomers is
significant and is seen primarily in reduced off-rates, which
result from multiple binding to two or more target antigens
and to rebinding when one Fv dissociates. When such schv
molecules associate into multimers, they can be designed
with either high avidity to a single target antigen or with
multiple specificities to different target antigens. Multiple
binding to antigens is dependent on correct alignment and
orientation in the Fv modules. For full avidity in multivalent
ScFVs target, the antigen binding sites must point towards the
same direction. If multiple binding is not sterically possible
then apparent gains in functional affinity are likely to be due
the effect of increased rebinding, which is dependent on dif
fusion rates and antigen concentration. Antibodies conju
US 2010/0028334 A1
gated with moieties that improve their properties are also
contemplated for the instant invention. For example, antibody
conjugates with PEG that increases their half-life in vivo can
be used for the present invention. Immune libraries are pre
pared by Subjecting the genes encoding variable antibody
fragments from the B lymphocytes of naive or immunized
animals or patients to PCR amplification. Combinations of
oligonucleotides which are specific for immunoglobulin
genes or for the immunoglobulin gene families are used.
Immunoglobulin germ line genes can be used to prepare
semisynthetic antibody repertoires, with the complementa
rily-determining region of the variable fragments being
amplified by PCR using degenerate primers. These single-pot
libraries have the advantage that antibody fragments againsta
large number of antigens can be isolated from one single
library. The phage-display technique can be used to increase
the affinity of antibody fragments, with new libraries being
prepared from already existing antibody fragments by ran
dom, codon-based or site-directed mutagenesis, by shuffling
the chains of individual domains with those of fragments
from naive repertoires or by using bacterial mutator strains.
0155 Alternatively, a SCID-humouse, for example the
model developed by Genpharm, can be used to produce anti
bodies, or fragments thereof. In one embodiment, a new type
of high avidity binding molecule, termed peptabody, created
by harnessing the effect of multivalent interaction is contem
plated. A short peptide ligand was fused via a semirigid hinge
region with the coiled-coil assembly domain of the cartilage
oligomeric matrix protein, resulting in a pentameric multiva
lent binding molecule. In preferred embodiment of this inven
tion, ligands and/or chimeric inhibitors can be targeted to
tissue- or tumor-specific targets by using bispecific antibod
ies, for example produced by chemical linkage of an anti
ligand antibody (Ab) and an Ab directed toward a specific
target. To avoid the limitations of chemical conjugates,
molecular conjugates of antibodies can be used for produc
tion of recombinant bispecific single-chain Abs directing
ligands and/or chimeric inhibitors at cell Surface molecules.
Alternatively, two or more active agents and or inhibitors
attached to targeting moieties can be administered, wherein
each conjugate includes a targeting moiety, for example, a
different antibody. Each antibody is reactive with a different
target site epitope (associated with the same or a different
target site antigen). The different antibodies with the agents
attached accumulate additively at the desired target site. Anti
body-based or non-antibody-based targeting moieties may be
employed to deliver a ligand or the inhibitor to a target site.
Preferably, a natural binding agent for an unregulated antigen
is used for this purpose. For example, diseases such as
hepatoma or myeloma are generally characterized by unregu
lated IL-6 receptors for which IL-6 acts as an autocrine or
paracrine moiety with respect to rapid proliferation of these
target cell types. For the treatment of Such ailments, IL-6 may
therefore be employed as a targeting moiety in a targeting
protocol of the present invention.
0156 Nucleic Acid Inhibitors
0157. It will be appreciated by those of skill that the genes
identified herein and those identified by the methods of the
present invention can be readily manipulated to alter the
amino acid sequence of a protein. Genes for example, but not
limited agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB,
cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK.
Feb. 4, 2010
yihV, yfhC), yibn and/or ynjD or homologues or variants
thereof can be manipulated by a variety of well known tech
niques for in vitro mutagenesis, among others, to produce
variants of the naturally occurring human protein or fragment
thereof, herein referred to as muteins, may be used in accor
dance with the invention.
0158. The variation in primary structure of muteins of
agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA,
cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA,
nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV. y?hO, yibn and/orynjDare useful in the invention, for
instance, may include deletions, additions and Substitutions.
The Substitutions may be conservative or non-conservative.
The differences between the natural protein and the mutein
generally conserve desired properties, mitigate or eliminate
undesired properties and add desired or new properties.
0159. Similarly, techniques for making small oligopep
tides and polypeptides that inactivate and/or function as
dominant negative versions (i.e. inactive versions) of larger
proteins from which they are derived are well known and have
become routine in the art. Thus, peptide analogs of gene
products of the invention that inactivate the gene product also
are useful in the invention.
(0160. In some embodiments, RNA interference or
“RNAi can be used as enhancers of antimicrobial agents. In
Such an embodiment, a RNAi molecule that negatively regu
lates the expression of the gene products of the invention, for
example but not limited to agaA, atpA, atpC, atpB, atp),
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE,
rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV. y?hC), yibn and/or ynjD or homo
logues or variants thereof, can be used as enhancers of anti
microbial peptides in the present invention. RNAi is a term
initially coined by Fire and co-workers to describe the obser
vation that double-stranded RNA (dsRNA) can block gene
expression when it is introduced into worms (Fire et al. (1998)
Nature 391, 806-811). dsRNA directs gene-specific, post
transcriptional silencing in many organisms, including verte
brates, and has provided a new tool for studying gene func
tion. RNAi involves mRNA degradation of a target gene.
Results showed that RNAi is ATP-dependent yet uncoupled
from mRNA translation. That is, protein synthesis is not
required for RNAi in vitro. In the RNAi reaction, both strands
(sense and antisense) of the dsRNA are processed to small
RNA fragments or segments of from about 21 to about 23
nucleotides (nt) in length (RNAS with mobility in sequencing
gels that correspond to markers that are 21-23 nt in length,
optionally referred to as 21-23 nt RNA). Processing of the
dsRNA to the small RNA fragments does not require the
targeted mRNA, which demonstrates that the small RNA
species is generated by processing of the dsRNA and not as a
product of dsRNA-targeted mRNA degradation. The mRNA
is cleaved only within the region of identity with the dsRNA.
Cleavage occurs at sites 21-23 nucleotides apart, the same
interval observed for the dsRNA itself, suggesting that the
21-23 nucleotide fragments from the dsRNA are guiding
mRNA cleavage. Isolated RNA molecules (double-stranded;
single-stranded) of from about 21 to about 23 nucleotides
mediate RNAi. That is, the isolated RNAs mediate degrada
tion of mRNA of a gene to which the mRNA corresponds
(mediate degradation of mRNA that is the transcriptional
product of the gene, which is also referred to as a target gene).
US 2010/0028334 A1
Isolated RNA molecules specific to G6PD mRNA, which
mediate RNAi are antagonists useful in the method of the
present invention. Alternative nucleic acid and nucleic acid
analogues can be used as enhancers of antimicrobial peptides,
for example oligonucleotides, antisense nucleic acid con
structs, siRNA, microRNA, shRNA etc.
0161. In some embodiments of the invention suitable
enhancers of antimicrobial agents may be administered to the
Subject in a vector. The vector may be a plasmid vector, a viral
vector, or any other suitable vehicle adapted for the insertion
and foreign sequence and for the introduction into eukaryotic
cells. The vector can be an expression vector capable of
directing the transcription of the DNA sequence of the agonist
or antagonist nucleic acid molecules into RNA. Viral expres
sion vectors can be selected from a group comprising, for
example, retroviruses, lentiviruses, Epstein Barr virus-,
bovine papilloma virus, adenovirus- and adeno-associated
based vectors or hybrid virus of any of the above. In one
embodiment, the vector is episomal. The use of a suitable
episomal vector provides a means of maintaining the agonist
orantagonist nucleic acid molecule in the Subject in high copy
number extra chromosomal DNA thereby eliminating poten
tial effects of chromosomal integration.
0162 Another embodiment of the invention, suitable
enhancers of antimicrobial agents can be achieved by intro
ducing catalytic antisense nucleic acid constructs, such as
ribozymes, which are capable of cleaving RNA transcripts
and thereby preventing the production of wildtype protein.
Ribozymes are targeted to and anneal with a particular
sequence by virtue of two regions of sequence complemen
tary to the target flanking the ribozyme catalytic site. After
binding the ribozyme cleaves the target in a site specific
manner. The design and testing of ribozymes which specifi
cally recognize and cleave sequences of the gene products
described herein, for example but not limited to agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or
ynjD or homologues or variants thereof can be achieved by
techniques well known to those in the art (for example Lleber
and Strauss, (1995) Mol Cell Biol 15:540.551, the disclosure
of which is incorporated herein by reference).
0163 Uses of Antimicrobial Agents and Enhancers
Thereof
0164. Some bacteria, including P. aeruginosa, actively
form tightly arranged multi-cell structures in vivo known as
biofilm. The production of biofilm is important for the per
sistence of infectious processes such as seen in pseudomonal
lung-infections in patients with cystic fibrosis and diffuse
panbronchiolitis and many other diseases. Biofilm is resistant
to phagocytosis by host immune cells and the effectiveness of
antibiotics at killing bacteria in biofilm structures may be
reduced by 10 to 1000 fold. Biofilm production and arrange
ment is governed by quorum sensing systems. The disruption
of the quorum sensing system in bacteria Such as P. aerugi
nosa is an important anti-pathogenic activity as it disrupts the
biofilm formation and also inhibits alginate production
0.165. The term “microorganism’ includes any micro
scopic organism or taxonomically related macroscopic
organism within the categories algae, bacteria, fungi, yeast
and protozoa or the like. The microorganisms targeted in the
Feb. 4, 2010
first aspect of the present invention are multi-drug resistant
microorganisms. In some embodiments, gram-negative
microorganisms are targeted
(0166 Bacterial infections include, but are not limited to,
infections caused by Bacillus cereus, Bacillus anthracis,
Bacillus cereus, Bacillus anthracis, Clostridium botulinum,
Clostridium difficle, Clostridium tetani, Clostridium perfrin
gens, Corynebacteria diptheriae, Enterococcus (Streptococ
cus D), Listeria monocytogenes, Pneumococcal infections
(Streptococcus pneumoniae), Staphylococcal infections and
Streptococcal infections/Gram-negative bacteria including
Bacteroides, Bordetella pertussis, Brucella, Campylobacter
infections, enterohaemorrhagic Escherichia coli (EHEC/E.
coli O157:17) enteroinvasive Escherichia coli (EIEC), entero
toxigenic Escherichia coli (ETEC), Haemophilus influenzae,
Helicobacter pylori, Klebsiella pneumoniae, Legionella spp.,
Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria
meningitidis, Proteus spp., Pseudomonas aeruginosa, Sal
monella spp., Shigella spp., Vibrio cholera and Yersinia, acid
fast bacteria including Mycobacterium tuberculosis, Myco
bacterium avium-intracellulars, Mycobacterium johnei,
Mycobacterium leprae, atypical bacteria, Chlamydia, Myo
plasma, Rickettsia, Spirochetes, Treponema pallidum, Borre
lia recurrentis, Borrelia burgdorfii and Leptospira icterohe
morrhagiae and other miscellaneous bacteria, including
Actinomyces and Nocardia.
(0167. In some embodiments, the microbial infection is
caused by gram-negative bacterium, for example, P aerugi
nosa, A. baumannii, Salmonella spp., Klebsiella pneumonia,
Shigella spp. and/or Stenotrophomonas maltophilia.
0168 Examples of microbial infections include bacterial
wound infections, mucosal infections, enteric infections, sep
tic conditions, pneumonia, trachoma, onithosis, trichomonia
sis and salmonellosis, especially in Veterinary practice.
0169. Examples of infections caused by P. aeruginosa
include: A) Nosocomial infections: 1. Respiratory tract infec
tions in cystic fibrosis patients and mechanically-ventilated
patients; 2. Bacteraemia and sepsis; 3. Wound infections,
particularly in burn wound patients; 4. Urinary tract infec
tions: 5. Post-surgery infections on invasive devises 5.
Endocarditis by intravenous administration of contaminated
drug Solutions; 7, Infections in patients with acquired immu
nodeficiency syndrome, cancer chemotherapy, steroid
therapy, hematological malignancies, organ transplantation,
renal replacement therapy, and other situations with severe
neutropenia.
0170 B) Community-acquired infections: 1. Community
acquired respiratory tract infections; 2. Meningitis; 3. Folli
culitis and infections of the ear canal caused by contaminated
waters; 4. Malignant otitis externa in the elderly and diabet
ics; 5. Osteomyelitis of the caleaneus in children: Eye infec
tions commonly associated with contaminated contact lens;
6. Skin infections such as nail infections in people whose
hands are frequently exposed to water, 7. Gatrointestinal tract
infections; 8. Muscoskeletal system infections.
0171 Examples of infections caused by A. baumannii
include: A) Nosocomial infections 1. Bacteraemia and sepsis,
2. respiratory tract infections in mechanically ventilated
patients; 3. Post-Surgery infections on invasive devices; 4.
wound infectious, particularly in bum wound patients; 5.
infection in patients with acquired immunodeficiency syn
drome, cancer chemotherapy, steroid therapy, hematological
malignancies, organ transplantation, renal replacement
therapy, and other situations with severe neutropenia; 6. uri
US 2010/0028334 A1
20
nary tract infections; 7. Endocarditis by intravenous admin
istration of contaminated drug Solutions; 8. Cellulitis.
0172 B) Community-acquired infections; a. community
acquired pulmonary infections; 2. Meningitis; Cheratitis
associated with contaminated contact lens; 4. War-Zone com
munity-acquired infections.
0173 C) Atypical infections: 1. Chronic gastritis.
0.174 Examples of infections caused by Stenotrophomo
nas maltophilia include Bacteremia, pneumonia, meningitis,
wound infections and urinary tract infections. Some hospital
breaks are caused by contaminated disinfectant Solutions,
respiratory devices, monitoring instruments and ice
machines. Infections usually occur in debilitated patients
with impaired host defense mechanisms.
0175 Examples of infections caused by Klebsiella pneu
moniae include community-acquired primary lobar pneumo
nia, particularly in people with compromised pulmonary
function and alcoholics. It also caused wound infections, soft
tissue infections and urinary tract infections.
0176 Examples of infections caused by Salmonella app.
are acquired by eating contaminated food products. Infec
tions include enteric fever, enteritis and bacteremia.
0177 Examples of infections caused by Shigella spp.
include gastroenteristis (shigellosis).
0.178 The antimicrobial agent and/or enhancer of antimi
crobial agent components of the invention may also be used in
various fields as where antiseptic treatment or disinfection of
materials it required, for example, Surface disinfection.
0179 The antimicrobial agent and enhancers of antimi
crobial peptides described herein can be used to treat micro
organisms infecting a cell, group of cells, or a multi-cellular
organism.
0180. In one embodiment, the antimicrobial agent and
enhancers of antimicrobial agent described herein can be
used to reduce the rate of proliferation and/or growth of
microorganisms. In some embodiments, the microorganism
are either or both gram-positive or gram-negative bacteria,
whether such bacteria are cocci (spherical), rods, vibrio
(comma shaped), or spiral.
0181. Of the cocci bacteria, micrococcusandstaphylococ
cus species are commonly associated with the skin, and Strep
tococcus species are commonly associated with tooth enamel
and contribute to tooth decay. Of the rods family, bacteria
Bacillus species produce endospores seen in various stages of
development in the photograph and B. cereus cause a rela
tively mild food poisoning, especially due to reheated fried
food. Of the vibrio species, V. cholerae is the most common
bacteria and causes cholera, a severe diarrhea disease result
ing from a toxin produced by bacterial growth in the gut. Of
the spiral bacteria, rhodospirillum and Treponema pallidum
are the common species to cause infection (e.g., Treponema
pallidum causes syphilis). Spiral bacteria typically grow in
shallow anaerobic conditions and can photosynthesize to
obtain energy from Sunlight.
0182 Moreover, the present invention relates to antimi
crobial agent and enhancers of antimicrobial agent that can be
used to reduce the rate of growth and/or kill either gram
positive, gram negative, or mixed flora bacteria or other
microorganisms.
0183 Such bacteria are for example, but are not limited to,
listed in Table 3. Further examples of bacteria are, for
example but not limited to Baciccis Antracis, Enterococcus
faecalis, Corynebacterium, diphtheriae, Escherichia coli,
Streptococcus coelicolor, Streptococcus pyogenes, Strepto
Feb. 4, 2010
bacillus Oniliformis, Streptococcus agalactiae, Streptococ
cus pneumoniae, Salmonella typhi; Salmonella paratyphi;
Salmonella schottmulleri, Salmonella hirshieldii; Staphylo
coccus epidermidis, Staphylococcus aureus, Klebsiella
pneumoniae, Legionella pneumophila, Helicobacter pylori;
Mycoplasma pneumonia, Mycobacterium tuberculosis,
Mycobacterium leprae, Yersinia enterocolitica, Yersinia pes
tis, Vibrio cholerae, Vibrio parahaemolyticus, Rickettsia pro
wOzekii, Rickettsia rickettsii, Rickettsia akari; Clostridium
difficile, Clostridium tetani, Clostridium perfingens,
Clostridianz novyi, Clostridianz Septicum, Clostridium
botulinum, Legionella pneumophila, Hemophilus influenzue;
Hemophilus parainfluenzue, Hemophilus aegyptus, Chlamy
dia psittaci, Chlamydia trachonzatis, Bordetella pertesis,
Shigella spp., Campylobacter jejuni; Proteus spp., Citro
bacter spp.; Enterobacter spp., Pseudomonas aeruginosa,
Propionibacterium spp.; Bacillus anthracis, Pseudomonas
syringae, Spirrillum minus, Neisseria meningitidis, Listeria
monocytogenes, Neisseria gonorrhoeae, Treponema palli
dum, Francisella tularensis, Brucella spp.; Borrelia recur
rentis, Borrelia hermsii; Borrelia turicatue, Borrelia burg
dorferi, Mycobacterium avium, Mycobacterium smegmatis,
Methicillin-resistant Staphylococcus aureus, Vanomycin-re
sistant enterococcus, and multi-drug resistant bacteria (e.g.,
bacteria that are resistant to more than 1, more than 2, more
than 3, or more than 4 different drugs).
TABLE 3
Examples of bacteria.
Gram positive bacteria
Staphylococcusatiretts
Bacilius anthracis
Bacilius cereus
Bacilius subtiis
Streptococci is pneumonia
Streptococci is pyogenes
Cliostridium tetani
Listeria monocytogenes
Mycobacterium tuberculosis
Staphylococci is epidermidis
Gram negative bacteria
Neisseria meningitidis
Neisseria gonorrhoeae
Vibrio cholerae
Escherichia coli K12
Bartoneia henseiae
Haemophilus influenzae
Salmonella typhi
Shigella dysenteriae
Yersinia pestis
Pseudomonas aeruginosa
Helicobacter pylori
Legionella pneumophila
Others
Borrelia burgdorferi
Ehrlichia chafeensis
Treponema pallidiin
Chlamydia trachomatis
0184. In some embodiments, antimicrobial agent and
enhancers of antimicrobial agent described herein is used to
treat an already drug resistant bacterial Strain such as Methi
cillin-resistant Staphylococcus aureus (MRSA) or Vancomy
cin-resistant enterococcus (VRE) of variants thereof.
0185. The present invention also contemplates the use of
antimicrobial agent and enhancers of antimicrobial agent
US 2010/0028334 A1
described herein in combinations with other antibiotics to
fight Gram-positive bacteria that cannot maintain resistance
to certain drugs.
0186 AS Such, antimicrobial agents and enhancers of anti
microbial agents herein may be used to treat infections, for
example bacterial infections and other conditions such as
urinary tract infections, ear infections, sinus infections, bac
terial infections of the skin, bacterial infections of the lungs,
sexually transmitted diseases, tuberculosis, pneumonia, lyme
disease, and Legionnaire's disease. Thus any of the above
conditions and other conditions resulting microorganism
infections, for example bacterial infections may be prevented
or treated by the compositions of the invention herein.
0187. In another example, an antimicrobial agents and
enhancers of antimicrobial agents are used to inhibit resis
tance to an antiprotozoan agent selected from the group con
sisting of: Chloroquine; Pyrimethamine; Mefloquine
Hydroxychloroquine; Metronidazole; Atovaquone; Imi
docarb; Malarone; Febendazole; Metronidazole; Ivomec;
Iodoquinol; Diloxanide Furoate; and Ronidazole. Examples
of protozoan organisms whose growth is reduced or inhibited
by antimicrobial agents and enhancers of antimicrobial
agents described herein include but are not limited to, Acan
thameba; Actinophrys. Amoeba, Anisonema; Anthophysa;
Ascaris lumbricoides, Bicosoeca: Blastocystis hominis,
Codonella; Coleps: Cothurina; Cryptosporidia Difflugia;
Entamoeba histolytica (a cause of amebiasis and amebic dys
entery); Entosipilon; Epalxis; Epistylis; Euglypha; Flukes;
Giardia lambia, Hookworm Leishmania spp.; Mayorella;
Monosiga: Naegleria Hartmannella; Paruroleptus; Plasmo
dium spp. (a cause of Malaria) (e.g., Plasmodium falciparum,
Plasmodium malariae, Plasmodium vivax and Plasmodium
ovale); Pneumocystis carinii (a common cause of pneumonia
in immunodeficient persons); microfilariae; Podophya;
Raphidiophys; Rhynchomonas; Salpingoeca; Schistosoma
japonicum, Schistosoma haematobium, Schistosoma Man
SOnii Stentor; Strongyloides; Stylonychia; Tapeworms; Tri
chomonas spp. (e.g., Trichuris trichiuris and Trichomonas
vaginalis (a cause of vaginal infection)); Tipanosoma spp.
and Vorticella.
0188 In another example, antimicrobial agents and
enhancers of antimicrobial agents used to inhibit antifungal
agent selected from the group consisting of imidazoles (e.g.,
clotrimazole, miconazole; econazole, ketonazole, oxicona
Zole, Sulconazole), ciclopiroZ, butenafine, and allylamines.
Examples of fungus infections growth is reduced or inhibited
by antimicrobial agents and enhancers of antimicrobial
agents described herein include but are not limited to, tinea;
athlete's foot, jock itch; and candida.
0189 Pharmaceutical Formulations.
0190. The present invention contemplates pharmaceutical
formulations comprising an antimicrobial agent and an
enhancer to such an antimicrobial agent in an effective
amount to achieve a therapeutic or prophylactic effect and a
pharmaceutically effective carrier.
0191 The actual effective amount will depend upon the
condition being treated, the route of administration, the type
and class of the antimicrobial agent and enhancer of the
antimicrobial agent used to treat the condition, and the medi
cal history of the patient. Determination of the effective
amount is well within the capabilities of those skilled in the
art. The effective amountforuse inhumans can be determined
from animal models. For example, a dose for humans can be
formulated to achieve circulating concentrations that have
Feb. 4, 2010
been found to be effective in animals. The effective amount of
an antimicrobial agent and an enhancer to Such an antimicro
bial agent can vary if the antimicrobial agent and an enhancer
of antimicrobial agent is coformulated with another therapeu
tic agents (for example, antibiotics, antiviral agents, antipro
toZoan agents). It is contemplated that lower dosages will be
needed in Such cases as a result of a synergistic effect of all
active ingredients.
0.192 In some embodiments, an effective amount of an
active ingredient (e.g., an antimicrobial agent and an
enhancer of antimicrobial agent and/or additional therapeutic
agent(s)) is from about 0.0001 mg to about 500 mg active
agent per kilogram body weight of a patient, in Some embodi
ments from about 0.001 to about 250 mg active agent per
kilogram body weight of the patient, in further embodiments
from about 0.01 mg to about 100 mg active agent per kilo
gram body weight of the patient, yet still more embodiments
from about 0.5 mg to about 50 mg active agent per kilogram
body weight of the patient, and in another embodiment from
about 1 mg to about 15 mg active agent per kilogram body
weight of the patient. In terms of weight percentage, a phar
maceutical formulation of an active agent (e.g., an antimicro
bial agent and an enhancer of antimicrobial agent or addi
tional therapeutic agent(s)) can, in some embodiments,
comprises of an amount from about 0.0001 wt.% to about 10
wt.%, and in alternative embodiments, from about 0.001 wt.
% to about 1 wt.%, and in further embodiments from about
0.01 wt.% to about 0.5 wt.%.
(0193 In any of the formulations herein antimicrobial
agents and enhancers of antimicrobial agents can be formu
lated as a salt, a prodrug, or a metabolite. Such formulations
can also include additional therapeutic agent(s) such as, for
example, antibiotics, antiviral agents, antifungal agents, and/
or antiprotozoan agents.
0194 Examples of antibiotics that may be coformulated
antimicrobial agents and enhancers of antimicrobial agents
include, for example, aminoglycosides, carbapenems, cepha
losporins, cephems, glycopeptides, fluoroquinolones/quino
lones, oxazolidinones, penicillins, streptogramins, Sulfona
mides, and tetracyclines.
0.195 Aminoglycosides are a group of antibiotics found to
be effective against gram-negative. Aminoglycosides are
used to treat complicated urinary tract infections, septicemia,
peritonitis and other severe intra-abdominal infections,
severe pelvic inflammatory disease, endocarditis, mycobac
terium infections, neonatal sepsis, and various ocular infec
tions. They are also frequently used in combination with
penicillins and cephalosporins to treat both gram-positive and
gram-negative bacteria. Examples of aminoglycosides
include amikacin, gentamycin, tobramycin, netromycin,
streptomycin, kanamycin, paromomycin, and neomycin.
0196. Carbapenems are a class of broad spectrum antibi
otics that are used to fight gram-positive, gram-negative, and
anaerobic microorganisms. Carbapenems are available for
intravenous administration, and as Such are used for serious
infections which oral drugs are unable to adequately address.
For example, carbapenems are often used to treat serious
single or mixed bacterial infections, such as lower respiratory
tract infections, urinary tract infections, intra-abdominal
infections, gynecological and postpartum infections, septice
mia, bone and joint infections, skin and skin structure infec
tions, and meningitis. Examples of carbapenems include imi
penem/cilastatin Sodium, meropenem, ertapenem, and
panipenem/betamipron.
US 2010/0028334 A1
22
0.197 Cephalosporins and cephems are broad spectrum
antibiotics used to treat gram-positive, gram-negative, and
spirochaetal infections. Cephems are considered the next
generation Cephalosporins with newer drugs being stronger
against gram negative and older drugs better against gram
positive. Cephalosporins and cephems are commonly Substi
tuted for penicillin allergies and can be used to treat common
urinary tract infections and upper respiratory infections (e.g.,
pharyugitis and tonsillitis).
0198 Cephalosporins and cephems are also used to treat
otitis media, Some skin infections, bronchitis, lower respira
tory infections (pneumonia), and bone infection (certain;
members), and are a preferred antibiotic for Surgical prophy
laxis. Examples of Cephalosporins include cefixime, cefpo
doxime, ceftibuten, cefdinir, cefaclor, cefprozil, loracarbef,
cefadroxil, cephalexin, and cephradineze. Examples of ceph
ems include cefepime, cefpirome, cefataxidime pentahy
drate, ceftazidime, ceftriaxone, ceftazidime, cefotaxime,
cefteram, cefotiam, cefuroxime, cefamandole, cefuroxime
axetil, cefotetan, cefazolin Sodium, cefazolin, cefalexin.
0199 Fluroquinolones/quinolones are antibiotics used to
treat gram-negative infections, though some newer agents
have activity against gram-positive bacteria and anaerobes.
Fluroquinolones/quinolones are often used to treat conditions
Such as urinary tract infections, sexually transmitted diseases
(e.g., gonorrhea, chlamydial urethritisficerviciitis, pelvic
inflammatory disease), gram-negative gastrointestinal infec
tions, Soft tissue infections, pphthalmic infections, dermato
logical infections, sinusitis, and respiratory tract infections
(e.g., bronchitis, pneumonia, and tuberculosis). Fluroquino
lones/quinolones are used in combination with other antibi
otics to treat conditions, such as multi-drug resistant tubercu
losis, neutropenic cancer patients with fever, and potentially
anthrax. Examples of fluoroquinolones/quinolones include
ciproflaxacin, levofloxacin, and ofloxacin, gatifloxacin, nor
floxacin, lomefloxacin, trovafloxacin, moxifloxacin, spar
floxacin, gemifloxacin, and paZufloxacin.
0200 Glycopeptides and streptogramins represent antibi
otics that are used to treat bacteria that are resistant to other
antibiotics, such as methicillin-resistant staphylococcus
aureus (MRSA). They are also be used for patients who are
allergic to penicillin. Examples of glycopeptides include van
comycin, teicoplanin, and daptomycin.
0201 Carbapenems are used to treatgram-positive, gram
negative, and/or anaerobes.
0202 Oxazolidinones are commonly administered to treat
gram-positive infections. Oxazolidinones are commonly
used as an alternative to other antibiotic classes for bacteria
that have developed resistance. Examples of oxazolidinones
include linezolid.
0203 Penicillins are broad spectrum used to treat gram
positive, gram-negative, and spirochaetal infections. Condi
tions that are often treated with penicillins include pneumo
coccal and meningococcal meningitis, dermatological
infections, ear infections, respiratory infections, urinary tract
infections, acute sinusitis, pneumonia, and lyme disease.
Examples of penicillins include penicillin, amoxicillin,
amoxicillin-clavulanate, amplicillin, ticarcillin, piperacillin
taZobactam, carbenicillin, piperacillin, meZocillin, benzathin
penicillin G. penicillin V potassium, methicillin, nafcillin,
oxacillin, cloxacillin, and dicloxacillin.
0204 Streptogramins are antibiotics developed in
response to bacterial resistance that diminished effectiveness
of existing antibiotics. Streptogramins are a very Small class
Feb. 4, 2010
of drugs and are currently very expensive. Examples of strep
togramins include quinupristin/dafopristin and pristinamy
cin.
0205 Sulphonamides are broad spectrum antibiotics that
have had reduced usage due to increase in bacterial resistance
to them. Suphonamides are commonly used to treat recurrent
attacks of rheumatic fever, urinary tract infections, prevention
of infections of the throat and chest, traveler's diarrhea,
whooping cough, meningococcal disease, sexually transmit
ted diseases, toxoplasmosis, and rhinitis. Examples of Sul
fonamides include co-trimoxazole, Sulfamethoxazole trime
thoprim, Sulfadiazine, Sulfadoxine, and trimethoprim.
0206 Tetracyclines are broad spectrum antibiotics that are
often used to treat gram-positive, gram-negative, and/or spi
rochaetal infections. Tetracyclines are often used to treat
mixed infections, such as chronic bronchitis and peritonitis,
urinary tract infections, rickets, chlamydia, gonorrhea, lyme
disease, and periodontal disease. Tetracyclines are an alter
native therapy to penicillin in syphilis treatment and are also
used to treat acne and anthrax. Examples of tetracyclines
include tetracycline, demeclocycline, minocycline, and
doxycycline.
0207. Other antibiotics contemplated herein (some of
which may be redundant with the list above) include, but are
not limited to; abrifam; acrofloxacin, aptecin, amoxicillin
plus clavulonic acid; apalcillin, apramycin; astromicin;
arbekacin; aspoxicillin; azidozillin; azlocillin; aztreonam,
bacitracin; benzathine penicillin; benzylpenicillin: clarithro
mycin, carbencillin: cefaclor, cefadroxil, cefalexin; cefaman
dole; cefaparin, cefatrizine, cefazolin, cefbuperaZone, cef
capene; cefdinir, cefditoren, cefepime, cefetamet, cefixime;
cefnetazole; cefiminox, cefoperaZone, ceforanide; cefo
taxime; cefotetan, cefotiam, cefoxitin; ce?pimizole; ce?pira
mide; cefpodoxime; cefprozil; cefradine, cefroxadine, cefsu
lodin, ceftazidime; ceftriaxone, cefuroxime; cephalexin;
chloramphenicol; chlortetracycline; ciclacillin; cinoxacin;
clemizole penicillin; cleocin, cleocin-T, cloxacillin; corifam;
daptomycin; daptomycin; demeclocycline; descuinolone;
dibekacin; dicloxacillin; dirithromycin; doxycycline; enoxa
cin; epicillin; ethambutol; gemifloxacin; fenampicin;
finamicina; fleroxacin; flomoxef flucloxacillin; flumequine;
flurithromycin; fosfomycin; fosmidomycin; fusidic acid;
gatifloxacin; gemifloxaxin, isepamicin; isoniazid; josamy
cin; kanamycin; kasugamycin; kitasamycin; kairifam, lata
moxef levofloxacin, levofloxacing lincomycin; lineZolid;
lomefloxacin; loracarbaf, lymecycline; mecillinam; meth
acycline; methicillin; metronidazole; mezlocillin; mideca
mycin; minocycline; miokamycin; moxifloxacin, nafcillin;
nafcillin, nalidixic acid; neomycin; netilmicin; norfloxacin;
novobiocin, oflaxacin; oleandomycin; oxacillin, oxolinic
acid; oxytetracycline; paromycin; paZufloxacin; pefloxacin;
penicillin g; penicillin V; phenethicillin; phenoxymethyl
penicillin; pipemidic acid; piperacillin and taZobactam com
bination; piromidic acid; procaine penicillin; propicillin;
pyrimethamine; rifadin: rifabutin: rifamide; rifampin: rifap
entene; rifomycin; rimactane, rofact, rokitamycin; rollitetra
cycline; roXithromycin; rufloxacin; sitafloxacin; sparfloxa
cin; spectinomycin; spiramycin; Sulfadiazine; Sulfadoxine;
Sulfamethoxazole; sisomicin; streptomycin; Sulfamethox
azole; Sulfisoxazole; quinupristan-dalfopristan; teicoplanin;
temocillin; gatifloxacin, tetracycline; tetroXoprim; tellithro
mycin; thiamphenicol; ticarcillin; tigecycline; tobramycin;
to Sufloxacin; trimethoprin; trimetrexate; trovafloxacin; Van
comycin; Verdamicin; azithromycin; and lineZolid.
US 2010/0028334 A1
0208. A “pharmaceutical acceptable carrier is a pharma
ceutically acceptable solvent, Suspending agent or vehicle for
delivering the antimicrobial agents and/or an enhancers anti
microbial agents of the present invention to an animal or
human. The carrier may be, for example, gaseous, liquid or
Solid and is selected with the planned manner of administra
tion in mind.
0209 Examples of pharmaceutically acceptable carriers
for oral pharmaceutical formulations include: lactose,
Sucrose, gelatin, agar and bulk powders. Examples of suitable
liquid carriers include water, pharmaceutically acceptable
fats and oils, alcohols or other organic solvents, including
esters, emulsions, syrups or elixirs, Suspensions, Solutions
and/or suspensions, and solution and or Suspensions recon
stituted from non-effervescent granules and effervescent
preparations reconstituted from effervescent granules. Such
liquid carriers may contain, for example, Suitable solvents,
preservatives, emulsifying agents, Suspending agents, dilu
ents, Sweeteners, thickeners, and melting agents. In some
embodiments, the carriers are edible oils, for example, corn or
canola oils. Polyethylene glycols, e.g. PEG, are also encom
passed in the invention as carriers.
0210 Examples of pharmaceutically acceptable carriers
for topical formulations include: ointments, cream, Suspen
sions, lotions, powder, Solutions, pastes, gels, spray, aerosol
or oil. Alternately, a formulation may comprise a transdermal
patch or dressing Such as a bandage impregnated with active
ingredients (e.g., antimicrobial agents and/or an enhancers of
antimicrobial agents) and optionally one or more carriers or
diluents. The topical formulations may include a compound
that enhances absorption or penetration of the active ingredi
ent through the skin or other affected areas. Examples of such
dermal penetration enhancers include dimethylsulfoxide and
related analogues.
0211 To be administered in the form of a transdermal
delivery system, the dosage administration can be continuous
rather than intermittent throughout the dosage regimen.
0212 Formulations suitable for parenteral administration
include aqueous and non-aqueous formulations isotonic with
the blood of the intended recipient; and aqueous and non
aqueous sterile Suspensions which may include Suspending
systems designed to target the compound to blood compo
nents or one or more organs. The formulations may be pre
sented in unit-dose or multi-dose sealed containers, for
example, ampoules or vials. Extemporaneous injection solu
tions and Suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
Parenteral and intravenous formulation may include minerals
and other materials to make them compatible with the type of
injection or delivery system chosen.
0213 Commonly used pharmaceutically acceptable carri
ers for parenteral administration includes, water, a suitable
oil, saline, aqueous dextrose (glucose), or related Sugar Solu
tions and glycols such as propylene glycol or polyethylene
glycols.
0214 Solutions for parenteral administration preferably
contain a water soluble salt of the active ingredient, suitable
stabilizing agents and, if necessary, buffer Substances. Anti
oxidizing agents. Such as sodium bisulfite, Sodium Sulfite, or
ascorbic acid, either alone or combined, are suitable stabiliz
ing agents. Citric acid salts and sodium EDTA may also be
used as carriers. In addition, parenteral Solutions may contain
preservatives, such as benzalkonium chloride, methyl- or pro
Feb. 4, 2010
pyl-paraben, or chlorobutanol. Suitable pharmaceutical car
riers are described in Remington, cited above.
0215. The present invention additionally contemplates
antimicrobial agent and enhancers of antimicrobial agents
formulated for veterinary administration by methods conven
tional in the art.
0216. The antimicrobial agents and enhancers of antimi
crobial agents described herein can also be formulated for
industrial applications with, for example, a cleaning product,
Such as Soap, laundry detergent, shampoo, dishwashing soap,
toothpaste, and other house cleaning detergents.
0217 Selection of Subjects Administered the Composi
tions
0218. In some embodiments, the subjects administered a
composition comprising antimicrobial agents and/or enhanc
ers of antimicrobial agents are selected based on the desired
treatment regime. For instance,
0219. Accordingly, in some embodiments, subjects are
administered the antimicrobial agents, such as colistin, and/or
enhancers of antimicrobial agents, such as the inhibitors of
the genes as disclosed in Table 1 and Table 4 herein. In some
embodiments, an enhancer of an antimicrobial agent can be
an antibiotic. In such embodiments, an antibiotic is adminis
tered to the subject for the purpose of its gene inhibiting
ability, as compared to its normal medical use as an anti
pathogenic or to decrease bacteria viability. Accordingly, the
administration of the antibiotics (with the antimicrobial
agent) as disclosed herein is different from the normal admin
istration of antibiotics for medical use. As disclosed herein,
the administration of an antibiotic is administered according
to a treatment regimen which is determined by the desired
duration of the treatment or administration with the antimi
crobial agent. Accordingly, as disclosed herein, the antibiotic
is administered for its gene inhibitory function as compared
to its ability to kill bacteria. For example, in general, the
medically appropriate administration of an antibiotic is to kill
bacteria, and administration is for a specific amount of time
which is determined by its ability to kill all the bacteria to a
maximum efficacy but to minimize the development of bac
terial or viral resistance. As such, antibiotics typically are
administered to a subject for a specific period of time, such as,
for example, for 5-7 days if used at a high dose, or 10 to 14
days if the antibiotic is used at a lower dose, which results in
efficient elimination of the bacteria but does not allow devel
opment of bacterial resistance. Extended administration of an
antibiotic beyond the time the bacteria is killed results in
deleterious results or undesirable side effects. Accordingly,
the inventors have discovered that, due to their gene inhibitor
functions, antibiotics can be administered to a subjects for a
prolonged period of time, which is determined by the duration
of administration of the antimicrobial activity, and impor
tantly, not by the antibiotics ability to eliminate bacteria. As
Such, the antibiotic administration as disclosed herein is
counter to the general medical advice on administration of
antibiotics to eliminate bacterial or other infections.
0220 Accordingly, in Some embodiments, a Subject is
selected for the administration with the compositions as dis
closed herein by identifying a Subject that needs a specific
treatment regimen of antimicrobial agent, and is administered
concurrently an enhancer of a antimicrobial agent such as an
antibiotic. As an exemplary example, where the Subject is a
subject with cystic fibrosis, the subject would be administered
a antimicrobial agent to avoid chronic endobronchial infec
tions, such as those caused by pseudomonas aeruginosis or
US 2010/0028334 A1
24
stentrophomonas maltophilia. One Such antimicrobial agent
is colistin. However, administration of colistin at the doses
and the duration required to efficiently prevent such endo
bronchial infections in Subjects is highly toxic and in some
instances fatal. Accordingly, in Some embodiments of the
present invention, the Subject is selected for a treatment regi
men of an antimicrobial agent. Such as colistin, and an
enhancer of an antimicrobial agent, Such as an antibiotic, and
the Subject is treated with the compositions as disclosed
herein for a specific duration of time. Administration of the
compositions as disclosed herein are not directed by the kill
ing of bacteria, but by the need for antimicrobial treatment,
and can be, for example more than one week, more than 2
weeks, more than 3 weeks, a month, 2 months, 3 months, 6
months or 12 months or longer.
0221) Importantly, as disclosed herein, the enhancer of the
antimicrobial agent as disclosed herein is not selected for its
effect on decreasing cell viability, it is selected based on its
ability to enhance the effect of the antimicrobial agent. In
Some embodiments therefore, an enhancer of the antimicro
bial agent may not have any anti-pathogenic effects or
decrease cell viability when used by themselves, and thus will
have no antibiotic or no antimicrobial activity on their own,
but when used concurrently with an antimicrobial agent as
disclosed herein, Such as for example with colistin, the
enhancer of the antimicrobial agent functions to enhance the
activity of the antimicrobial agent.
0222 Administration
0223 The antimicrobial agents and/or enhancers of anti
microbial agents components may be administered topically,
including local delivery to the gastrointestinal tract and other
membrane Surfaces including aerosol delivery for adminis
tration to lungs or nasal cavity, parenterally or orally in dos
age unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants, and
vehicles. The term parenteral as used herein includes subcu
taneous injections, intravenous, intramuscular, intrathecal,
intraventricular, intracranial injection or infusion techniques.
The present invention also provides Suitable topical,
parenteral and oral pharmaceutical formulations for use in the
novel methods of treatment of the present invention.
0224. The compositions and pharmaceutical formulation
herein can be administered to an organism by any means
known in the art. Routes for administering the compositions
and pharmaceutical formulations hereinto an animal. Such as
a human, include parenterally, intravenously, intramuscu
larly, orally, by inhalation, topically, vaginally, rectally,
nasally, buccally, transdermally, or by an implanted reservoir
external pump or catheter. When administered to a plan, Such
means can be by spray or via irrigation.
0225. Although any route of administration may be used,
parenteral administration, i.e., administration by injection, is
preferred. Injectable formulations can be prepared in conven
tional forms, eitheras liquid solutions or Suspensions; as Solid
forms suitable for Solubilization or Suspension in liquid prior
to injection; or as emulsions. Preferably, sterile injectable
Suspensions are formulated according to techniques known in
the art using Suitable pharmaceutically acceptable carriers
and other optional components as discussed above.
0226. The combination of antimicrobial agents and/or
enhancers of antimicrobial agents components may be
administered orally as tablets, Suspensions, lozenges, tro
ches, powders, granules, emulsions, capsules, syrups or elix
irs. The composition for oral use may contain one or more
Feb. 4, 2010
agents selected from the group of Sweetening agents, flavor
ing agents, coloring agents and preserving agents in order to
produce pharmaceutically elegant and palatable preparations.
0227 Parenteral administration may be carried out in any
number of ways, but it is preferred that the use of a syringe,
catheter, or similar device, be used to effect parenteral admin
istration of the formulations described herein. The formula
tion may be injected systemically such that the active agent
travels substantially throughout the entire bloodstream.
0228. In addition, the formulation may also be injected
locally to a target site, e.g., injected to a specific portion of the
body for which inhibition of mutagenesis is desired. An
advantage of local administration via injection is that it limits
or avoids exposure of the entire body to the active agent(s)
(e.g., antimicrobial peptide and an enhancer of such a peptide
and/or other therapeutic agents). It must be noted that in the
present context, the term local administration includes
regional administration, e.g., administration of a formulation
directed to a portion of the body through delivery to a blood
vessel serving that body Zone. Local delivery may be direct,
e.g., intratumoral. Local delivery may also be nearly direct,
i.e. intralesional or intraperitoneal, that is, to an area that is
Sufficiently close to a site of infection so that the active agent
(s) exhibit the desired pharmacological activity. Thus, when
local delivery is desired, the pharmaceutical formulations are
preferably delivered intralesionally, intratumorally, or intra
peritoneally.
0229. It is intended that, by local delivery of the presently
described pharmaceutical formulations, a higher concentra
tion of the active agent may be directed to the target site.
There are several advantages to having high concentrations
delivered directly at the target site. First, since the active agent
is more localized, there is less potential for toxicity to the
patient since minimal systemic exposure occurs. Second,
drug efficacy is improved since the target site is exposed to
higher concentrations of the drug. Third, relatively fast deliv
ery minimizes solubility and stability liabilities of the active
agent before reaching its target site.
0230 Preferably the pharmaceutical compositions are in
unit dosage form. In such form, the composition is divided
into unit doses containing appropriate quantities of the active
component. The unit dosage form can be a packaged prepa
ration, the package containing discrete quantities of the
preparations, for example, packeted tablets, capsules, and
powders in vials or ampoules. The unit dosage form can also
be a capsule, cachet, or tablet, or it can be the appropriate
number of any of these packaged forms.
0231. Useful pharmaceutical dosage formulations for
administration of the compounds of the present invention are
illustrated as follows:
0232 Capsules: A large number of unit capsules are pre
pared by filling standard two-piece hard gelatin capsules each
with 1-100 milligrams of powdered active ingredient, milli
grams of lactose, 50 milligrams of cellulose, and 6 milligrams
magnesium Stearate.
0233 Soft Gelatin Capsules: A mixture of active ingredi
ent in a digestible oil such as Soybean oil, cottonseed oil or
olive oil is prepared and injected by means of a positive
displacement pump into gelatin to form soft gelatin capsules
containing 1-100 milligrams of the active ingredient. The
capsules are washed and dried.
0234 Tablets: A large number of tablets are prepared by
conventional procedures so that the dosage unit was 1-100
milligrams of active ingredient, 0.2 milligrams of colloidal
US 2010/0028334 A1
silicon dioxide, 5-6 milligrams of magnesium Stearate, 275
milligrams of microcrystalline cellulose, 11 milligrams of
starch and 98.8 milligrams of lactose. Appropriate coatings
can be applied to increase palatability or delay absorption.
0235 Injectable: A parenteral composition suitable for
administration by injection is prepared by stirring 0.5-1.5%
by weight of active ingredient in 10% by volume propylene
glycol and water. The Solution is made isotonic with sodium
chloride and sterilized.
0236 Suspension: An aqueous Suspension is prepared for
oral administration so that each 5 ml contains 1-100 mg of
finely divided active ingredient, 200 mg of sodium carboxym
ethyl cellulose, 5 mg of sodium benzoate, 1 g of sorbitol
solution, U.S.P., and 0.02 ml of vanillin.
0237 Antimicrobial agents and enhancers of antimicro
bial agents of the present invention may be administered in
the form of liposome delivery systems. Such as Small unila
mellar vesicles, large unilamellar vesicles, and multilamellar
vesicles. Liposomes can be formed from a variety of phos
pholipids, such as cholesterol, Stearylamine, orphosphatidyl
cholines.
0238 Antimicrobial agents and enhancers of antimicro
bial agents of the present invention may be coupled with
soluble polymers as targetable drug carriers. Such polymers
can include polyvinylpyrrolidone, pyran copolymer, polyhy
droxylpropylmethacrylamide-phenol, polyhydroxyethylas
partamidephenol, or polyethyleneoxide-polylysine Substi
tuted with palmitoyl residues. Furthermore, the compounds
of the present invention can be coupled to a class of biode
gradable polymers useful in achieving controlled release of
the drug, for example, polylactic acid, polyglycolic acid,
copolymers of polylactic and polyglycolic acid, polyepsilon
caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydibydropyrians, polycyanoacylates, and
crosslinked or amphipathic block copolymers of hydrogels.
0239. In some embodiment of this invention, antimicro
bial agents and enhancers of antimicrobial agents can be
incorporated into a biodistribution directing moiety, such as a
polymer, to direct the biodistribution and/or to allow for con
tinuous release of thereof. Alternatively, microparticulate or
nanoparticulate polymeric bead dosage forms may be
employed. In this case, the antimicrobial agents and/or
enhancers of antimicrobial agents will be encapsulated in the
particulate dosage forms. In this manner, the antimicrobial
agents and/or enhancers of antimicrobial agents are released
over time to provide a sustained therapeutic benefit. These
Sustained release dosage forms are also useful with regard to
other active agents useful in the practice of the present inven
tion, Such other therapeutic agents discussed below, for
example anti-bacterial agents, antibiotics, anti-fungal agents,
anti-protozoan agents etc. Release of the active agents (anti
microbial agents and/or enhancers of antimicrobial agents
and/or other therapeutic agents) from the particulate dosage
forms of the present invention can occur as a result of both
diffusion and particulate matrix erosion. Biodegradation rate
directly impacts active agent release kinetics.
0240 Controlled release parenteral formulations of the
agonists and/or antagonists of the present invention can be
made as implants, oily injections, or as particulate systems.
Particulate systems include: microspheres, microparticles,
microcapsules, nanocapsules, nanospheres, and nanopar
ticles. Microcapsules contain the therapeutic protein as a
central core. In microspheres the therapeutic is dispersed
Feb. 4, 2010
throughout the particle. Liposomes can be used for controlled
release as well as drug targeting of entrapped drug.
0241 Antimicrobial agents and enhancers of antimicro
bial agents of the present invention can be administered to any
organism (eukaryotic or prokaryotic) to prevent or treat drug
resistance. Antimicrobial agent and enhancers of such agents
can also be administered to a first organism in order to target
a second organism associated with the first organism. For
example, an antimicrobial agent and enhancers of the antimi
crobial agent can be administered to a mammal infected by
bacteria or virus or other pathogen, or for example the com
positions as disclosed herein can be administered to a plant
infected by a fungus or other pathogen. In some embodi
ments, the methods and compositions as disclosed herein can
be used to prevent the development of resistance of a micro
organism to the antimicrobial agent, Such as colistin. Without
being bound by theory, concurrent administration of an
enhancer of the microbial agent and an antimicrobial agent as
disclosed in the compositions and methods herein, enables
targeting of at least two different pathways, Such as, for e.g.
parallel or downstream pathways, and thus delays or
decreases the microorganism’s ability to accumulate sponta
neous mutations in the genes involved in the pathways tar
geted by the enhancer of antimicrobial agent and/or antimi
crobial agent, and thus decreases the ability of the
microorganism to circumvent the ability of the antimicrobial
agent to kill the microorganism by spontaneous mutations.
0242 Antimicrobial agents and enhancers of antimicro
bial agents of the present invention can be administered as a
monotherapy or in combination with additional therapeutic
agents (e.g., antibiotic, antiviral, antifungal, antiprotozoan
agents etc.) When administered as part of a combination
therapy, antimicrobial agents and enhancers of antimicrobial
agents herein can be administered serially or simultaneously
with the additional agent(s). In some embodiments, antimi
crobial agents and enhancers of antimicrobial agents are
administered prior to the administration of additional thera
peutic agent(s). In other embodiments, antimicrobial agents
and enhancers of antimicrobial agents are administered after
the administration of additional therapeutic agent(s). For
example, for prophylactic benefit, antimicrobial agents and
enhancers of antimicrobial agents may be co-administered
(concurrent) to a Subject at risk of developing an infection, for
example a bacterial infection. In some embodiments, the
antimicrobial agents and enhancers of antimicrobial agents
are administered prior to, or after, the administration of the
additional therapeutic agents.
0243 The antimicrobial agent and enhancers of antimi
crobial agent components of the present invention may addi
tionally be combined with other medicaments to provide an
operative combination. It is intended to include any chemi
cally compatible combination of pharmacologically-active
agents, as long as the combination does not eliminate the
activity of the antimicrobial peptides and/or macrollide com
ponents.
0244. It will be appreciated that the antimicrobial agents
and/or enhancers of antimicrobial agents components of the
invention and the other medicament may be administered
separately, sequentially or simultaneously.
0245. Other medicaments which may be used when treat
ing bacterial infections include salbutamol, ipratropium, dor
nase alpha, for example, for use in inhalation for respiratory
infections such as cystic fibrosis.
US 2010/0028334 A1
26
0246 Screening to Identify Gene Products that when Inac
tivated Enhance the Activity of Antimicrobial Agents.
0247 The invention relates to a method of enhancing
function of antimicrobial agents by inhibiting (inactivating)
the function of a gene product. For example, ifa gene product
inhibits and/or Suppresses the function of an antimicrobial
agent and/or enhances the activity of a microorganism, its
function decreases the effectiveness of the antimicrobial
agent at that dose. Inactivation of specific gene products
enhances the effectiveness of the antimicrobial agent.
Accordingly, inactivation of gene products enables use of
antimicrobial agents for various implications, for example at
lower doses, thus reducing possible associated toxicity.
0248. These principles can be used to determine if a given
gene product Suppresses antimicrobial agent, and thus an
inhibitor to Such a gene product acts as an enhancer of anti
microbial agent and therefore is a potent candidate in the
development of drug to enhance antimicrobial agents.
0249. In one embodiment, a gene product is genetically
inactivated using known gene disruption techniques. After
Such a disruption event, the locus that encoded the gene
product would now be unable to produce the gene product and
the cell would lack the function of that gene product. Various
known mutability assays are used to assess the effect of the
gene disruption event on a cell's mutability. See Friedberg, E
C. Walker, GC, Siede, W. DNA Repair and Mutagenesis (ed.
Friedberg, E. C.) American Society of Microbiology, Wash
ington D.C., 1995. For example, an adaptation of the so
called Stressful Lifestyle Associated Mutation (or SLAM)
assay (wherein the evolution of resistance to an antibiotic of
choice is measured) or a forward mutation or reversion assay
can be used. See Bull, HJ, Lombardo, MJ, Rosenberg, SM:
Stationary-phase mutation in the bacterial chromosome:
recombination protein and DNA polymerase IV dependence.
Bull H.J., Proc. Natl. Acad. Sci. USA (2001) 98: 8334-8341:
Friedberg, E. C. et al. DNA Repair and Mutagenesis (ea.
Friedberg, E. C.) American Society of Microbiology, Wash
ington D.C., 1995: Crouse, GF: Methods (2000) 22:116-119:
Rosenberg, S M Nature. Rev. Genet. (2001) 2:504-515;
Rosche, W. A., Methods (2000) 20:4-17; end roster, PL.
BioEssays (2000) 22:1067-1074.
0250. In one embodiment, bacterial cells with an inacti
vated gene or a wild-type gene are exposed to one or more
antibacterial agent. The number of cells that grow and/or
Survive in the presence of the antibacterial agentis quantified
in both cells with the inactive gene and cells with the wild
type gene. A decrease in the number of cells that grow in the
inactivated gene group compared with those with the wild
type gene Suggests that the inhibitors to the gene being tested
are potential enhancers of antimicrobial agent.
0251 Numerous techniques are known in the art to inac
tivate genes, many of which could be used to inactivate a test
gene of interest. These techniques include the direct inacti
Vation of the test gene, for example via mutation of the test
gene via homologous recombination. Another useful tech
nique is the indirect activation of the test gene, for example
via mutation of a gene whose gene product modulates the
activity of the test gene.
0252 Typically, the test gene is inactivated via one or
more mutations such that the resulting protein encoded by the
test gene is inactive. Alternatively, the entire gene (or a large
portion of the gene's open reading frame) is deleted from the
genome. Mutation of the test gene may be carried out using
numerous mutagenesis techniques known in the art. At the
Feb. 4, 2010
genetic level, the mutants ordinarily are prepared by site
directed mutagenesis of the DNA encoding the gene. The
mutants can be substitution mutants, deletion mutants, or
insertion mutants.
0253. In some embodiments, the enhancer to the antimi
crobial agent is an inhibitor or a binding agent of the gene
product of the following examples of genes: agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yecY, yiaY, yidK, yihV, yfhC), yibN and/or
yn D. In some embodiments, the genes are homologous or
Substantially homologous, analogues or variants thereof of
agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA,
cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA,
nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV. y?hO, yibn and/orynjD, in particular the genes atpA.
atpF, atpH, betB, guaA, guaB, lip A, lySA, rpiA and/or trXA.
Methods for identifying binding agents are known in the art
and include yeast two hybrid systems, etc.
0254 Screening for Small Molecules that Inhibit the Gene
Product
0255 Enhancers to antimicrobial agents can be identified
by a number of methods including screening libraries of
chemical compounds. Combinatorial libraries and methods
for searching Such libraries are known in the art and include:
biological libraries, natural products libraries, spatially
addressable parallel solid phase or solution phase libraries,
synthetic library methods requiring deconvolution, the one
bead one-compound library method, and synthetic library
methods using affinity chromatography selection. The bio
logical library approach is largely limited to polypeptide
libraries, while the other four approaches are applicable to
polypeptide, non-peptide oligomer or Small molecule librar
ies of compounds. See Lam, K. S. (1997) Anticancer Drug
DeS. 12:145.
0256 In one embodiment, enhancers to antimicrobial
agents are screened using Automated Ligand Identification
System (referred to herein as “ALIS). See, e.g., U.S. Pat.
Nos. 6,721,665, 6,714,875, 6,694,267, 6,691,046, 6,581,013,
6,207,861, and 6,147,344, which are incorporated herein by
reference for all intended purposes. ALIS is a high-through
put technique for the identification of small molecules that
bind to proteins of interest (e.g., agaA, atp A, atpC, atpB.
atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB,
iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L.
pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA,
visB, yeeY, yiaY, yidK, yihV.yfhO,ybN and/orynjD). Small
molecules found to bind tightly to a protein can then be tested
for their ability to inhibit the biochemical activity of that
protein.
0257 Thus, in some embodiments, a target protein (e.g.
atpA, atpF etc) is mixed with pools of small molecules. Pref
erably, more than 1,000 pools are used, more preferably more
than 2,000 pools are used, more preferably more than 3,000
pools are used, or more preferably, more than 10,000 pools
are used. Each pool contains approximately, 1,000 com
pounds, more preferably approximately 2,500 compounds, or
more preferably approximately 5,000 compounds that are
mass encoded, meaning that their precise molecular struc
ture can be determined using only their mass and knowledge
of the chemical library.
US 2010/0028334 A1
27
0258. The small molecules and proteins are mixed
together and allowed to come to equilibrium (they are incu
bated together for 30 minutes at room temperature). The
mixture is rapidly cooled to trap bound complexes and Subject
to rapid size exclusion; chromatography (SEC). Small mol
ecules that bind tightly to the protein of interest will be co
excluded with the protein during SEC. Mass spectroscopic
analysis is performed to determine the masses of all Small
molecules found to bind the protein. Measurement of these
masses allows for the rapid determination of the molecular
structures of the small molecules.
0259 Compounds that bind to a target gene product (e.g.,
atpA, atpF etc) in ALIS can then be tested for their ability to
inhibit atpA function in vitro. Molecules with potent in vitro
inhibitory properties can be tested using a modified Stressful
Lifestyle Adaptation and Mutation (referred to herein as
“SLAM) assays, to assess their function as an enhancer of
antimicrobial peptide (i.e., the ability to reduce or inhibit the
growth and/or survival of E. coli or colistin-resistant E. coli
grown on colistin, see Examples). Molecules that function to
increase the activity of colistin, for example function as an
enhancer of antimicrobial peptide activity in SLAM assays
can be selected for further tested.
0260. In one embodiment, a chemical collection of com
pounds is screened in a format similar to the SLAM assay to
identify molecules that function as an enhancer of antimicro
bial peptide. Bacterial cells are exposed to either one test
compound or a library of compounds and the number of cells
that grown over a period of time in the presence of an anti
microbial peptide is determined in the presence and absence
of the test compound. A decrease in the number of cells
indicates increased inhibition of growth and/or decreased
Survival of the cells, and thus indicates the test compound or
compound functions as an enhancer the antimicrobial pep
tide. The number of cells is determined both before and after
bacteria are exposed to the inhibitor, drug and/or compound.
The number of cells is quantified using known assays. See
Friedberg, E. C. Walker, G C, Siede, W. DNA Repair and
Mztagertesis (ea. Friedberg, E. C.) (American Society of
Microbiology, Washington D.C., 1995); Crouse, Methods
(2000) 22: 116-119.
0261. In yet another embodiment, the bacterial cells are
exposed to an antimicrobial agent and the number of cells
generated is quantified in the presence and absence of the test
compound.
0262. In another example of a method to screen for
enhancers of antimicrobial agent, purified gene products, for
example agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH,
betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK.
yihV, yfhC), yibn and/or ynjD or homologues or variants
thereofare exposed to test compounds. In the presence of the
test compounds, inhibitor and/or drug, their function in vitro
is assessed, and a reduction in activity identifies a potential
enhancer of antimicrobial agent. The inhibition of different
gene products described herein by potential enhancers of
antimicrobial agent can be quantified using standard meth
ods.
0263. Alternatively, high-throughput assays can be used to
screen through large compound libraries to identify potential
enhancers of antimicrobial peptides. Such assays rely on
Feb. 4, 2010
arraying the reaction mixtures in 96-well plates, where each
well also contains a different enhancer of antimicrobial
agents.
0264 Fluorophore labeled nucleoside triphosphates or
oligonucleotide primers or templates can be used in conjunc
tion with standard plate handling and visualization proce
dures to determine which molecules effectively inhibited the
activity of agene product of the invention, for example but not
limited to agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH,
betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK.
yihV, yfhC), yibn and/or ynjD or homologues or variants
thereof. In one embodiment, libraries can be screened in the
presence of one or more of the genes identified, for example
agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA,
cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA,
nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV, yfhC), yibn and/or ynjD or homologues or variants
thereof, in order to identify drugs, compounds and/or mol
ecules that would most efficiently potentate the effectiveness
of an antimicrobial agent synergistically, and thus function as
enhancers of antimicrobial agents.
0265 Structure-Based Design Methods to Create Small
Molecule Enhancers of Antimicrobial Agents:
0266. In some embodiments, the enhancers of the antimi
crobial agents identified herein, and those yet not identified
can be modified using molecular modeling software tools to
create realistic 3-D models of how molecules are shaped.
Such methods include the use of for example, molecular
graphics (i.e., 3D representations) and computational chem
istry (e.g., calculations of the physical and chemical proper
ties).
0267 Using Such molecular modeling, rational drug
design programs can predict which of a collection of different
drug like compounds may fit into the active site of an enzyme,
and by computationally adjusting their bound conformation,
decide which compounds actually might fit the active site
well. See William Bains, Biotechnology from A to Z. 2nd
edition, Oxford University Press, 1998, at 259.
0268 For basic information on molecular modeling, see,
e.g., M. Schlecht, Molecular Modeling on the PC, 1998, John
Wiley & Sons: Gans et al., Fundamental Principals of
Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen
(editor), Guidebook on Molecular Modeling in Drug Design,
1996, Academic Press; and W. B. Smith, Introduction to
Theoretical Organic Chemistry and Molecular Modeling,
1996. U.S. Patents which provide detailed information on
molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080,
576; 5,612,894; 5,583,973; 5,030,103: 4,906,122; and 4,812,
12.
0269. The present invention permits the use of molecular
and computer modeling techniques to design, and select com
pounds (e.g., enhancers to antimicrobial agents) that bind and
inhibit agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB,
cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK.
yihV.yfhO, yibn and/or other gene products that suppress the
activity of antimicrobial peptides. Thus, the invention
enables, for example, the use of atomic coordinates deposited
at the RCSB Protein Data Bank which can be readily identi
fied by persons skilled in the art, to design compounds that
US 2010/0028334 A1
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interact with Such gene products (e.g., agaA, atp A, atpC,
atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA,
guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH.
phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH,
uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD
or homologues or variants thereof). For example, this inven
tion enables the design of compounds that act as competitive
inhibitors agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH,
betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK.
yihV, yfhC), yibn and/or ynjD or homologues or variants
thereof by binding to, all or a portion of the active site of these
gene products or the gene products listed in Table 4.
0270. This invention also enables the design of com
pounds that act as uncompetitive inhibitors of agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or
ynjD or homologues or variants thereof. These inhibitors may
bind to, all or a portion of the active site of agaA, atpA, atpC,
atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA,
guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH.
phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH,
uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD
or homologues or variants thereof. Similarly, non-competi
tive inhibitors; that bind to either agaA, atp A, atpC, atpB.
atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB,
iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L.
pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA,
visB, yeeY, yiaY, yidK, yihV, yfhO, yibn and/or homologues
or variants thereof (whether or not bound to another chemical
entity) may be designed using the atomic coordinates of each
gene respectively of this invention.
0271 Alternatively, the atomic coordinates provided by
the present invention are useful in designing improved ana
logues of known inhibitors of the gene products identified
herein; for example, but not limited to (e.g., mefloquine,
Venturicidin A, diayquinoline, betaine aldehyde chloride,
acivein, psicofuraine, buthionine Sulfoximine, diaminope
melic acid, 4-phospho-D-erythronhydroxamic acid, motexa
fin gadolinium and/or Xycitrin or homologs thereof, and frag
ments thereof) or to design novel classes of inhibitors. This
provides a novel route for designing potent and selective
inhibitors.
0272. In alternative embodiments, the present invention
also includes the designing of improved analogues of antimi
crobial agents, wherein the analogue comprises both the anti
microbial agent and at least one or more enhancer of antimi
crobial agents as identified herein. The analogue of the
antimicrobial agent and enhancer of antimicrobial agents can
be joined by chemical linkage by any means known by per
sons skilled in the art. In additional embodiments, the anti
microbial agent-enhancer of antimicrobial agent molecule
can be subjected to molecule modeling as described above for
optimal configuration of the joined molecules.
0273. The availability of both protein crystals and of
atomic coordinates determined by X-ray diffraction studies
enables soaking experiments with agaA, atpA, atpC, atpB.
atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB,
iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L.
pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA,
visB, yeeY, yiaY, yidK, yihV, yfhO, yibn and etc. crystals
Feb. 4, 2010
with molecules composed of a variety of different chemical
entities to identify potential sites for interaction of candidate
inhibitors. For example, high resolution X-ray diffraction
data collected from crystals saturated with solvent allows the
determination of where each type of solvent molecule binds
the protein. Small molecules that bind tightly to those sites
can then be tested for their ability to inhibit induced mutation
(Travis, J., Science (1993) 262: 1374).
0274 Moreover, the present invention enables computa
tional screening of Small molecule databases for chemical
entities, agents, or compounds that can bind in whole, or in
part, to agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB,
cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK.
yihV, yfhC), yibn and/or the genes listed in Table 4 and,
thereby enhance antimicrobial agent function.
0275. In this screening technique, the quality of fit of such
entities or compounds to the binding site may be judged either
by shape complementarily or by estimated interaction energy.
See Meng, E. C. et al. J. Coma. Chem., 13: 505-524 (1992).
The design of compounds that bind to or inhibit agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibN and/or
the genes listed in Table 4 according to this invention gener
ally involves consideration of two factors. First, the com
pound must be capable of physically associating with agaA,
atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB.
fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC,
papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB.
ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn
and/oryn D etc. Inhibition of proteins associated with agaA,
atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB.
fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC,
papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB.
ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn
and/or yn D and/or the genes listed in Table 4 required for
their function and/or non-covalent molecular interactions
other molecules important in their function, includehydrogen
bonding, van der Waals and hydrophobic interactions is also
encompassed in this invention. Second, the inhibitor must be
able to assume a conformation that allows it to associate with
agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA,
cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA,
nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV, yfhC), yibn and/orynjD etc. or other protein required
for their function. Although certain portions of the inhibitor
will not directly participate in this association with agaA,
atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB.
fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC,
papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB.
ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn
and/orynjD etc. or associated proteins thereof, those portions
may still influence the overall conformation of the molecule.
This, in turn, may have a significant impact on potency.
0276 Such conformational requirements include the over
all three-dimensional structure and orientation of the chemi
cal entity or compound in relation to all or a portion of the
active site of agaA, atp A, atpC, atpB, atpD, atpE, atpG, atpH,
betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA,
US 2010/0028334 A1
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tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK.
yihV, yfhC), yibn and/orynjD etc. or other protein required
for their function or the spacing between functional groups of
a compound comprising several chemical entities that
directly interact with agaA, atpA, atpC, atpB, atp), atpE.
atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK,
lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA,
sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.
yiaY, yidK, yihV, yfhC), yibn and/orynjD etc., or other pro
tein; required their function.
(0277. The potential inhibitory or binding effect of a
chemical compound on inhibiting the gene products identi
fied herein, and other potential gene products that Suppress
the activity of antimicrobial agents may be analyzed prior to
its actual synthesis and by the use of computer modeling
techniques. If the theoretical structure of the given compound
precludes any potential association between it and agaA,
atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB.
fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC,
papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB.
ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn
and/orynjD etc., or other protein required for their function,
synthesis and testing of the compound is obviated.
0278 However, if computer modeling Suggests a strong
interaction is possible, the molecule may then be synthesized
and tested for its ability to interact with a agaA, atp A, atpC,
atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA,
guaB, iscS, kdgK, lip A, lySA, mnmA, nuVC, papa, pdxH.
phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH,
uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD
etc., or other protein required for their function and act as an
enhancer of antimicrobial agents of the invention. In this
manner, synthesis of inactive compounds may be avoided.
0279. One skilled in the art may use one of several meth
ods to screen chemical entities fragments, compounds, or
agents for their ability to associate with agaA, atpA, atpC,
atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA,
guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH.
phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH,
uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD
etc., or other protein required for their function and more
particularly with the individual binding pockets of such gene
products, or associated proteins required for their function.
This process may begin by visual inspection of, for example,
the active site on the computer screen based on agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or
ynjD etc., or other protein required for their function coordi
nates deposited in the RCSB Protein Data Bank. Selected
chemical entities, compounds, or agents may then be posi
tioned in a variety of orientations, or docked, within an indi
vidual binding pocket of agaA, atp A, atpC, atpB, atp), atpE.
atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK,
lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA,
sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.
yiaY, yidK, yihV, yfhC), yibn and/orynjD etc., or other pro
tein required for their function as defined above. Docking
may be accomplished using software Such as Quanta and
Sybyl, followed by energy minimization and molecular
dynamics with standard molecular mechanics force fields,
Such as CHARMM or AMBER.
Feb. 4, 2010
0280 Specialized computer programs also assist in the
process of selecting chemical entities. These include but are
not limited to GRID (Goodford, P. J. J. Med. Chem. (1985)
28, 849-857). GRID is available from Oxford University,
Oxford, UK; MCSS (Miranker, A. et al., Structure, Function
and Genetics, (1991) Vol. 11, 29-34), MCSS is available from
Molecular Simulations, Burlington, Mass. AUTODOCK
(Goodsell, D. S. and A. J. Olsen, “Automated Docking of
Substrates to Proteins by Simulated Annealing Proteins:
Structure, Function, and Genetics, 8, 195-202 (1990)).
(0281 AUTODOCK is available from Scripps Research
Institute, La Jolla, Calif.; DOCK (Kuntz, I. D. et al., “A
Geometric Approach to Macromolecule-Ligand Interac
tions' J. Mol. Biol., (1982) 161,269-288). DOCK is available
from University of California, San Francisco, Calif.
0282. Once suitable chemical entities, compounds, or
agents have been selected, they can be assembled into a single
compound or inhibitor. Assembly may proceed by visual
inspection of the relationship of the fragments to each other
on the three-dimensional image displayed on a computer
screen in relation to the atomic coordinates of agaA, atpA,
atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC,
guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa,
pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE,
ubiH, uncA, visB, yecY, yiaY, yidK, yihV, yfhC), yibN and/or
yn D etc., or other protein required for their function. This
would be followed by manual model building using software
Such as Quanta or Sybyl. Useful programs to aid one of skill
in the art in connecting the individual chemical entities, com
pounds, or agents include but are not limited to CAVEAT
(Bartlett, P. A. et al., “CAVEAT: A Program to Facilitate the
Structure-Derived Design of Biologically Active Molecules”.
In Molecular Recognition in Chemical and Biological Prob
lems”. Special Pub., Royal Chem. Soc., 78, pp. 82-196
(1989)).
(0283 CAVEAT is available from the University of Cali
fornia, Berkeley, Calif.; 3D Database systems such as
MACCS-3D (MDL Information Systems, San Leandro,
Calif.). This area is reviewed in Martin, Y.C., “3D Database
Searching in Drug Design”. J. Med. Chem., 35, pp. 2145
2154 (1992); also HOOK (available from Molecular Simula
tions, Burlington, Mass.).
0284. Instead of designing an inhibitor of atpA, atpF,
atpH, betB, guaA, guaB, lipA, lySA, rpiA and/or trXA etc., or
other protein required for their in a step-wise fashion one
chemical moiety at a time as described above, inhibitors of
atpA, atpF, atpH, betB, guaA, guaB, lip A, lySA, rpiA and/or
trXA etc., or other protein required for their function may be
designed as a whole or “de novo” using either an empty
binding site or optionally including some portion(s) of known
inhibitor(s). These methods include LUDI (Bohm, H.-J.,
“The Computer Program LUDI: A New Method for the De
Novo Design of Enzyme Inhibitors'. J. ComR. Aid. Molec.
Design, (1992) 6, 61-78). LUDI is available from Biosym
Technologies, San Diego, Calif. and LEGEND (Nishibata, Y.
and A. Itai, Tetrahedron, (1991) 47, p. 8985). LEGEND is
available from Molecular Simulations, Burlington, Mass. and
LeapFrog (available from Tripos Associates, St. Louis, Mo.).
0285) Other molecular modeling techniques may also be
employed in accordance with this invention. See, e.g., Cohen,
N. C. et al., “Molecular Modeling Software and; Methods for
Medicinal Chemistry,” J. Med. Chem., (1990) 33, 883-894.
See also, Navia, M. A. and M. A. Murcko, "The Use of
US 2010/0028334 A1
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Structural Information in Drug Design. Current Opinions
Structural Biology, (1992) 2, 202-210.
0286 Once a compound has been designed or selected by
the above methods, the efficiency with which that compound
may bind to agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH,
betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lip A, lySA,
mnmA, nuvC, papa, pdxH. phn L. potE. rpiA, such3, trx A,
tusB (YheL), tus, ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK,
yihV, y?hC), yibN and/orynjD etc., or other protein required
for their function may be tested and optimized by computa
tional evaluation. An effective antimicrobial agent and/or
enhancer of antimicrobial agent must preferably demonstrate
a relatively small difference in energy between its bound and
free states (i.e., a small deformation energy of binding). Thus,
the most efficient inhibitors of agaA, atpA, atpC, atpB, atpl).
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lysA, mmm A. nuvC, papa, pdxH. phn L. potE.
rpiA, such3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV.yfhO, yibN and/orynjD etc., or other
proteins should preferably be designed with deformation
energy of binding of not greater than about 10 kcal/mole, or
more preferably, not greater than 7 kcal/mole.
0287. Inhibitors of agaA, atpA, atpC, atpB, atp), atpE.
atpG. atpH, betB, csdA, csdB, fepC. gua.A. guaB, iscS, kdgK.
lip A. lys A, mnmA. nuvC, papa, pdxH. phn L. potE. rpiA.
such}, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.
yiaY, yidK, yihV, yfhO, yibN and/orynjD etc., or other pro
teins may interact with their target in more than one confor
mation that is similarin overall binding energy. In those cases,
the deformation energy of binding is taken to be the difference
between the energy of the free compound and the average
energy of the conformations observed when the inhibitor
binds to agaA, atpA, atpC, atpB, atpl), atpE, atpG, atpH,
betB, csdA, csdB, fepC. guaA, guaB, iscS, kdgK, lip A, lySA,
mnmA. nuvC, papa, pdxH. phn L. potE. rpiA, such3, trx A,
tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY.yidK,
yihV. y?hO, yjbN and/orynjDA etc.
0288 A compound designed or selected, as binding to
agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, betB, csdA,
csdB, fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA,
nuvC, papa, pdxH, phn L. potE. rpiA, such3, trXA, tusB
(YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK,
yihV, yfhO, yjbN and/orynjD etc. can be further computa
tionally optimized so that in its bound state it would prefer
ably lack repulsive electrostatic interaction with the target.
Such non-complementary (e.g., electrostatic) interactions
include repulsive charge-charge, dipole-dipole and charged
dipole interactions. Specifically, the sum of all electrostatic
interactions between the inhibitor and the enzyme when the
inhibitor is bound to its target (e.g., agaA, atpA, atpC, atpB.
atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB,
iscS, kdgK, lip A, lysA, mnmA, nuvC, papa, pdxH, phn L.
potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE., ubiH, uncA,
visB, yeeY, yiaY, yidK, yihV.yfhO, yibN and/orynjD etc. or
other protein required for their function), make a neutral or
favorable contribution to the enthalpy of binding.
0289 Specific computer software is available in the art to
evaluate compound deformation energy and electrostatic
interaction. Examples of programs designed for such uses
include: Gaussian 92, revision C (M.J. Frisch, Gaussian, Inc.,
Pittsburgh, Pa., 1992); AMBER, version 4.0 (P. A. Kollman,
University of California at San Francisco, 1994); QUANTA/
CHARMM (Molecular Simulations, Inc., Burlington, Mass.
1994); and Insight II/Discover (Biosysm Technologies Inc.,
Feb. 4, 2010
San Diego, Calif., 1994). These programs may be imple
mented, for instance, using a Silicon Graphics workstation,
IRIS 4D/35 or IBM RISC/6000 workstation model 550.
Other hardware systems and software packages will be
known to those skilled in the art.
0290. Once an inhibitor of agaA, atpA, atpC, atpB, atpD.
atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS,
kdgK, lip A, lysA, mnmA. nuvC, papa, pdxH. phn L. potE.
rpiA, such3, trxA, tusB (YheL), tusB. ubiE., ubiH, uncA, visB,
yeeY, yiaY, yidK, yihV.yfhO, yibNetc. or other proteins that
suppress antimicrobial agents has been optimally selected or
designed, as described above, substitutions may then be made
in some of its atoms or side groups to improve or modify its
binding properties. Generally, initial substitutions are conser
vative, e.g., the replacement group will have approximately
the same size, shape, hydrophobicity and charge as the origi
nal group. It should, of course, be understood that compo
nents known in the art to alter conformation should be
avoided. Such substituted chemical compounds may then be
analyzed for efficiency of fit into the 3-D structures of agaA,
atpA, atpC, atpB, atplD, atpE, atpG, atpH, betB, csdA, csdB,
fepC, guaA, guaB, iscS, kdgK, lip A. lySA, mnmA. nuvC,
papa, pdxH. phn L. potE. rpiA. Suchs, trx A, tusB (YheL), tusB.
ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhO, yjbN
etc. or other proteins by the same computer methods
described in detail, above.
0291. The compounds designed by any of the above meth
ods are useful for inhibiting genes and/or gene products
which when inactivated potentiate activity of antimicrobial
agent and thus are useful as therapeutic agents with antimi
crobial agents to synergistically inhibit the growth and/or kill
microorganisms.
Examples
0292. The examples presented herein relate to composi
tions comprising antimicrobial agents and enhancers of anti
microbial agents. In the examples, antimicrobial peptides are
used as exemplary antimicrobial agents. Further, colistin is
used as an exemplary antimicrobial agent, in particular as an
exemplary antimicrobial peptide, although the methods and
compositions of the invention are applicable to any antimi
crobial agent. Further, mefloquine and potassium tellurite are
used as an exemplary enhancer of antimicrobial agents,
although the methods and compositions of the invention are
applicable to any enhancer of the antimicrobial agent, or any
molecule which inhibits a gene listed in Table 1 or Table 4.
Throughout this application, various publications are refer
enced. The disclosures of all of the publications and those
references cited within those publications in their entireties
are hereby incorporated by reference into this application in
order to more fully describe the state of the art to which this
invention pertains. The following examples are not intended
to limit the scope of the claims to the invention, but are rather
intended to be exemplary of certain embodiments. Any varia
tions in the exemplified methods which occur to the skilled
artisan are intended to fall within the scope of the present
invention.
0293 Methods
(0294 All experiments were performed in Luria-Bertani
(LB) medium (Fisher Scientific, Pittsburgh, Pa.). kanamycin
(Fisher Scientific), colistin sulfate (Sigma-Aldrich), and
potassium tellurite. Kanamycin (50 mg/ml), colistin (25
mg/ml), potassium tellurite (10 mg/ml) stocks on water.
0295 Straind
US 2010/0028334 A1
0296 BW25113 (lacIqrrnBT14 AlaczWJ16 hsdR514
AaraBADAH33 ArhabADLD78)
0297 Growth of deletion strains ubiH and iscS are con
tained in a BW251 13 deletion library (Baba et al., 2006).
0298 Mutant Deletion Screen
0299. Each plate of knockout strain (11 in total) was first
pre-grown in 50 ug/mL of Kanamycin in LB broth, then
re-grown in LB broth without Kanamycin.
0300 Twelve 384 well plates were filled with 50 uLLB
broth supplemented with 50 ug/mL of Kanamycin in each
well. Using a Beckman Multimek pipetting robot. The plates
were inoculated from KO library frozen stocks into the newly
filled plates using 384 well pin stamper. The stamper was
sterilized between each stamps by allowing the pin stamperto
sit in 70% Ethanol for 10-15 seconds, briefly placed into
100% Ethanol, then placed over a blame to bum off remaining
ethanol. The freshly inoculated plates were incubated for 24
hours at 37 C.
0301 A sample of these re-grown cultures was then
diluted 1/250 and stamped onto an LBagar plate. The remain
ing culture was then treated with colistin 20 ug/mL for 1.5
Hours. After treatment a second sample is then taken and
diluted 1/250 insterilized H2O before being stamped onto an
agar plate in duplicate. After 24 hours of growth at 37°C. the
plates are photographed and stored for further image analysis.
The digital images of treated vs. non treated plates are auto
matically overlapped using a developed image analysis pro
gram to measure size and density of the resulting colonies.
0302 Colony Formation Unit Assay
0303 For CFU measurements, 50 ul of stationary phase
culture was inoculated into 5 ml of LB. 240 or 245 ul of the
freshly diluted cells were placed into wells of a 96 well plate
(Costar), 5ul of colistin and or potassium tellurite were added
into each well to obtain a final volume of 250 ul. The 96 well
plates were then incubated at 37° C. At the 0, 3 and 6 hours
time points, 20 ul of culture was collected and then serially
diluted in 180 ul of 1xPBS, pH 7.2 (Fisher). A 10ul portion of
each dilution was plated onto LBagar (Fisher)), and the plate
was incubated overnight at 37° C. Dilutions that yielded
between 20 and 100 colonies were counted and CFU were
normalized to reflect the same amount of cells for the starting
cultures.
0304. The resistance or susceptibility of microorganism to
an antibiotic is classified by using defined minimum inhibi
tory concentration (MIC) breakpoints. MIC breakpoints for
an antibiotic are determined by the MIC distributions of
pathogenic in a clinical indication and its pharmacokinetics
and pharmacodynamics in humans. While a microorganism
may literally be susceptible to a high concentration of an
antibiotic in vitro, the microorganism may in fact be resistant
to that antibiotic at physiologically realistic concentrations if
the concentration of drug required to inhibit growth of or kill,
the microorganism is greater than the concentration that can
safely be achieved without toxicity Lo the subject, the micro
organism is considered to be resistant to the antibiotic. To
facilitate the identification of antibiotic resistance or suscep
tibility using In vitro test results, the National Committee for
Clinical Laboratory Standards (NCCLS, now known as Clini
cal and Laboratory Standards Institute) has formulated stan
dards for antibiotic susceptibility that correlate clinical out
come to in vitro determinations of the MIC antibiotics.
0305 Generally, MIC values indicate resistance or sus
ceptibility of a microorganism. For example, MIC valves of a
microorganism to colistin/polymyxin B are as follows:
Feb. 4, 2010
MIC<4 mg/L susceptible, MIC28 mg mg/L resistant and
MIC2128 mg/L-highly resistant
Example 1
0306 Over 4,000 single mutant E. coli were initially
screened in a cell based assay for ability to grow and/or
Survive in the presence of colistin, an antimicrobial peptide
(FIG.1). Approximately 92-95 E. colimutants were unable to
grow and Survive in the presence of colistin. The gene loci of
the mutations were analyzed and identified which are listed in
FIG. 2. The homology of the identified gene loci (herein
referred to as “gene products”) was analyzed with respect to
gene homology across different species of bacteria (FIG. 3),
and it was found that 24 of 66 gene products had human
homologues, listed in Table 1. Of these 24 gene products, 7
had known identified inhibitor which are listed in Table 2.
0307 The efficacy of colistin in inhibiting the growth and/
or Suppressing Survival of E. coli in the presence of one
inhibitor, mefloquine was assessed in a minimum inhibitory
concentration (MIC) assay. As shown in FIG. 4, in the
absence of mefloquine, the MIC of colistin is 6.3, whereas in
the presence of 50, 100 and 200 ug/ml of mefloquine, the MIC
of colistin required to suppress growth is 6.3, 3.1 and 0.8
respectively. Therefore, there is a dose-dependent potentia
tion of colistin efficacy by mefloquine at the concentrations
above 50 ug/ml or greater. Thus, mefloquine was demon
strated to act as a potent enhancer to the antimicrobial peptide
colistin. The inventors have also demonstrated that mefloqui
nine when used concurrently with colistin, potentiates colis
tin ability to decrease gram-positive bacteria (data not
shown).
0308 Accordingly, the inventors have demonstrated that
mefloquine is an exemplary example of an enhancer of an
antimicrobial agent, and was demonstrated to potentiate the
activity of an antimicrobial agent Such as colistin. Accord
ingly, the inventors have discovered that dose and use of
mefloquineas an enhancer of the antimicrobial agent is deter
mined by the therapeutic regimen (i.e. duration and adminis
tration) of the antimicrobial agent Such as colistin, as apposed
to any ability to decrease cell viability that mefloquine may
have when used by itself.
Example 2
0309 The inventors used a systems biology platform to
identify of several genes and therefore pathway, which once
inactivated can increase the killing effect of colistin. This
approach has its origin with the notion of synthetic lethality
screen in yeast where two genes non essential for viability on
their own are found to induce a non viable strain when com
bined and under specific growth condition(s) (Cottarel,
1997). With the access to the yeast deletion knockout library
(reviewed in Ooi et al., 2006) major efforts have been initiated
to create genetic mapping of gene interaction (Tong et al.,
2004). In a further step such reagents were used to link bio
active compounds to genetic pathways (Parsdons et al.,
2004). Reminiscent to this work, our screen was designed to
identify mutants exhibiting hyper sensitivity to antibacterials
to (i) map a chemogenomic response pathway and (ii) to
further identify and/or develop inhibitors which can mimic
the genetic observation. Indeed, such gene products can ulti
mately be the target of compounds which can in turn chemi
cally potentiate Colistin.
US 2010/0028334 A1 Feb. 4, 2010
32

0310. The inventors have identified herein using a systems

biology platform, several genes and pathways, which once TABLE 4-continued
inactivated, can increase the killing effect of colistin. For List of genes identified to exhibit sensitivity
example, the inventors have discovered a number of genes, to colistin from the primary screen.
such as those listed in Table 1 or Table 4, which normally
function in either secondary, parallel or downstream path Gene Function
ways to the target action of the antimicrobial agent, and when malG part of maltose permease, inner membrane
such pathways are inhibited or inactivated by inhibitors, mbha putative motility protein
potentiate the activity of antimicrobial agents, such as for mdoG periplasmic glucans biosynthesis protein
example colistin. The inventors have discovered, using an nei endonuclease VIII and DNAN-glycosylase with an AP lyase
activity
assay or screen designed to identify mutants exhibiting hyper nmpo. outer membrane porin protein; locus of qsr prophage
sensitivity to antibacterials to (i) map a chemogenomic nud
response pathway and (ii) to further identify and/or develop pdxH pyridoxinephosphate oxidase
inhibitors which can mimic the genetic phenotype. Indeed, phnB orf, hypothetical protein
Such gene products can ultimately be the target of compounds phnL ATP-binding component of phosphonate transport
phnO putative regulator, phn operon
which can in turn chemically potentiate colistin. pnuC required for NMN transport
0311. The Keio collection of E. coli mutants consist of potE putrescine transport protein
pshM putative general secretion
3,985 single gene knock-out of non essential for viability ptSA PEP-protein phosphotransferase system enzyme 1
genes (Baba et al., 2006). In the goal to identify mutants rhaT rhamnose transport
which exhibit increased sensitivity to antibiotics, colistin in rpiA ribosephosphate isomerase, constitutive
that case, the inventors developed a high-throughout assay rseA sigma-E factor, negative regulatory protein
which the screen basic scheme is shown on FIG. 1. The sbp periplasmic sulfate-binding protein
speA biosynthetic arginine decarboxylase
collection was re-formatted in a 384 well plate, from a 96 well SucB 2-oxoglutarate dehydrogenase
plate format, for ease of manipulation. Each identified can SugE Suppresses groEL, may be chaperone
didates found more than once in separated Screens were tdcE probable formate acetyltransferase 3
called positives. 73 Positives were then reformatted into a 96 tolC outer membrane channel
well plate for further testing (FIG. 2, Table 4). From these trxA hioredoxin 1
screen we selected the iscS (Lauhon, 2002) and the ubiH ubi 2-octaprenyl-6-methoxy-1,4-benzoquinone --> 2-octaprenyl
3-methyl-6-methoxy-1,4-benzoquinone
(Young et al., 1973) mutants for further studies. ubi 2-octaprenyl-6-methoxyphenol-->2-octaprenyl-6-methoxy
0312 Such inhibitors of the gene products as disclosed 1.4
herein are termed "enhancers of antimicrobial agents' herein, benzoquinone
ubiX 3-octaprenyl-4-hydroxybenzoate carboxy-lyase
and are selected based on their ability to inhibit a gene, such Xni
as any of those selected from the list of genes in Table 1 or ybb Y putative transport
Table 4. In particular, an inhibitor or enhancer of antimicro ycfM orf, hypothetical protein
bial agent is selected based on its gene inhibiting function yde orf, hypothetical protein
rather than any ability it may have to decrease cell viability yeeY putative transcriptional regulator LYSR-type
yfeT orf, hypothetical protein
when it is used by itself. ygaA putative 2-component transcriptional regulator
ygfZ orf, hypothetical protein
TABLE 4 yhdX putative transport system permease protein
yheL orf, hypothetical protein
List of genes identified to exhibit sensitivity yheM orf, hypothetical protein
to colistin from the primary screen. yiaY putative oxidoreductase
yid K putative cotransporter
Gene Function yihV putative kinase
ybN orf, hypothetical protein
agaA putative N-acetylgalactosamine-6-phosphate deacetylase yjcR putative membrane protein
atpA membrane-bound ATP synthase, F1 sector, alpha-subunit ycZ orf, hypothetical protein
atpF membrane-bound ATP synthase, F0 sector, subunit b
atpH membrane-bound ATP synthase, F1 sector, delta-subunit ygeC
betB NAD+-dependent betaine aldehyde dehydrogenase ygiH putative membrane protein
bg|F PTS system beta-glucosides, enzyme II, cryptic yrfA orf, hypothetical protein
cysE serine acetyltransferase
cysI Sulfite reductase, alpha Subunit The genes identified in bold are the qualified mutants after a secondary
fepG ATP-binding component of ferric enterobactin transport screen in a 96 well plate format.
fepD ferric enterobactin (enterochelin) transport
frvR. putative frv operon regulatory protein 0313 The cells were grown with or without colistin, ali
guaa GMP synthetase (glutamine-hydrolyzing) quots were taken at defined time points and serial dilution of
guaB IMP dehydrogenase
hoff putative general protein secretion protein the cells plated, colony count allowed to quantify the bacte
hsdS specificity determinant for hsdM and hsdR ricidal effect of colistin. We noticed that the iscS and ubi
iscS putative aminotransferase mutants were slow growers and formed Small size colonies
JW5075 compared to the parent strain.
JW 5227 0314. The used amount of colistin was chosen to not affect
JW 5257
UWS360
or little the parent strain, no significant decrease in viability
kdgK ketodeoxygluconokinase was detected when compared to the non treated cells at the 3
lip A lipoate synthesis, Sulfur insertion? and 5 hour time points. To the contrary, the mutants showed a
lySA diaminopimelate decarboxylase significant decrease in viability when exposed to colistin,
resulting to almost no colony detection at the 5 hour time
US 2010/0028334 A1
point (FIG. 5A). The mutants have a slower growth rate and
are characterized as forming Smaller size colonies (data not
shown), the reduced growth rate cannot on its own justify the
low colony formation in response to colistin.
0315. There are known inhibitors of the iscS gene product
such as iodoacetamine (Urbina et al., 2001) potassium tellu
rite (Rojas and Vasquez, 2005; Tantalean, 2003). The iscS
mutant was shown to be sensitive to potassium tellurite (Rojas
and Vasquez, 2005) while ectopic expression of iscS confers
resistance to potassium tellurite (Tantalean et al., 2003).
0316 Potassium tellurite is a heavy metal carrier (Te4+)
which is known to be toxic for bacteria. It is in fact a known
antibacterial agent not use as a therapeutic though and served
as a selective agent in microbiological culture medium (e.g.
Perez et al., 2007; Zanaroli et al., 2002). Potassium tellurite
mediates thiol oxidation in E. coli (Turneret al., 1999). How
ever, the inventors chose to continue experiments with Potas
sium tellurite based on its ability to inhibit the gene iscS, and
not due to any potential anti-bacterial activity it may have.
Thus, the inventors demonstrated that inhibition of a gene
listed in Table 4, in particular inhibition of iscS by potassium
tellurite potentiated colistin effects was a proof of principal
that inhibition of the genes listed in table 4 could potentiate
antimicrobial activity, such as potentiation of colistinactivity.
0317. The inventors used the CFU assay to observe that the
amount of colistin used was not affecting the parent strain, no
significant decrease in viability was detected when compared
to the non treated cells at the 3 hour time point (FIG. 5B). A
decrease in viability was noticed at the 5 hour time point.
Potassium tellurite at 1.6 ug/ml was not or little affecting the
CFU at the 3 hour time point while it shows a stronger growth
inhibition at 6 hours. The inventors observed that the combi
nation of colistin and potassium tellurite resulted in a signifi
cant decrease in viability closed to a 2 log decrease in average.
0318. The inventors have therefore designed systems biol
ogy platform that can be used to discover and identify E. coli
mutants which exhibit increased sensitivity to colistin. From
the pool of mutant candidates the inventors qualified the
strains disrupted on the ubiH and iscS loci. These gene prod
ucts are incorporated into two major cellular processes. ubiH
is involved the process to generate ATP while the iscS gene
product is involved the transfer of iron-sulfur clusters.
0319 Iron-Sulfur Fe—S clusters are prosthetic groups
which are required for crucial biological processes such as
electron transfer, iron/sulfur storage, gene regulation, tRNA
modification and enzyme activity. The iscS gene product, a
cysteine desulfurase, acts mostly as a Sulfur donor for other
proteins involved in diverse cellular regulatory pathways.
iscS obtains sulfur from cysteine which is then converted to
alanine and serves as Sulfur donor for Fe—S cluster assem
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 42
<21 Os SEQ ID NO 1
&211s LENGTH: 167
212s. TYPE: PRT
<213> ORGANISM: Escherichia coli
<4 OOs SEQUENCE: 1
Feb. 4, 2010
bly. The role of these genes to potentiate colistin is likely
related to a more global protein functionality aspect. These
Fe—S cluster groups are important for many cellular pro
cesses Such as thNA modification, regulation of enzyme
activity, Substrate binding and activation as well as regulation
of gene expression. The Fe—S clusters main biochemical
roles are (1) as acceptor and donor of electrons and (2) as
binder of the oxygen nitrogen groups of diverse Substrates
(review in Inlay, 2006). Consequently their roles are linked
into the process to synthesize ATP
0320 Such an approach can lead to a combination therapy
involving colistin and selected compounds which can ulti
mately reduce the toxic adverse effect of colistin either by
reducing the colistin dosage and/or by reducing the treatment
length.
REFERENCES
0321. The references cited herein and throughout the
application are incorporated herein by reference.
0322 Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y.
Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. Mol
Syst Biol. 2:2006.0008 (2006).
0323 Falagas ME and Kasakiou S K Clin Infect Dis 40:
1333-1341 (2005).
0324 Imlay J A. Mol Microbiol. 59:1073-82 (2006).
0325 Lauhon J Bacteriol. 184:6820-6829 (2002).
0326 Li J, Nation RL, Turnidge JD, Milne RW, Coulth
ard K. Rayner C R. Paterson D. L. Lancet Infect Dis. 6:589
601 (2006).
0327 Perez J. M. Calderon L, Arenas FA, Fuentes DE,
Pradenas GA, Fuentes EL, Sandoval JM, Castro ME, Elias
AO, Vasquez C C. PLoS ONE. 14:2:e211 (2007).
0328. Rojas DM, Vasquez C C. Res Microbiol. 156:465
71 (2005).
0329 Song JY, Kee SY. Hwang IS, Seo Y B, Jeong H.W.
Kim WJ, Cheong HJ.J Antimicrob Chemother. 60:317-322
(2007).
0330 Tan TY, Ng L S. Tan E. Huang G. J. Antimicrob
Chemother. 60:421-423 (2007).
0331 Tantalean J C, Araya MA, Saavedra C P Fuentes D
E. Perez J. M. Calderon I L. Youderian P. Vasquez C C. J
Bacteriol. 185:5831-7 (2003).
0332 Turner RJ, Weiner J. H. Taylor D E. Microbiology.
145:2549-2557 (1999).
0333 Urbina HD, Silberg JJ, Hoff KG, Vickery L. E. J.
Biol. Chem., 276:44521-44526 (2001).
0334 Young IG, Stroobant P. Macdonald CG, Gibson F.
J Bacteriol. 114:42-52 (1973).
0335 Zanaroli G, Fedi S, Carnevali M. Fava F. Zannoni D.
Res Microbiol. 153:353–360 (2002).
US 2010/0028334 A1 Feb. 4, 2010
34

- Continued
Met His Cys Tyr Asn Gly Met Thr Gly Lieu. His His Arg Glu Pro Gly
1. 5 1O 15

Met Val Gly Ala Gly Lieu. Thir Asp Lys Arg Ala Trp Lieu. Glu Lieu. Ile
2O 25 3O

Ala Asp Gly His His Val His Pro Ala Ala Met Ser Lieu. Cys Cys Cys
35 4O 45

Cys Ala Lys Glu Arg Ile Val Lieu. Ile Thr Asp Ala Met Glin Ala Ala
SO 55 6O

Gly Met Pro Asp Gly Arg Tyr Thr Lieu. Cys Gly Glu Glu Val Glin Met
65 70 7s 8O

His Gly Gly Val Val Arg Thr Ala Ser Gly Gly Lieu Ala Gly Ser Thr
85 90 95

Lieu. Ser Val Asp Ala Ala Val Arg Asn Met Val Glu Lieu. Thr Gly Val
1OO 105 11 O

Thr Pro Ala Glu Ala Ile His Met Ala Ser Lieu. His Pro Ala Arg Met
115 12 O 125

Lieu. Gly Val Asp Gly Val Lieu. Gly Ser Lieu Lys Pro Gly Lys Arg Ala
13 O 135 14 O

Arg Val Val Ala Lieu. Asp Ser Gly Lieu. His Val Glin Glin Ile Trp Ile
145 150 155 160

Glin Gly Glin Lieu Ala Ser Phe

1.65

<210s, SEQ ID NO 2
&211s LENGTH: 513
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2

Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile
1. 5 1O 15

Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val
2O 25 3O

Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met
35 4O 45

Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu.
SO 55 6O

Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala
65 70 7s 8O

Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu
85 90 95

Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly
1OO 105 11 O

Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala
115 12 O 125

Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin
13 O 135 14 O

Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly
145 150 155 160

Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr
1.65 17O 17s

Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys
18O 185 19 O
US 2010/0028334 A1 Feb. 4, 2010
35

- Continued

Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val
195 2OO 2O5

Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val
21 O 215 22O

Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr
225 23 O 235 24 O

Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala
245 250 255

Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin
26 O 265 27 O

Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly
27s 28O 285

Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val
29 O 295 3 OO

Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys
3. OS 310 315 32O

Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp
3.25 330 335

Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin
34 O 345 35. O

Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val
355 360 365

Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys
37 O 375 38O

Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr
385 390 395 4 OO

Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala
4 OS 41O 415

Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys
42O 425 43 O

Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu.
435 44 O 445

Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile
450 45.5 460

Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala
465 470 47s 48O

Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile
485 490 495

Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser
SOO 505 51O

Trp

<210s, SEQ ID NO 3
&211s LENGTH: 271
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 3

Met Ala Ser Glu Asn Met Thr Pro Glin Asp Tyr Ile Gly His His Leu
1. 5 1O 15

Asn Asn Lieu. Glin Lieu. Asp Lieu. Arg Thr Phe Ser Lieu Val Asp Pro Glin
2O 25 3O
US 2010/0028334 A1 Feb. 4, 2010
36

- Continued

Asn Pro Pro Ala Thr Phe Trp Thir Ile Asin Ile Asp Ser Met Phe Phe
35 4O 45

Ser Val Val Lieu. Gly Lieu. Lieu. Phe Lieu Val Lieu. Phe Arg Ser Val Ala
SO 55 6O

Lys Lys Ala Thr Ser Gly Val Pro Gly Llys Phe Glin Thr Ala Ile Glu
65 70 7s 8O

Lieu Val Ile Gly Phe Val Asn Gly Ser Val Lys Asp Met Tyr His Gly
85 90 95

Llys Ser Lys Lieu. Ile Ala Pro Lieu Ala Lieu. Thir Ile Phe Val Trp Val
1OO 105 11 O

Phe Lieu Met Asn Lieu Met Asp Lieu. Lieu Pro Ile Asp Lieu. Lieu Pro Tyr
115 12 O 125

Ile Ala Glu. His Val Lieu. Gly Lieu Pro Ala Lieu. Arg Val Val Pro Ser
13 O 135 14 O

Ala Asp Val Asn Val Thr Lieu. Ser Met Ala Lieu. Gly Val Phe Ile Lieu
145 150 155 160

Ile Leu Phe Tyr Ser Ile Llys Met Lys Gly Ile Gly Gly Phe Thr Lys
1.65 17O 17s

Glu Lieu. Thir Lieu Gln Pro Phe Asn His Trp Ala Phe Ile Pro Val Asn
18O 185 19 O

Lieu. Ile Lieu. Glu Gly Val Ser Lieu. Lieu. Ser Llys Pro Val Ser Lieu. Gly
195 2OO 2O5

Lieu. Arg Lieu. Phe Gly Asn Met Tyr Ala Gly Glu Lieu. Ile Phe Ile Lieu.
21 O 215 22O

Ile Ala Gly Lieu Lleu Pro Trp Trp Ser Glin Trp Ile Lieu. Asn Val Pro
225 23 O 235 24 O

Trp Ala Ile Phe His Ile Lieu. Ile Ile Thr Lieu. Glin Ala Phe Ile Phe
245 250 255

Met Val Lieu. Thir Ile Val Tyr Lieu Ser Met Ala Ser Glu Glu. His
26 O 265 27 O

<210s, SEQ ID NO 4
&21.1 L ENGTH: 139
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4

Met Ala Met Thr Tyr His Lieu. Asp Val Val Ser Ala Glu Glin Glin Met
1. 5 1O 15

Phe Ser Gly Lieu Val Glu Lys Ile Glin Val Thr Gly Ser Glu Gly Glu
2O 25 3O

Lieu. Gly Ile Tyr Pro Gly His Ala Pro Leu Lieu. Thir Ala Ile Llys Pro
35 4O 45

Gly Met Ile Arg Ile Val Lys Gln His Gly His Glu Glu Phe Ile Tyr
SO 55 6O

Lieu. Ser Gly Gly Ile Lieu. Glu Val Glin Pro Gly Asn Val Thr Val Lieu.
65 70 7s 8O

Ala Asp Thir Ala Ile Arg Gly Glin Asp Lieu. Asp Glu Ala Arg Ala Met
85 90 95

Glu Ala Lys Arg Lys Ala Glu Glu. His Ile Ser Ser Ser His Gly Asp
1OO 105 11 O

Val Asp Tyr Ala Glin Ala Ser Ala Glu Lieu Ala Lys Ala Ile Ala Glin
US 2010/0028334 A1 Feb. 4, 2010
37

- Continued
115 12 O 125

Lieu. Arg Val Ile Glu Lieu. Thir Lys Lys Ala Met
13 O 135

<210s, SEQ ID NO 5
&211s LENGTH: 460
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 5

Met Ala Thr Gly Lys Ile Val Glin Val Ile Gly Ala Val Val Asp Wall
1. 5 1O 15

Glu Phe Pro Glin Asp Ala Val Pro Arg Val Tyr Asp Ala Lieu. Glu Val
2O 25 3O

Glin Asn Gly Asn. Glu Arg Lieu Val Lieu. Glu Val Glin Glin Glin Lieu. Gly
35 4O 45

Gly Gly Ile Val Arg Thir Ile Ala Met Gly Ser Ser Asp Gly Lieu. Arg
SO 55 6O

Arg Gly Lieu. Asp Wall Lys Asp Lieu. Glu. His Pro Ile Glu Val Pro Val
65 70 7s 8O

Gly Lys Ala Thr Lieu. Gly Arg Ile Met Asin Val Lieu. Gly Glu Pro Val
85 90 95

Asp Met Lys Gly Glu Ile Gly Glu Glu Glu Arg Trp Ala Ile His Arg
1OO 105 11 O

Ala Ala Pro Ser Tyr Glu Glu Lieu. Ser Asn. Ser Glin Glu Lieu. Lieu. Glu
115 12 O 125

Thr Gly Ile Llys Val Ile Asp Lieu Met Cys Pro Phe Ala Lys Gly Gly
13 O 135 14 O

Llys Val Gly Lieu. Phe Gly Gly Ala Gly Val Gly Llys Thr Val Asn Met
145 150 155 160

Met Glu Lieu. Ile Arg Asn. Ile Ala Ile Glu. His Ser Gly Tyr Ser Val
1.65 17O 17s

Phe Ala Gly Val Gly Glu Arg Thr Arg Glu Gly Asn Asp Phe Tyr His
18O 185 19 O

Glu Met Thr Asp Ser Asn Val Ile Asp Llys Val Ser Lieu Val Tyr Gly
195 2OO 2O5

Glin Met Asn. Glu Pro Pro Gly Asn Arg Lieu. Arg Val Ala Lieu. Thr Gly
21 O 215 22O

Lieu. Thir Met Ala Glu Lys Phe Arg Asp Glu Gly Arg Asp Val Lieu. Lieu.
225 23 O 235 24 O

Phe Val Asp Asn Ile Tyr Arg Tyr Thr Lieu Ala Gly Thr Glu Val Ser
245 250 255

Ala Lieu. Leu Gly Arg Met Pro Ser Ala Val Gly Tyr Gln Pro Thr Lieu.
26 O 265 27 O

Ala Glu Glu Met Gly Val Lieu. Glin Glu Arg Ile Thr Ser Thr Lys Thr
27s 28O 285

Gly Ser Ile Thir Ser Val Glin Ala Val Tyr Val Pro Ala Asp Asp Lieu.
29 O 295 3 OO

Thr Asp Pro Ser Pro Ala Thr Thr Phe Ala His Lieu. Asp Ala Thr Val
3. OS 310 315 32O

Val Lieu. Ser Arg Glin Ile Ala Ser Lieu. Gly Ile Tyr Pro Ala Val Asp
3.25 330 335
US 2010/0028334 A1 Feb. 4, 2010
38

- Continued
Pro Lieu. Asp Ser Thr Ser Arg Glin Lieu. Asp Pro Lieu Val Val Gly Glin
34 O 345 35. O

Glu. His Tyr Asp Thr Ala Arg Gly Val Glin Ser Ile Lieu. Glin Arg Tyr
355 360 365

Glin Glu Lieu Lys Asp Ile Ile Ala Ile Lieu. Gly Met Asp Glu Lieu. Ser
37 O 375 38O

Glu Glu Asp Llys Lieu Val Val Ala Arg Ala Arg Lys Ile Glin Arg Phe
385 390 395 4 OO

Lieu. Ser Glin Pro Phe Phe Val Ala Glu Val Phe Thr Gly Ser Pro Gly
4 OS 41O 415

Llys Tyr Val Ser Lieu Lys Asp Thir Ile Arg Gly Phe Lys Gly Ile Met
42O 425 43 O

Glu Gly Glu Tyr Asp His Leu Pro Glu Glin Ala Phe Tyr Met Val Gly
435 44 O 445

Ser Ile Glu Glu Ala Val Glu Lys Ala Lys Llys Lieu.
450 45.5 460

<210s, SEQ ID NO 6
&211s LENGTH: 79
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 6

Met Glu Asn Lieu. ASn Met Asp Lieu. Lieu. Tyr Met Ala Ala Ala Val Met
1. 5 1O 15

Met Gly Lieu Ala Ala Ile Gly Ala Ala Ile Gly Ile Gly Ile Lieu. Gly
2O 25 3O

Gly Llys Phe Lieu. Glu Gly Ala Ala Arg Glin Pro Asp Lieu. Ile Pro Lieu.
35 4O 45

Lieu. Arg Thr Glin Phe Phe Ile Val Met Gly Lieu Val Asp Ala Ile Pro
SO 55 6O

Met Ile Ala Val Gly Lieu. Gly Lieu. Tyr Val Met Phe Ala Wall Ala
65 70 7s

<210s, SEQ ID NO 7
&211s LENGTH: 287
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 7

Met Ala Gly Ala Lys Glu Ile Arg Ser Lys Ile Ala Ser Val Glin Asn
1. 5 1O 15

Thr Gln Lys Ile Thr Lys Ala Met Glu Met Val Ala Ala Ser Lys Met
2O 25 3O

Arg Llys Ser Glin Asp Arg Met Ala Ala Ser Arg Pro Tyr Ala Glu Thr
35 4O 45

Met Arg Llys Val Ile Gly. His Lieu Ala His Gly Asn Lieu. Glu Tyr Lys
SO 55 6O

His Pro Tyr Lieu. Glu Asp Arg Asp Wall Lys Arg Val Gly Tyr Lieu Val
65 70 7s 8O

Val Ser Thir Asp Arg Gly Lieu. Cys Gly Gly Lieu. Asn. Ile Asn Lieu. Phe
85 90 95

Llys Llys Lieu. Lieu Ala Glu Met Lys Thir Trp Thr Asp Llys Gly Val Glin
1OO 105 11 O
US 2010/0028334 A1 Feb. 4, 2010
39

- Continued
Cys Asp Leu Ala Met Ile Gly Ser Lys Gly Val Ser Phe Phe Asin Ser
115 12 O 125

Val Gly Gly Asn Val Val Ala Glin Val Thr Gly Met Gly Asp Asn Pro
13 O 135 14 O

Ser Lieu. Ser Glu Lieu. Ile Gly Pro Val Llys Val Met Lieu. Glin Ala Tyr
145 150 155 160

Asp Glu Gly Arg Lieu. Asp Llys Lieu. Tyr Ile Val Ser Asn Llys Phe Ile
1.65 17O 17s

Asn. Thir Met Ser Glin Wall Pro Thir Ile Ser Glin Leu Lleu Pro Leu Pro
18O 185 19 O

Ala Ser Asp Asp Asp Asp Lieu Lys His Llys Ser Trp Asp Tyr Lieu. Tyr
195 2OO 2O5

Glu Pro Asp Pro Lys Ala Lieu. Lieu. Asp Thir Lieu. Lieu. Arg Arg Tyr Val
21 O 215 22O

Glu Ser Glin Val Tyr Glin Gly Val Val Glu Asn Lieu Ala Ser Glu Glin
225 23 O 235 24 O

Ala Ala Arg Met Val Ala Met Lys Ala Ala Thr Asp Asn Gly Gly Ser
245 250 255

Lieu. Ile Lys Glu Lieu. Glin Lieu Val Tyr Asn Lys Ala Arg Glin Ala Ser
26 O 265 27 O

Ile Thr Glin Glu Lieu. Thr Glu Ile Val Ser Gly Ala Ala Ala Val
27s 28O 285

<210s, SEQ ID NO 8
&211s LENGTH: 177
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 8

Met Ser Glu Phe Ile Thr Val Ala Arg Pro Tyr Ala Lys Ala Ala Phe
1. 5 1O 15

Asp Phe Ala Val Glu. His Glin Ser Val Glu Arg Trp Glin Asp Met Lieu
2O 25 3O

Ala Phe Ala Ala Glu Val Thir Lys Asn. Glu Gln Met Ala Glu Lieu. Lieu
35 4O 45

Ser Gly Ala Lieu Ala Pro Glu Thir Lieu Ala Glu Ser Phe Ile Ala Val
SO 55 6O

Cys Gly Glu Gln Lieu. Asp Glu Asin Gly Glin Asn Lieu. Ile Arg Val Met
65 70 7s 8O

Ala Glu Asn Gly Arg Lieu. Asn Ala Lieu Pro Asp Val Lieu. Glu Glin Phe
85 90 95

Ile His Lieu. Arg Ala Val Ser Glu Ala Thr Ala Glu Val Asp Val Ile
1OO 105 11 O

Ser Ala Ala Ala Lieu. Ser Glu Glin Glin Lieu Ala Lys Ile Ser Ala Ala
115 12 O 125

Met Glu Lys Arg Lieu. Ser Arg Llys Wall Lys Lieu. Asn. Cys Lys Ile Asp
13 O 135 14 O

Llys Ser Wal Met Ala Gly Val Ile Ile Arg Ala Gly Asp Met Val Ile
145 150 155 160

Asp Gly Ser Val Arg Gly Arg Lieu. Glu Arg Lieu Ala Asp Val Lieu. Glin
1.65 17O 17s

Ser
US 2010/0028334 A1 Feb. 4, 2010
40

- Continued

<210s, SEQ ID NO 9
&211s LENGTH: 490
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 9

Met Ser Arg Met Ala Glu Gln Gln Leu Tyr Ile His Gly Gly Tyr Thr
1. 5 1O 15

Ser Ala Thr Ser Gly Arg Thr Phe Glu Thir Ile Asn Pro Ala Asn Gly
2O 25 3O

Asn Val Lieu Ala Thr Val Glin Ala Ala Gly Arg Glu Asp Val Asp Arg
35 4O 45

Ala Wall Lys Ser Ala Glin Glin Gly Glin Lys Ile Trp Ala Ser Met Thr
SO 55 6O

Ala Met Glu Arg Ser Arg Ile Lieu. Arg Arg Ala Val Asp Ile Lieu. Arg
65 70 7s 8O

Glu Arg Asn Asp Glu Lieu Ala Lys Lieu. Glu Thir Lieu. Asp Thr Gly Lys
85 90 95

Ala Tyr Ser Glu Thir Ser Thr Val Asp Ile Val Thr Gly Ala Asp Val
1OO 105 11 O

Lieu. Glu Tyr Tyr Ala Gly Lieu. Ile Pro Ala Lieu. Glu Gly Ser Glin Ile
115 12 O 125

Pro Leu Arg Glu Thir Ser Phe Val Tyr Thr Arg Arg Glu Pro Leu Gly
13 O 135 14 O

Val Val Ala Gly Ile Gly Ala Trp Asn Tyr Pro Ile Glin Ile Ala Lieu
145 150 155 160

Trp Llys Ser Ala Pro Ala Lieu Ala Ala Gly Asn Ala Met Ile Phe Lys
1.65 17O 17s

Pro Ser Glu Val Thr Pro Leu. Thir Ala Leu Lys Lieu Ala Glu Ile Tyr
18O 185 19 O

Ser Glu Ala Gly Lieu Pro Asp Gly Val Phe Asin Val Lieu Pro Gly Val
195 2OO 2O5

Gly Ala Glu Thr Gly Glin Tyr Lieu. Thr Glu. His Pro Gly Ile Ala Lys
21 O 215 22O

Val Ser Phe Thr Gly Gly Val Ala Ser Gly Lys Llys Val Met Ala Asn
225 23 O 235 24 O

Ser Ala Ala Ser Ser Lieu Lys Glu Val Thr Met Glu Lieu. Gly Gly Lys
245 250 255

Ser Pro Lieu. Ile Val Phe Asp Asp Ala Asp Lieu. Asp Lieu Ala Ala Asp
26 O 265 27 O

Ile Ala Met Met Ala Asn Phe Phe Ser Ser Gly Glin Val Cys Thr Asn
27s 28O 285

Gly Thr Arg Val Phe Val Pro Ala Lys Cys Lys Ala Ala Phe Glu Glin
29 O 295 3 OO

Lys Ile Lieu Ala Arg Val Glu Arg Ile Arg Ala Gly Asp Val Phe Asp
3. OS 310 315 32O

Pro Gln Thr Asn Phe Gly Pro Leu Val Ser Phe Pro His Arg Asp Asn
3.25 330 335

Val Lieu. Arg Tyr Ile Ala Lys Gly Lys Glu Glu Gly Ala Arg Val Lieu
34 O 345 35. O

Cys Gly Gly Asp Val Lieu Lys Gly Asp Gly Phe Asp Asn Gly Ala Trp
355 360 365
US 2010/0028334 A1 Feb. 4, 2010
41

- Continued

Val Ala Pro Thr Val Phe Thr Asp Cys Ser Asp Asp Met Thr Ile Val
37 O 375 38O

Arg Glu Glu Ile Phe Gly Pro Val Met Ser Ile Lieu. Thr Tyr Glu Ser
385 390 395 4 OO

Glu Asp Glu Val Ile Arg Arg Ala Asn Asp Thr Asp Tyr Gly Lieu Ala
4 OS 41O 415

Ala Gly Ile Val Thr Ala Asp Lieu. Asn Arg Ala His Arg Val Ile His
42O 425 43 O

Gln Leu Glu Ala Gly Ile Cys Trp Ile Asn. Thir Trp Gly Glu Ser Pro
435 44 O 445

Ala Glu Met Pro Val Gly Gly Tyr Lys His Ser Gly Ile Gly Arg Glu
450 45.5 460

Asn Gly Val Met Thr Lieu. Glin Ser Tyr Thr Glin Val Lys Ser Ile Glin
465 470 47s 48O

Val Glu Met Ala Lys Phe Glin Ser Ile Phe
485 490

<210s, SEQ ID NO 10
&211s LENGTH: 4 O1
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 10

Met Asin Val Phe Asn Pro Ala Glin Phe Arg Ala Glin Phe Pro Ala Leu
1. 5 1O 15

Glin Asp Ala Gly Val Tyr Lieu. Asp Ser Ala Ala Thr Ala Lieu Lys Pro
2O 25 3O

Glu Ala Val Val Glu Ala Thr Glin Glin Phe Tyr Ser Leu Ser Ala Gly
35 4O 45

Asn. Wal His Arg Ser Glin Phe Ala Glu Ala Glin Arg Lieu. Thir Ala Arg
SO 55 6O

Tyr Glu Ala Ala Arg Glu Lys Val Ala Glin Lieu. Lieu. Asn Ala Pro Asp
65 70 7s 8O

Asp Llys Thir Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met
85 90 95

Val Ala Glin Cys Tyr Ala Arg Pro Arg Lieu. Glin Pro Gly Asp Glu Ile
1OO 105 11 O

Ile Val Ser Val Ala Glu. His His Ala Asn Lieu Val Pro Trp Lieu Met
115 12 O 125

Val Ala Glin Glin Thr Gly Ala Lys Val Val Lys Lieu Pro Lieu. Asn Ala
13 O 135 14 O

Glin Arg Lieu Pro Asp Wall Asp Lieu. Lieu Pro Glu Lieu. Ile Thr Pro Arg
145 150 155 160

Ser Arg Ile Lieu Ala Lieu. Gly Glin Met Ser Asn Val Thr Gly Gly Cys
1.65 17O 17s

Pro Asp Lieu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val
18O 185 19 O

Val Met Val Asp Gly Ala Glin Gly Ala Wal His Phe Pro Ala Asp Wall
195 2OO 2O5

Glin Glin Lieu. Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Llys Lieu. Tyr
21 O 215 22O

Gly Pro Thr Gly Ile Gly Val Lieu. Tyr Gly Lys Ser Glu Lieu. Lieu. Glu
US 2010/0028334 A1 Feb. 4, 2010
42

- Continued
225 23 O 235 24 O

Ala Met Ser Pro Trp Lieu. Gly Gly Gly Lys Met Val His Glu Val Ser
245 250 255

Phe Asp Gly Phe Thr Thr Glin Ser Ala Pro Trp Llys Lieu. Glu Ala Gly
26 O 265 27 O

Thr Pro Asn. Wall Ala Gly Val Ile Gly Lieu. Ser Ala Ala Lieu. Glu Trp
27s 28O 285

Lieu Ala Asp Tyr Asp Ile Asn Glin Ala Glu Ser Trp Ser Arg Ser Lieu.
29 O 295 3 OO

Ala Thr Lieu Ala Glu Asp Ala Lieu Ala Lys Arg Pro Gly Phe Arg Ser
3. OS 310 315 32O

Phe Arg Cys Glin Asp Ser Ser Lieu. Lieu Ala Phe Asp Phe Ala Gly Val
3.25 330 335

His His Ser Asp Met Val Thir Lieu. Lieu Ala Glu Tyr Gly Ile Ala Lieu.
34 O 345 35. O

Arg Ala Gly Glin His Cys Ala Glin Pro Lieu. Lieu Ala Glu Lieu. Gly Val
355 360 365

Thr Gly. Thir Lieu. Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp
37 O 375 38O

Val Asp Ala Lieu Val Asn Ala Val Asp Arg Ala Lieu. Glu Lieu. Lieu Val
385 390 395 4 OO

Asp

<210s, SEQ ID NO 11
&211s LENGTH: 646
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 11

Met Met Ser Tyr Val Asp Trp Pro Pro Leu. Ile Lieu. Arg His Thr Tyr
1. 5 1O 15

Tyr Met Ala Glu Phe Glu Thir Thr Phe Ala Asp Lieu. Gly Lieu Lys Ala
2O 25 3O

Pro Ile Lieu. Glu Ala Lieu. Asn Asp Lieu. Gly Tyr Glu, Llys Pro Ser Pro
35 4O 45

Ile Glin Ala Glu. Cys Ile Pro His Lieu. Lieu. Asn Gly Arg Asp Val Lieu.
SO 55 6O

Gly Met Ala Glin Thr Gly Ser Gly Lys Thr Ala Ala Phe Ser Leu Pro
65 70 7s 8O

Lieu. Lieu. Glin Asn Lieu. Asp Pro Glu Lieu Lys Ala Pro Glin Ile Lieu Val
85 90 95

Lieu Ala Pro Thir Arg Glu Lieu Ala Val Glin Val Ala Glu Ala Met Thr
1OO 105 11 O

Asp Phe Ser Lys His Met Arg Gly Val Asin Val Val Ala Lieu. Tyr Gly
115 12 O 125

Gly Glin Arg Tyr Asp Val Glin Lieu. Arg Ala Lieu. Arg Glin Gly Pro Glin
13 O 135 14 O

Ile Val Val Gly Thr Pro Gly Arg Lieu. Lieu. Asp His Lieu Lys Arg Gly
145 150 155 160

Thir Lieu. Asp Lieu. Ser Llys Lieu. Ser Gly Lieu Val Lieu. Asp Glu Ala Asp
1.65 17O 17s

Glu Met Leu Arg Met Gly Phe Ile Glu Asp Val Glu Thir Ile Met Ala
US 2010/0028334 A1 Feb. 4, 2010
43

- Continued
18O 185 19 O

Glin Ile Pro Glu Gly His Glin Thr Ala Leu Phe Ser Ala Thr Met Pro
195 2OO 2O5

Glu Ala Ile Arg Arg Ile Thr Arg Arg Phe Met Lys Glu Pro Glin Glu
21 O 215 22O

Val Arg Ile Glin Ser Ser Val Thir Thr Arg Pro Asp Ile Ser Glin Ser
225 23 O 235 24 O

Tyr Trp Thr Val Trp Gly Met Arg Lys Asn. Glu Ala Lieu Val Arg Phe
245 250 255

Lieu. Glu Ala Glu Asp Phe Asp Ala Ala Ile Ile Phe Val Arg Thr Lys
26 O 265 27 O

Asn Ala Thir Lieu. Glu Val Ala Glu Ala Lieu. Glu Arg Asn Gly Tyr Asn
27s 28O 285

Ser Ala Ala Lieu. Asn Gly Asp Met Asn Glin Ala Lieu. Arg Glu Glin Thr
29 O 295 3 OO

Lieu. Glu Arg Lieu Lys Asp Gly Arg Lieu. Asp I e Lieu. Ile Ala Thr Asp
3. OS 310 3 5 32O

Val Ala Ala Arg Gly Lieu. Asp Val Glu Arg Ile Ser Lieu Val Val Asn
3.25 330 335

Tyr Asp Ile Pro Met Asp Ser Glu Ser Tyr Val His Arg Ile Gly Arg
34 O 345 35. O

Thr Gly Arg Ala Gly Arg Ala Gly Arg Ala Lieu. Lieu. Phe Val Glu ASn
355 360 365

Arg Glu Arg Arg Lieu. Lieu. Arg Asn. Ile Glu Arg Thr Met Lys Lieu. Thr
37 O 375 38O

Ile Pro Glu Val Glu Lieu Pro Asn Ala Glu Lieu. Lieu. Gly Lys Arg Arg
385 390 395 4 OO

Lieu. Glu Lys Phe Ala Ala Lys Val Glin Glin Gln Lieu. Glu Ser Ser Asp
4 OS 41O 415

Lieu. Asp Glin Tyr Arg Ala Lieu. Lieu. Ser Lys Ile Glin Pro Thir Ala Glu
42O 425 43 O

Gly Glu Glu Lieu. Asp Lieu. Glu Thir Lieu Ala Ala Ala Lieu. Lieu Lys Met
435 44 O 445

Ala Glin Gly Glu Arg Thr Lieu. Ile Val Pro Pro Asp Ala Pro Met Arg
450 45.5 460

Pro Lys Arg Glu Phe Arg Asp Arg Asp Asp Arg Gly Pro Arg Asp Arg
465 470 47s 48O

Asn Asp Arg Gly Pro Arg Gly Asp Arg Glu Asp Arg Pro Arg Arg Glu
485 490 495

Arg Arg Asp Val Gly Asp Met Glin Lieu. Tyr Arg Ile Glu Val Gly Arg
SOO 505 51O

Asp Asp Gly Val Glu Val Arg His Ile Val Gly Ala Ile Ala Asn. Glu
515 52O 525

Gly Asp Ile Ser Ser Arg Tyr Ile Gly Asn. Ile Llys Lieu. Phe Ala Ser
53 O 535 54 O

His Ser Thr Ile Glu Lieu Pro Lys Gly Met Pro Gly Glu Val Leu Gln
5.45 550 555 560

His Phe Thr Arg Thr Arg Ile Lieu. Asn Llys Pro Met Asn Met Glin Leu
565 st O sts

Lieu. Gly Asp Ala Glin Pro His Thr Gly Gly Glu Arg Arg Gly Gly Gly
58O 585 59 O
US 2010/0028334 A1 Feb. 4, 2010
44

- Continued

Arg Gly Phe Gly Gly Glu Arg Arg Glu Gly Gly Arg Asn. Phe Ser Gly
595 6OO 605

Glu Arg Arg Glu Gly Gly Arg Gly Asp Gly Arg Arg Phe Ser Gly Glu
610 615 62O

Arg Arg Glu Gly Arg Ala Pro Arg Arg Asp Asp Ser Thr Gly Arg Arg
625 630 635 64 O

Arg Phe Gly Gly Asp Ala

645

<210s, SEQ ID NO 12
&211s LENGTH: 406
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 12

Met Ile Phe Ser Val Asp Llys Val Arg Ala Asp Phe Pro Val Lieu. Ser
1. 5 1O 15

Arg Glu Val Asn Gly Lieu Pro Lieu Ala Tyr Lieu. Asp Ser Ala Ala Ser
2O 25 3O

Ala Glin Llys Pro Ser Glin Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg
35 4O 45

His Gly Tyr Ala Ala Wal His Arg Gly Ile His Thr Lieu. Ser Ala Glin
SO 55 6O

Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Lieu. Phe Ile
65 70 7s 8O

Asn Ala Arg Ser Ala Glu Glu Lieu Val Phe Val Arg Gly. Thir Thr Glu
85 90 95

Gly Ile Asn Lieu Val Ala Asn. Ser Trp Gly Asn. Ser Asn Val Arg Ala
1OO 105 11 O

Gly Asp Asn. Ile Ile Ile Ser Glin Met Glu. His His Ala Asn. Ile Val
115 12 O 125

Pro Trp Gln Met Lieu. Cys Ala Arg Val Gly Ala Glu Lieu. Arg Val Ile
13 O 135 14 O

Pro Leu. Asn Pro Asp Gly Thr Lieu Gln Leu Glu Thir Lieu Pro Thr Lieu
145 150 155 160

Phe Asp Glu Lys Thr Arg Lieu. Lieu Ala Ile Thr His Val Ser Asn. Wall
1.65 17O 17s

Lieu. Gly. Thr Glu Asn Pro Lieu Ala Glu Met Ile Thr Lieu Ala His Glin
18O 185 19 O

His Gly Ala Lys Val Lieu Val Asp Gly Ala Glin Ala Wal Met His His
195 2OO 2O5

Pro Val Asp Val Glin Ala Lieu. Asp Cys Asp Phe Tyr Val Phe Ser Gly
21 O 215 22O

His Llys Lieu. Tyr Gly Pro Thr Gly Ile Gly Ile Lieu. Tyr Val Lys Glu
225 23 O 235 24 O

Ala Lieu. Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile
245 250 255

Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp
26 O 265 27 O

Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Lieu. Gly
27s 28O 285

Ala Ala Lieu. Glu Tyr Val Ser Ala Lieu. Gly Lieu. Asn. Asn. Ile Ala Glu
US 2010/0028334 A1 Feb. 4, 2010
45

- Continued
29 O 295 3 OO

Tyr Glu Glin Asn Lieu Met His Tyr Ala Lieu. Ser Glin Lieu. Glu Ser Val
3. OS 310 315 32O

Pro Asp Lieu. Thir Lieu. Tyr Gly Pro Glin Asn Arg Lieu. Gly Val Ile Ala
3.25 330 335

Phe Asn Lieu. Gly Llys His His Ala Tyr Asp Val Gly Ser Phe Lieu. Asp
34 O 345 35. O

Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu
355 360 365

Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met
37 O 375 38O

Tyr Asn. Thir His Glu Glu Val Asp Arg Lieu Val Thr Gly Lieu. Glin Arg
385 390 395 4 OO

Ile His Arg Lieu. Lieu. Gly

4 OS

<210s, SEQ ID NO 13
&211s LENGTH: 271
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 13

Met Thr Glu Ser Val Ala Arg Lieu. Arg Gly Glu Glin Lieu. Thir Lieu. Gly
1. 5 1O 15

Tyr Gly Lys Tyr Thr Val Ala Glu Asn Lieu. Thr Val Glu Ile Pro Asp
2O 25 3O

Gly His Phe Thr Ala Ile Ile Gly Pro Asn Gly Cys Gly Lys Ser Thr
35 4O 45

Lieu. Lieu. Arg Thir Lieu. Ser Arg Lieu Met Thr Pro Ala His Gly His Val
SO 55 6O

Trp Lieu. Asp Gly Glu. His Ile Glin His Tyr Ala Ser Lys Glu Val Ala
65 70 7s 8O

Arg Arg Ile Gly Lieu. Lieu Ala Glin Asn Ala Thir Thr Pro Gly Asp Ile
85 90 95

Thr Val Glin Glu Lieu Val Ala Arg Gly Arg Tyr Pro His Glin Pro Leu
1OO 105 11 O

Phe Thr Arg Trp Arg Lys Glu Asp Glu Glu Ala Val Thir Lys Ala Met
115 12 O 125

Glin Ala Thr Gly Ile Thr His Lieu Ala Asp Glin Ser Val Asp Thir Lieu.
13 O 135 14 O

Ser Gly Gly Glin Arg Glin Arg Ala Trp Ile Ala Met Val Lieu Ala Glin
145 150 155 160

Glu Thir Ala Ile Met Leu Lleu. Asp Glu Pro Thir Thr Trp Lieu. Asp Ile
1.65 17O 17s

Ser His Glin Ile Asp Lieu. Lieu. Glu Lieu. Lieu. Ser Glu Lieu. Asn Arg Glu
18O 185 19 O

Lys Gly Tyr Thir Lieu Ala Ala Val Lieu. His Asp Lieu. Asn Glin Ala Cys
195 2OO 2O5

Arg Tyr Ala Ser His Lieu. Ile Ala Lieu. Arg Glu Gly Lys Ile Val Ala
21 O 215 22O

Glin Gly Ala Pro Llys Glu Ile Val Thir Ala Glu Lieu. Ile Glu Arg Ile
225 23 O 235 24 O
US 2010/0028334 A1 Feb. 4, 2010
46

- Continued
Tyr Gly Lieu. Arg Cys Met Ile Ile Asp Asp Pro Val Ala Gly Thr Pro
245 250 255

Lieu Val Val Pro Leu Gly Arg Thr Ala Pro Ser Thr Ala Asn Ser
26 O 265 27 O

<210s, SEQ ID NO 14
&211s LENGTH: 525
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 14

Met Thr Glu Asn. Ile His Llys His Arg Ile Lieu. Ile Lieu. Asp Phe Gly
1. 5 1O 15

Ser Glin Tyr Thr Glin Lieu Val Ala Arg Arg Val Arg Glu Lieu. Gly Val
2O 25 3O

Tyr Cys Glu Lieu. Trp Ala Trp Asp Val Thr Glu Ala Glin Ile Arg Asp
35 4O 45

Phe Asin Pro Ser Gly Ile Ile Leu Ser Gly Gly Pro Glu Ser Thir Thr
SO 55 6O

Glu Glu Asn Ser Pro Arg Ala Pro Glin Tyr Val Phe Glu Ala Gly Val
65 70 7s 8O

Pro Val Phe Gly Val Cys Tyr Gly Met Glin Thr Met Ala Met Gln Leu
85 90 95

Gly Gly His Val Glu Ala Ser ASn Glu Arg Glu Phe Gly Tyr Ala Glin
1OO 105 11 O

Val Glu Val Val Asn Asp Ser Ala Lieu Val Arg Gly Ile Glu Asp Ala
115 12 O 125

Lieu. Thir Ala Asp Gly Llys Pro Lieu. Lieu. Asp Val Trp Met Ser His Gly
13 O 135 14 O

Asp Llys Val Thr Ala Ile Pro Ser Asp Phe Ile Thr Val Ala Ser Thr
145 150 155 160

Glu Ser Cys Pro Phe Ala Ile Met Ala Asn Glu Glu Lys Arg Phe Tyr
1.65 17O 17s

Gly Val Glin Phe His Pro Glu Val Thr His Thr Arg Glin Gly Met Arg
18O 185 19 O

Met Lieu. Glu Arg Phe Val Arg Asp Ile Cys Glin Cys Glu Ala Lieu. Trp
195 2OO 2O5

Thr Pro Ala Lys Ile Ile Asp Asp Ala Val Ala Arg Ile Arg Glu Glin
21 O 215 22O

Val Gly Asp Asp Llys Val Ile Lieu. Gly Lieu. Ser Gly Gly Val Asp Ser
225 23 O 235 24 O

Ser Val Thir Ala Met Lieu. Lieu. His Arg Ala Ile Gly Lys Asn Lieu. Thir
245 250 255

Cys Val Phe Val Asp Asn Gly Lieu. Lieu. Arg Lieu. Asn. Glu Ala Glu Glin
26 O 265 27 O

Val Lieu. Asp Met Phe Gly Asp His Phe Gly Lieu. Asn. Ile Val His Val
27s 28O 285

Pro Ala Glu Asp Arg Phe Lieu. Ser Ala Lieu Ala Gly Glu Asn Asp Pro
29 O 295 3 OO

Glu Ala Lys Arg Lys Ile Ile Gly Arg Val Phe Val Glu Val Phe Asp
3. OS 310 315 32O

Glu Glu Ala Lieu Lys Lieu. Glu Asp Wall Lys Trp Lieu Ala Glin Gly. Thir
3.25 330 335
US 2010/0028334 A1 Feb. 4, 2010
47

- Continued

Ile Pro Asp Wall Ile Glu Ser Ala Ala Ser Ala Thir Gly Lys Ala
34 O 345 35. O

His Wall Ile Ser His His Asn Wall Gly Gly Lell Pro Lys Glu Met
355 360 365

Met Gly Luell Wall Glu Pro Luell Glu Luell Phe Asp Glu Wall
37 O 375

Arg Ile Gly Lell Glu Lell Gly Luell Pro Tyr Asp Met Luell Tyr Arg
385 390 395 4 OO

His Pro Phe Pro Gly Pro Gly Luell Gly Wall Arg Wall Lell Gly Glu Wall
4 OS 415

Glu Tyr Cys Asp Lell Luell Arg Arg Ala Asp Ala Ile Phe Ile
425 43 O

Glu Glu Luell Arg Ala Asp Luell Tyr Asp Wall Ser Glin Ala Phe
435 44 O 445

Thir Wall Phe Luell Pro Wall Arg Ser Wall Gly Wall Met Gly Asp Gly Arg
450 45.5 460

Lys Asp Trp Wall Wall Ser Luell Arg Ala Wall Glu Thir Ile Asp Phe
465 470

Met Thir Ala His Trp Ala His Luell Pro Tyr Asp Phe Lell Gly Arg Wall
485 490 495

Ser Asn Arg Ile Ile Asn Glu Wall Asn Gly Ile Ser Arg Wall Wall Tyr
SOO 505 51O

Asp Ile Ser Gly Pro Pro Ala Thir Ile Glu Trp Glu
515 52O 525

<210s, SEQ ID NO 15
&211s LENGTH: 488
212. TYPE : PRT
&213s ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 15

Met Lieu. Arg Ile Ala Glu Ala Luell Thir Phe Asp Asp Wall Luell Luell
1. 5 15

Wall Pro Ala His Ser Thir Wall Luell Pro Asn Thir Ala Asp Luell Ser Thir
25

Glin Luell Thir Thir Ile Arg Luell Asn Ile Pro Met Lell Ser Ala Ala
35 4O 45

Met Asp Thir Wall Thir Glu Ala Arg Luell Ala Ile Ala Lell Ala Glin Glu
SO 55 6O

Gly Gly Ile Gly Phe Ile His Asn Met Ser Ile Glu Arg Glin Ala
65 70

Glu Glu Wall Arg Arg Wall His Glu Ser Gly Wall Wall Thir Asp
85 90 95

Pro Glin Thir Wall Lell Pro Thir Thir Thir Luell Arg Glu Wall Lys Glu Luell
105 11 O

Thir Glu Arg Asn Gly Phe Ala Gly Tyr Pro Wall Wall Thir Glu Glu Asn
115 12 O 125

Glu Luell Wall Gly Ile Ile Thir Gly Arg Asp Wall Arg Phe Wall Thir Asp
13 O 135 14 O

Lell Asn Glin Pro Wall Ser Wall Met Thir Pro Glu Arg Luell Wall
145 150 155 160

Thir Wall Arg Glu Gly Glu Ala Arg Glu Wall Wall Lell Ala Met His
US 2010/0028334 A1 Feb. 4, 2010
48

- Continued
1.65 17O 17s

Glu Lys Arg Val Glu Lys Ala Luell Wall Wall Asp Asp Glu Phe His Luell
18O 185 19 O

Ile Gly Met Ile Thr Val Lys Asp Phe Glin Ala Glu Arg Lys Pro
195

Asn Ala Cys Lys Asp Glu Glin Gly Arg Luell Arg Wall Gly Ala Ala Wall
21 O 215 22O

Gly Ala Gly Ala Gly Asn. Glu Glu Arg Wall Asp Ala Lell Wall Ala
225 23 O 235

Gly Val Asp Val Lieu. Lieu. Ile Asp Ser Ser His Gly His Ser Glu
245 250 255

Val Lieu. Glin Arg Ile Arg Glu Thir Arg Ala Tyr Pro Asp Luell
26 O 265 27 O

Ile Ile Gly Gly Asn Val Ala Thir Ala Ala Gly Ala Arg Ala Luell
27s 28O 285

Glu Ala Gly Cys Ser Ala Wall Wall Gly Ile Gly Pro Gly Ser
29 O 295 3 OO

Cys Thir Thr Arg Ile Val Thr Gly Wall Gly Wall Pro Glin Ile Thir
3. OS 310 315

Val Ala Asp Ala Val Glu Ala Luell Glu Gly Thir Gly Ile Pro Wall
3.25 330 335

Ala Asp Gly Gly Ile Arg Phe Ser Gly Asp Ile Ala Ala Ile
34 O 345 35. O

Ala Gly Ala Ser Ala Wal Met Wall Gly Ser Met Lell Ala Gly Thir
355 360 365

Glu Ser Pro Gly Glu Ile Glu Luell Glin Gly Arg Ser Ser
37 O 375

Tyr Arg Gly Met Gly Ser Lieu. Gly Ala Met Ser Lys Gly Ser Ser Asp
385 390 395 4 OO

Arg Tyr Phe Glin Ser Asp Asn Ala Ala Asp Lell Wall Pro Glu Gly
4 OS 415

Ile Glu Gly Arg Val Ala Tyr Gly Arg Luell Lys Glu Ile Ile His
42O 425 43 O

Glin Gln Met Gly Gly Lieu. Arg Ser Met Gly Lell Thir Gly Gly
435 44 O 445

Thir Ile Asp Glu Lieu. Arg Thr Ala Glu Phe Wall Arg Ile Ser Gly
450 45.5 460

Ala Gly Ile Glin Glu Ser His Wall His Asp Wall Thir Ile Thir Glu
465 470 47s 48O

Ser Pro Asn Tyr Arg Lieu. Gly Ser

485

<210s, SEQ ID NO 16
&211s LENGTH: 412
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 16

Met Met Tyr Gly Val Tyr Arg Ala Met Lys Lieu Pro Ile Tyr Lieu. Asp
1. 5 15

Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met
2O 25
US 2010/0028334 A1 Feb. 4, 2010
49

- Continued
Glin Phe Met Thr Met Asp Gly Thr Phe Gly ASn Pro Ala Ser Arg Ser
35 4O 45

His Arg Phe Gly Trp Glin Ala Glu Glu Ala Val Asp Ile Ala Arg Asn
SO 55 6O

Glin Ile Ala Asp Lieu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr
65 70 7s 8O

Ser Gly Ala Thr Glu Ser Asp Asn Lieu Ala Ile Lys Gly Ala Ala Asn
85 90 95

Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu. His
1OO 105 11 O

Lys Ala Val Lieu. Asp Thr Cys Arg Glin Lieu. Glu Arg Glu Gly Phe Glu
115 12 O 125

Val Thr Tyr Lieu Ala Pro Glin Arg Asn Gly Ile Ile Asp Lieu Lys Glu
13 O 135 14 O

Lieu. Glu Ala Ala Met Arg Asp Asp Thir Ile Lieu Val Ser Ile Met His
145 150 155 160

Val Asn. Asn. Glu Ile Gly Val Val Glin Asp Ile Ala Ala Ile Gly Glu
1.65 17O 17s

Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Glin Ser
18O 185 19 O

Val Gly Lys Lieu Pro Ile Asp Lieu. Ser Glin Lieu Lys Val Asp Lieu Met
195 2OO 2O5

Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu
21 O 215 22O

Tyr Val Arg Arg Llys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly
225 23 O 235 24 O

Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Lieu Pro Val His Glin
245 250 255

Ile Val Gly Met Gly Glu Ala Tyr Arg Ile Ala Lys Glu Glu Met Ala
26 O 265 27 O

Thr Glu Met Glu Arg Lieu. Arg Gly Lieu. Arg Asn Arg Lieu. Trp Asn Gly
27s 28O 285

Ile Lys Asp Ile Glu Glu Val Tyr Lieu. Asn Gly Asp Lieu. Glu. His Gly
29 O 295 3 OO

Ala Pro Asn. Ile Lieu. Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser
3. OS 310 315 32O

Lieu. Ile Met Ala Lieu Lys Asp Lieu Ala Val Ser Ser Gly Ser Ala Cys
3.25 330 335

Thir Ser Ala Ser Lieu. Glu Pro Ser Tyr Val Lieu. Arg Ala Lieu. Gly Lieu.
34 O 345 35. O

Asn Asp Glu Lieu Ala His Ser Ser Ile Arg Phe Ser Lieu. Gly Arg Phe
355 360 365

Thir Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Lieu Val Arg Llys Ser
37 O 375 38O

Ile Gly Arg Lieu. Arg Asp Lieu. Ser Pro Lieu. Trp Glu Met Tyr Lys Glin
385 390 395 4 OO

Gly Val Asp Lieu. Asn. Ser Ile Glu Trp Ala His His
4 OS 41O

<210s, SEQ ID NO 17
&211s LENGTH: 382
212. TYPE: PRT
US 2010/0028334 A1 Feb. 4, 2010
50

- Continued
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 17

Met Phe Llys Ser Asp Ala Ala Arg Thr Arg Glin Arg Lieu. Ser Ala Arg
1. 5 1O 15

Lys Gly Arg Thr Lieu Pro Pro Gly Arg Gly Llys Phe Pro His Ser Thr
2O 25 3O

Thr Glu Ser Phe Arg Asn Thr Phe Trp Leu Lys Lys Ile Met Glu. His
35 4O 45

Cys Phe Asn Met Val Asp Gln Gln Thr Thr Thr Ala Glin Thr Asn Ala
SO 55 6O

Asn Phe Leu Glin Ile Arg Phe Thr Thr Met Ser Lys Lys Ile Ala Val
65 70 7s 8O

Ile Gly Glu. Cys Met Ile Glu Lieu. Ser Glu Lys Gly Ala Asp Wall Lys
85 90 95

Arg Gly Phe Gly Gly Asp Thir Lieu. Asn. Thir Ser Val Tyr Ile Ala Arg
1OO 105 11 O

Glin Val Asp Pro Ala Ala Lieu. Thr Val His Tyr Val Thr Ala Lieu. Gly
115 12 O 125

Thir Asp Ser Phe Ser Glin Gln Met Lieu. Asp Ala Trp His Gly Glu Asn
13 O 135 14 O

Val Asp Thir Ser Lieu. Thr Glin Arg Met Glu Asn Arg Lieu Pro Gly Lieu
145 150 155 160

Tyr Tyr Ile Glu Thr Asp Ser Thr Gly Glu Arg Thr Phe Tyr Tyr Trp
1.65 17O 17s

Arg Asn. Glu Ala Ala Ala Lys Phe Trp Lieu. Glu Ser Glu Glin Ser Ala
18O 185 19 O

Ala Ile Cys Glu Glu Lieu Ala Asn. Phe Asp Tyr Lieu. Tyr Lieu. Ser Gly
195 2OO 2O5

Ile Ser Lieu Ala Ile Lieu. Ser Pro Thir Ser Arg Glu Lys Lieu. Lieu. Ser
21 O 215 22O

Lieu. Lieu. Arg Glu. Cys Arg Ala Asn Gly Gly Llys Val Ile Phe Asp Asn
225 23 O 235 24 O

Asn Tyr Arg Pro Arg Lieu. Trp Ala Ser Lys Glu Glu Thr Glin Glin Val
245 250 255

Tyr Glin Gln Met Lieu. Glu. Cys Thr Asp Ile Ala Phe Lieu. Thir Lieu. Asp
26 O 265 27 O

Asp Glu Asp Ala Lieu. Trp Gly Glin Glin Pro Val Glu Asp Val Ile Ala
27s 28O 285

Arg Thr His Asn Ala Gly Val Lys Glu Val Val Val Lys Arg Gly Ala
29 O 295 3 OO

Asp Ser Cys Lieu Val Ser Ile Ala Gly Glu Gly Lieu Val Asp Val Pro
3. OS 310 315 32O

Ala Wall Lys Lieu Pro Llys Glu Lys Val Ile Asp Thir Thr Ala Ala Gly
3.25 330 335

Asp Ser Phe Ser Ala Gly Tyr Lieu Ala Val Arg Lieu. Thr Gly Gly Ser
34 O 345 35. O

Ala Glu Asp Ala Ala Lys Arg Gly. His Lieu. Thir Ala Ser Thr Val Ile
355 360 365

Glin Tyr Arg Gly Ala Ile Ile Pro Arg Glu Ala Met Pro Ala
37 O 375 38O
US 2010/0028334 A1 Feb. 4, 2010
51

- Continued

<210s, SEQ ID NO 18
&211s LENGTH: 321
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 18

Met Ser Llys Pro Ile Val Met Glu Arg Gly Val Llys Tyr Arg Asp Ala
1. 5 1O 15

Asp Llys Met Ala Lieu. Ile Pro Wall Lys Asn Val Ala Thr Glu Arg Glu
2O 25 3O

Ala Lieu. Lieu. Arg Llys Pro Glu Trp Met Lys Ile Llys Lieu Pro Ala Asp
35 4O 45

Ser Thr Arg Ile Glin Gly Ile Lys Ala Ala Met Arg Lys Asn Gly Lieu.
SO 55 6O

His Ser Val Cys Glu Glu Ala Ser Cys Pro Asn Lieu Ala Glu. Cys Phe
65 70 7s 8O

Asn His Gly Thr Ala Thr Phe Met Ile Leu Gly Ala Ile Cys Thr Arg
85 90 95

Arg Cys Pro Phe Cys Asp Wall Ala His Gly Arg Pro Wall Ala Pro Asp
1OO 105 11 O

Ala Asn. Glu Pro Val Llys Lieu Ala Glin Thir Ile Ala Asp Met Ala Lieu
115 12 O 125

Arg Tyr Val Val Ile Thr Ser Val Asp Arg Asp Asp Lieu. Arg Asp Gly
13 O 135 14 O

Gly Ala Gln His Phe Ala Asp Cys Ile Thir Ala Ile Arg Glu Lys Ser
145 150 155 160

Pro Glin Ile Lys Ile Glu Thir Lieu Val Pro Asp Phe Arg Gly Arg Met
1.65 17O 17s

Asp Arg Ala Lieu. Asp Ile Lieu. Thir Ala Thr Pro Pro Asp Val Phe Asn
18O 185 19 O

His Asn Lieu. Glu Asn Val Pro Arg Ile Tyr Arg Glin Val Arg Pro Gly
195 2OO 2O5

Ala Asp Tyr Asn Trp Ser Lieu Lys Lieu. Lieu. Glu Arg Phe Lys Glu Ala
21 O 215 22O

His Pro Glu Ile Pro Thr Lys Ser Gly Lieu Met Val Gly Lieu. Gly Glu
225 23 O 235 24 O

Thir Asn. Glu Glu Ile Ile Glu Val Met Arg Asp Lieu. Arg Arg His Gly
245 250 255

Val Thr Met Lieu. Thir Lieu. Gly Glin Tyr Lieu Gln Pro Ser Arg His His
26 O 265 27 O

Lieu Pro Val Glin Arg Tyr Val Ser Pro Asp Glu Phe Asp Glu Met Lys
27s 28O 285

Ala Glu Ala Leu Ala Met Gly Phe Thr His Ala Ala Cys Gly Pro Phe
29 O 295 3 OO

Val Arg Ser Ser Tyr His Ala Asp Lieu. Glin Ala Lys Gly Met Glu Val
3. OS 310 315 32O

Lys

<210s, SEQ ID NO 19
&211s LENGTH: 42O
212. TYPE: PRT
<213> ORGANISM: Escherichia coli
US 2010/0028334 A1 Feb. 4, 2010
52

- Continued
<4 OOs, SEQUENCE: 19

Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Lieu. Thir Ala Glu Asn
1. 5 1O 15

Lieu. Lieu. Arg Lieu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp
2O 25 3O

Ala Glin Ile Ile Arg Arg Glin Ile Ala Ala Lieu Lys Glin Phe Asp Wall
35 4O 45

Val Arg Phe Ala Gln Lys Ala Cys Ser Asn. Ile His Ile Lieu. Arg Lieu
SO 55 6O

Met Arg Glu Glin Gly Val Llys Val Asp Ser Val Ser Lieu. Gly Glu Ile
65 70 7s 8O

Glu Arg Ala Lieu Ala Ala Gly Tyr Asn Pro Glin Thr His Pro Asp Asp
85 90 95

Ile Val Phe Thir Ala Asp Val Ile Asp Glin Ala Thr Lieu. Glu Arg Val
1OO 105 11 O

Ser Glu Lieu. Glin Ile Pro Val Asn Ala Gly Ser Val Asp Met Lieu. Asp
115 12 O 125

Glin Lieu. Gly Glin Val Ser Pro Gly His Arg Val Trp Lieu. Arg Val Asn
13 O 135 14 O

Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu
145 150 155 160

ASn Ser Lys His Gly Ile Trp Tyr Thr Asp Lieu. Pro Ala Ala Lieu. Asp
1.65 17O 17s

Val Ile Glin Arg His His Leu Gln Leu Val Gly Ile His Met His Ile
18O 185 19 O

Gly Ser Gly Val Asp Tyr Ala His Lieu. Glu Glin Val Cys Gly Ala Met
195 2OO 2O5

Val Arg Glin Val Ile Glu Phe Gly Glin Asp Lieu. Glin Ala Ile Ser Ala
21 O 215 22O

Gly Gly Gly Lieu. Ser Val Pro Tyr Glin Glin Gly Glu Glu Ala Val Asp
225 23 O 235 24 O

Thr Glu. His Tyr Tyr Gly Lieu. Trp Asn Ala Ala Arg Glu Glin Ile Ala
245 250 255

Arg His Lieu. Gly His Pro Wall Lys Lieu. Glu Ile Glu Pro Gly Arg Phe
26 O 265 27 O

Lieu Val Ala Glin Ser Gly Val Lieu. Ile Thr Glin Val Arg Ser Val Lys
27s 28O 285

Glin Met Gly Ser Arg His Phe Val Lieu Val Asp Ala Gly Phe Asn Asp
29 O 295 3 OO

Lieu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu
3. OS 310 315 32O

Ala Ala Asp Gly Arg Ser Lieu. Glu. His Ala Pro Thr Val Glu Thr Val
3.25 330 335

Val Ala Gly Pro Leu. Cys Glu Ser Gly Asp Val Phe Thr Glin Glin Glu
34 O 345 35. O

Gly Gly Asn Val Glu Thir Arg Ala Lieu Pro Glu Val Lys Ala Gly Asp
355 360 365

Tyr Lieu Val Lieu. His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser
37 O 375 38O

Asn Tyr Asn. Ser Arg Pro Lieu. Lieu Pro Glu Val Lieu. Phe Asp Asn Gly
385 390 395 4 OO
US 2010/0028334 A1 Feb. 4, 2010
53

- Continued

Glin Ala Arg Lieu. Ile Arg Arg Arg Glin Thir Ile Glu Glu Lieu. Lieu Ala
4 OS 41O 415

Lieu. Glu Lieu. Lieu.

<210s, SEQ ID NO 2 O
&211s LENGTH: 368
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2O

Met Ser Glu Thir Ala Lys Llys Val Ile Val Gly Met Ser Gly Gly Val
1. 5 1O 15

Asp Ser Ser Val Ser Ala Trp Lieu. Lieu. Glin Glin Glin Gly Tyr Glin Val
2O 25 3O

Glu Gly Lieu. Phe Met Lys Asn Trp Glu Glu Asp Asp Gly Glu Glu Tyr
35 4O 45

Cys Thr Ala Ala Ala Asp Lieu Ala Asp Ala Glin Ala Val Cys Asp Llys
SO 55 6O

Lieu. Gly Ile Glu Lieu. His Thr Val Asn. Phe Ala Ala Glu Tyr Trp Asp
65 70 7s 8O

Asn Val Phe Glu Lieu. Phe Lieu Ala Glu Tyr Lys Ala Gly Arg Thr Pro
85 90 95

Asn Pro Asp Ile Lieu. Cys Asn Lys Glu Ile Llys Phe Lys Ala Phe Lieu
1OO 105 11 O

Glu Phe Ala Ala Glu Asp Lieu. Gly Ala Asp Tyr Ile Ala Thr Gly. His
115 12 O 125

Tyr Val Arg Arg Ala Asp Wall Asp Gly Lys Ser Arg Lieu. Lieu. Arg Gly
13 O 135 14 O

Lieu. Asp Ser Asn Lys Asp Glin Ser Tyr Phe Lieu. Tyr Thr Lieu. Ser His
145 150 155 160

Glu Glin Ile Ala Glin Ser Lieu. Phe Pro Val Gly Glu Lieu. Glu Lys Pro
1.65 17O 17s

Glin Val Arg Lys Ile Ala Glu Asp Lieu. Gly Lieu Val Thr Ala Lys Llys
18O 185 19 O

Lys Asp Ser Thr Gly Ile Cys Phe Ile Gly Glu Arg Llys Phe Arg Glu
195 2OO 2O5

Phe Leu Gly Arg Tyr Lieu Pro Ala Glin Pro Gly Lys Ile Ile Thr Val
21 O 215 22O

Asp Gly Asp Glu Ile Gly Glu. His Glin Gly Lieu Met Tyr His Thir Lieu
225 23 O 235 24 O

Gly Glin Arg Lys Gly Lieu. Gly Ile Gly Gly. Thir Lys Glu Gly Thr Glu
245 250 255

Glu Pro Trp Tyr Val Val Asp Lys Asp Val Glu Asn. Asn. Ile Lieu Val
26 O 265 27 O

Val Ala Glin Gly His Glu. His Pro Arg Lieu Met Ser Val Gly Lieu. Ile
27s 28O 285

Ala Glin Gln Lieu. His Trp Val Asp Arg Glu Pro Phe Thr Gly Thr Met
29 O 295 3 OO

Arg Cys Thr Val Lys Thr Arg Tyr Arg Glin Thr Asp Ile Pro Cys Thr
3. OS 310 315 32O

Val Lys Ala Lieu. Asp Asp Asp Arg Ile Glu Val Ile Phe Asp Glu Pro
US 2010/0028334 A1 Feb. 4, 2010
54

- Continued
3.25 330 335

Val Ala Ala Val Thr Pro Gly Glin Ser Ala Val Phe Tyr Asn Gly Glu
34 O 345 35. O

Val Cys Lieu. Gly Gly Gly Ile Ile Glu Glin Arg Lieu Pro Lieu Pro Val
355 360 365

<210s, SEQ ID NO 21
&211s LENGTH: 404
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2

Met Lys Lieu Pro Ile Tyr Lieu. Asp Tyr Ser Ala Thr Thr Pro Val Asp
1. 5 1O 15

Pro Arg Val Ala Glu Lys Met Met Glin Phe Met Thr Met Asp Gly Thr
2O 25 3O

Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Glin Ala Glu
35 4O 45

Glu Ala Val Asp Ile Ala Arg Asn Glin Ile Ala Asp Lieu Val Gly Ala
SO 55 6O

Asp Pro Arg Glu Ile Val Phe Thir Ser Gly Ala Thr Glu Ser Asp Asn
65 70 7s 8O

Lieu Ala Ile Lys Gly Ala Ala Asn. Phe Tyr Glin Llys Lys Gly Llys His
85 90 95

Ile Ile Thir Ser Llys Thr Glu. His Lys Ala Val Lieu. Asp Thir Cys Arg
1OO 105 11 O

Glin Lieu. Glu Arg Glu Gly Phe Glu Val Thir Tyr Lieu Ala Pro Glin Arg
115 12 O 125

Asn Gly Ile Ile Asp Lieu Lys Glu Lieu. Glu Ala Ala Met Arg Asp Asp
13 O 135 14 O

Thir Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val
145 150 155 160

Glin Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile
1.65 17O 17s

Tyr His Val Asp Ala Thr Glin Ser Val Gly Lys Lieu Pro Ile Asp Lieu.
18O 185 19 O

Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr
195 2OO 2O5

Gly Pro Lys Gly Ile Gly Ala Lieu. Tyr Val Arg Arg Llys Pro Arg Val
21 O 215 22O

Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg
225 23 O 235 24 O

Ser Gly. Thir Lieu Pro Val His Glin Ile Val Gly Met Gly Glu Ala Tyr
245 250 255

Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Lieu. Arg Gly
26 O 265 27 O

Lieu. Arg Asn Arg Lieu. Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr
27s 28O 285

Lieu. Asn Gly Asp Lieu. Glu. His Gly Ala Pro Asn. Ile Lieu. Asn Val Ser
29 O 295 3 OO

Phe Asn Tyr Val Glu Gly Glu Ser Lieu. Ile Met Ala Lieu Lys Asp Lieu.
3. OS 310 315 32O
US 2010/0028334 A1 Feb. 4, 2010
55

- Continued
Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Lieu. Glu Pro Ser
3.25 330 335

Tyr Val Lieu. Arg Ala Lieu. Gly Lieu. Asn Asp Glu Lieu Ala His Ser Ser
34 O 345 35. O

Ile Arg Phe Ser Lieu. Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr
355 360 365

Thir Ile Glu Lieu Val Arg Llys Ser Ile Gly Arg Lieu. Arg Asp Lieu. Ser
37 O 375 38O

Pro Lieu. Trp Glu Met Tyr Lys Glin Gly Val Asp Lieu. Asn. Ser Ile Glu
385 390 395 4 OO

Trp Ala His His

<210s, SEQ ID NO 22
&211s LENGTH: 513
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 22

Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile
1. 5 1O 15

Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val
2O 25 3O

Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met
35 4O 45

Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu.
SO 55 6O

Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala
65 70 7s 8O

Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu
85 90 95

Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly
1OO 105 11 O

Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala
115 12 O 125

Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin
13 O 135 14 O

Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly
145 150 155 160

Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr
1.65 17O 17s

Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys
18O 185 19 O

Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val
195 2OO 2O5

Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val
21 O 215 22O

Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr
225 23 O 235 24 O

Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala
245 250 255

Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin
26 O 265 27 O
US 2010/0028334 A1 Feb. 4, 2010
56

- Continued
Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly
27s 28O 285

Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val
29 O 295 3 OO

Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys
3. OS 310 315 32O

Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp
3.25 330 335

Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin
34 O 345 35. O

Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val
355 360 365

Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys
37 O 375 38O

Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr
385 390 395 4 OO

Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala
4 OS 41O 415

Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys
42O 425 43 O

Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu.
435 44 O 445

Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile
450 45.5 460

Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala
465 470 47s 48O

Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile
485 490 495

Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser
SOO 505 51O

Trp

<210s, SEQ ID NO 23
&211s LENGTH: 218
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 23

Met Ser Asp Asin Asp Glu Lieu. Glin Glin Ile Ala His Lieu. Arg Arg Glu
1. 5 1O 15

Tyr Thr Lys Gly Gly Lieu. Arg Arg Arg Asp Lieu Pro Ala Asp Pro Lieu.
2O 25 3O

Thir Lieu. Phe Glu Arg Trp Lieu. Ser Glin Ala Cys Glu Ala Lys Lieu Ala
35 4O 45

Asp Pro Thr Ala Met Val Val Ala Thr Val Asp Glu. His Gly Glin Pro
SO 55 6O

Tyr Glin Arg Ile Val Lieu Lleu Lys His Tyr Asp Glu Lys Gly Met Val
65 70 7s 8O

Phe Tyr Thr Asn Lieu. Gly Ser Arg Lys Ala His Glin Ile Glu Asn. Asn
85 90 95

Pro Arg Val Ser Lieu. Leu Phe Pro Trp His Thr Lieu. Glu Arg Glin Val
1OO 105 11 O
US 2010/0028334 A1 Feb. 4, 2010
57

- Continued
Met Val Ile Gly Lys Ala Glu Arg Lieu. Ser Thr Lieu. Glu Val Met Lys
115 12 O 125

Tyr Phe His Ser Arg Pro Arg Asp Ser Glin Ile Gly Ala Trp Val Ser
13 O 135 14 O

Lys Glin Ser Ser Arg Ile Ser Ala Arg Gly Ile Lieu. Glu Ser Llys Phe
145 150 155 160

Lieu. Glu Lieu Lys Glin Llys Phe Glin Glin Gly Glu Val Pro Lieu Pro Ser
1.65 17O 17s

Phe Trp Gly Gly Phe Arg Val Ser Lieu. Glu Glin Ile Glu Phe Trp Glin
18O 185 19 O

Gly Gly Glu. His Arg Lieu. His Asp Arg Phe Lieu. Tyr Glin Arg Glu Asn
195 2OO 2O5

Asp Ala Trp Llys Ile Asp Arg Lieu Ala Pro
21 O 215

<210s, SEQ ID NO 24
&211s LENGTH: 226
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 24

Met Ile Asin Val Glin Asn Val Ser Lys Thr Phe Ile Lieu. His Glin Glin
1. 5 1O 15

ASn Gly Val Arg Lieu. Pro Val Lieu. ASn Arg Ala Ser Lieu. Thr Val Asn
2O 25 3O

Ala Gly Glu. Cys Val Val Lieu. His Gly His Ser Gly Ser Gly Lys Ser
35 4O 45

Thir Lieu. Lieu. Arg Ser Lieu. Tyr Ala Asn Tyr Lieu Pro Asp Glu Gly Glin
SO 55 6O

Ile Glin Ile Llys His Gly Asp Glu Trp Val Asp Lieu Val Thir Ala Pro
65 70 7s 8O

Ala Arg Llys Val Val Glu Ile Arg Llys Thir Thr Val Gly Trp Val Ser
85 90 95

Glin Phe Lieu. Arg Val Ile Pro Arg Ile Ser Ala Lieu. Glu Val Val Met
1OO 105 11 O

Glin Pro Lieu. Lieu. Asp Thr Gly Val Pro Arg Glu Ala Cys Ala Ala Lys
115 12 O 125

Ala Ala Arg Lieu. Lieu. Thir Arg Lieu. Asn Val Pro Glu Arg Lieu. Trp His
13 O 135 14 O

Lieu Ala Pro Ser Thr Phe Ser Gly Gly Glu Gln Glin Arg Val Asn Ile
145 150 155 160

Ala Arg Gly Phe Ile Val Asp Tyr Pro Ile Lieu Lleu Lieu. Asp Glu Pro
1.65 17O 17s

Thir Ala Ser Lieu. Asp Ala Lys Asn. Ser Ala Ala Val Val Glu Lieu. Ile
18O 185 19 O

Arg Glu Ala Lys Thr Arg Gly Ala Ala Ile Val Gly Ile Phe His Asp
195 2OO 2O5

Glu Ala Val Arg Asn Asp Wall Ala Asp Arg Lieu. His Pro Met Gly Ala
21 O 215 22O

Ser Ser
225

<210s, SEQ ID NO 25
US 2010/0028334 A1 Feb. 4, 2010
58

- Continued
&211s LENGTH: 439
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 25

Met Ser Glin Ala Lys Ser Asn Llys Met Gly Val Val Glin Lieu. Thir Ile
1. 5 1O 15

Lieu. Thir Met Val Asn Met Met Gly Ser Gly Ile Ile Met Leu Pro Thr
2O 25 3O

Lys Lieu Ala Glu Val Gly. Thir Ile Ser Ile Ile Ser Trp Leu Val Thr
35 4O 45

Ala Val Gly Ser Met Ala Lieu Ala Trp Ala Phe Ala Lys Cys Gly Met
SO 55 6O

Phe Ser Arg Llys Ser Gly Gly Met Gly Gly Tyr Ala Glu Tyr Ala Phe
65 70 7s 8O

Gly Lys Ser Gly Asn Phe Met Ala Asn Tyr Thr Tyr Gly Val Ser Lieu.
85 90 95

Lieu. Ile Ala Asn. Wall Ala Ile Ala Ile Ser Ala Val Gly Tyr Gly Thr
1OO 105 11 O

Glu Lieu. Lieu. Gly Ala Ser Lieu. Ser Pro Val Glin Ile Gly Lieu Ala Thr
115 12 O 125

Ile Gly Val Lieu. Trp Ile Cys Thr Val Ala Asn. Phe Gly Gly Ala Arg
13 O 135 14 O

Ile Thr Gly Glin Ile Ser Ser Ile Thr Val Trp Gly Val Ile Ile Pro
145 150 155 160

Val Val Gly Lieu. Cys Ile Ile Gly Trp Phe Trp Phe Ser Pro Thr Lieu.
1.65 17O 17s

Tyr Val Asp Ser Trp Asn Pro His His Ala Pro Phe Phe Ser Ala Val
18O 185 19 O

Gly Ser Ser Ile Ala Met Thr Lieu. Trp Ala Phe Leu Gly Lieu. Glu Ser
195 2OO 2O5

Ala Cys Ala Asn. Thir Asp Val Val Glu Asn Pro Glu Arg Asn Val Pro
21 O 215 22O

Ile Ala Val Lieu. Gly Gly Thr Lieu. Gly Ala Ala Val Ile Tyr Ile Val
225 23 O 235 24 O

Ser Thr Asn Val Ile Ala Gly Ile Val Pro Asn Met Glu Lieu Ala Asn
245 250 255

Ser Thr Ala Pro Phe Gly Lieu Ala Phe Ala Gln Met Phe Thr Pro Glu
26 O 265 27 O

Val Gly Llys Val Ile Met Ala Leu Met Val Met Ser Cys Cys Gly Ser
27s 28O 285

Lieu. Leu Gly Trp Glin Phe Thr Ile Ala Glin Val Phe Lys Ser Ser Ser
29 O 295 3 OO

Asp Glu Gly Tyr Phe Pro Lys Ile Phe Ser Arg Val Thr Llys Val Asp
3. OS 310 315 32O

Ala Pro Val Glin Gly Met Lieu. Thir Ile Val Ile Ile Glin Ser Gly Lieu.
3.25 330 335

Ala Lieu Met Thir Ile Ser Pro Ser Lieu. Asn. Ser Glin Phe Asn. Wall Lieu.
34 O 345 35. O

Val Asn Lieu Ala Val Val Thr Asn Ile Ile Pro Tyr Ile Leu Ser Met
355 360 365

Ala Ala Lieu Val Ile Ile Glin Llys Val Ala Asn Val Pro Pro Ser Lys
US 2010/0028334 A1 Feb. 4, 2010
59

- Continued
37 O 375 38O

Ala Lys Val Ala Asn Phe Val Ala Phe Val Gly Ala Met Tyr Ser Phe
385 390 395 4 OO

Tyr Ala Leu Tyr Ser Ser Gly Glu Glu Ala Met Leu Tyr Gly Ser Ile
4 OS 41O 415

Val Thr Phe Leu Gly Trp Thr Lieu. Tyr Gly Lieu Val Ser Pro Arg Phe
42O 425 43 O

Glu Lieu Lys Asn Llys His Gly

435

<210s, SEQ ID NO 26
&211s LENGTH: 219
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 26

Met Thr Glin Asp Glu Lieu Lys Lys Ala Val Gly Trp Ala Ala Lieu. Glin
1. 5 1O 15

Tyr Val Glin Pro Gly. Thir Ile Val Gly Val Gly Thr Gly Ser Thr Ala
2O 25 3O

Ala His Phe Ile Asp Ala Lieu. Gly. Thir Met Lys Gly Glin Ile Glu Gly
35 4O 45

Ala Val Ser Ser Ser Asp Ala Ser Thr Glu Lys Lieu Lys Ser Lieu. Gly
SO 55 6O

Ile His Val Phe Asp Lieu. Asn. Glu Val Asp Ser Lieu. Gly Ile Tyr Val
65 70 7s 8O

Asp Gly Ala Asp Glu Ile Asn Gly. His Met Gln Met Ile Lys Gly Gly
85 90 95

Gly Ala Ala Lieu. Thir Arg Glu Lys Ile Ile Ala Ser Val Ala Glu Lys
1OO 105 11 O

Phe Ile Cys Ile Ala Asp Ala Ser Lys Glin Val Asp Ile Lieu. Gly Lys
115 12 O 125

Phe Pro Leu Pro Val Glu Val Ile Pro Met Ala Arg Ser Ala Val Ala
13 O 135 14 O

Arg Glin Lieu Val Llys Lieu. Gly Gly Arg Pro Glu Tyr Arg Glin Gly Val
145 150 155 160

Val Thr Asp Asn Gly Asn Val Ile Lieu. Asp Wal His Gly Met Glu Ile
1.65 17O 17s

Lieu. Asp Pro Ile Ala Met Glu Asn Ala Ile Asn Ala Ile Pro Gly Val
18O 185 19 O

Val Thr Val Gly Lieu. Phe Ala Asn Arg Gly Ala Asp Wall Ala Lieu. Ile
195 2OO 2O5

Gly Thr Pro Asp Gly Val Lys Thr Ile Val Lys
21 O 215

<210s, SEQ ID NO 27
&211s LENGTH: 405
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 27

Met Ser Ser Val Asp Ile Lieu Val Pro Asp Lieu Pro Glu Ser Val Ala
1. 5 1O 15

Asp Ala Thr Val Ala Thir Trp His Llys Llys Pro Gly Asp Ala Val Val
US 2010/0028334 A1 Feb. 4, 2010
60

- Continued
2O 25 3O

Arg Asp Glu Val Lieu Val Glu Ile Glu Thir Asp Llys Val Val Lieu. Glu
35 4O 45

Val Pro Ala Ser Ala Asp Gly Ile Lieu. Asp Ala Val Lieu. Glu Asp Glu
SO 55 6O

Gly. Thir Thr Val Thir Ser Arg Glin Ile Lieu. Gly Arg Lieu. Arg Glu Gly
65 70 7s 8O

Asn Ser Ala Gly Lys Glu Thir Ser Ala Lys Ser Glu Glu Lys Ala Ser
85 90 95

Thr Pro Ala Glin Arg Glin Glin Ala Ser Lieu. Glu Glu Glin Asn. Asn Asp
1OO 105 11 O

Ala Lieu. Ser Pro Ala Ile Arg Arg Lieu. Lieu Ala Glu. His Asn Lieu. Asp
115 12 O 125

Ala Ser Ala Ile Lys Gly Thr Gly Val Gly Gly Arg Lieu. Thir Arg Glu
13 O 135 14 O

Asp Val Glu Lys His Lieu Ala Lys Ala Pro Ala Lys Glu Ser Ala Pro
145 150 155 160

Ala Ala Ala Ala Pro Ala Ala Glin Pro Ala Lieu Ala Ala Arg Ser Glu
1.65 17O 17s

Lys Arg Val Pro Met Thr Arg Lieu. Arg Lys Arg Val Ala Glu Arg Lieu.
18O 185 19 O

Lieu. Glu Ala Lys Asn Ser Thr Ala Met Lieu. Thir Thr Phe Asin Glu Val
195 2OO 2O5

Asn Met Llys Pro Ile Met Asp Lieu. Arg Lys Glin Tyr Gly Glu Ala Phe
21 O 215 22O

Glu Lys Arg His Gly Ile Arg Lieu. Gly Phe Met Ser Phe Tyr Val Lys
225 23 O 235 24 O

Ala Val Val Glu Ala Lieu Lys Arg Tyr Pro Glu Val Asn Ala Ser Ile
245 250 255

Asp Gly Asp Asp Val Val Tyr His Asn Tyr Phe Asp Wal Ser Met Ala
26 O 265 27 O

Val Ser Thr Pro Arg Gly Lieu Val Thr Pro Val Lieu. Arg Asp Val Asp
27s 28O 285

Thir Lieu. Gly Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Lieu Ala Val
29 O 295 3 OO

Lys Gly Arg Asp Gly Llys Lieu. Thr Val Glu Asp Lieu. Thr Gly Gly Asn
3. OS 310 315 32O

Phe Thir Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser Thr Pro
3.25 330 335

Ile Ile Asn Pro Pro Glin Ser Ala Ile Lieu. Gly Met His Ala Ile Llys
34 O 345 35. O

Asp Arg Pro Met Ala Val Asn Gly Glin Val Glu Ile Leu Pro Met Met
355 360 365

Tyr Lieu Ala Lieu. Ser Tyr Asp His Arg Lieu. Ile Asp Gly Arg Glu Ser
37 O 375 38O

Val Gly Phe Lieu Val Thir Ile Lys Glu Lieu. Lieu. Glu Asp Pro Thr Arg
385 390 395 4 OO

Lieu. Lieu. Lieu. Asp Wall

4 OS

<210s, SEQ ID NO 28
US 2010/0028334 A1 Feb. 4, 2010
61

- Continued
&211s LENGTH: 127
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 28

Met Lieu. His Glin Glin Arg Asn. Glin His Ala Arg Lieu. Ile Pro Val Glu
1. 5 1O 15

Lieu. Tyr Met Ser Asp Llys Ile Ile His Lieu. Thir Asp Asp Ser Phe Asp
2O 25 3O

Thir Asp Val Lieu Lys Ala Asp Gly Ala Ile Lieu Val Asp Phe Trp Ala
35 4O 45

Glu Trp Cys Gly Pro Cys Llys Met Ile Ala Pro Ile Lieu. Asp Glu Ile
SO 55 6O

Ala Asp Glu Tyr Glin Gly Llys Lieu. Thr Val Ala Lys Lieu. Asn. Ile Asp
65 70 7s 8O

Gln Asn Pro Gly Thr Ala Pro Llys Tyr Gly Ile Arg Gly Ile Pro Thr
85 90 95

Lieu. Lieu. Lieu. Phe Lys Asn Gly Glu Val Ala Ala Thr Llys Val Gly Ala
1OO 105 11 O

Lieu. Ser Lys Gly Glin Lieu Lys Glu Phe Lieu. Asp Ala Asn Lieu Ala
115 12 O 125

<210s, SEQ ID NO 29
&211s LENGTH: 95
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 29

Met Lieu. His Thr Lieu. His Arg Ser Pro Trp Lieu. Thr Asp Phe Ala Ala
1. 5 1O 15

Lieu. Lieu. Arg Lieu Lleu Ser Glu Gly Asp Glu Lieu Lleu Lleu Lieu. Glin Asp
2O 25 3O

Gly Val Thir Ala Ala Val Asp Gly Asn Arg Tyr Lieu. Glu Ser Lieu. Arg
35 4O 45

Asn Ala Pro Ile Llys Val Tyr Ala Lieu. Asn. Glu Asp Lieu. Ile Ala Arg
SO 55 6O

Gly Lieu. Thr Gly Glin Ile Ser Asn Asp Ile Ile Lieu. Ile Asp Tyr Thr
65 70 7s 8O

Asp Phe Val Arg Lieu. Thr Val Llys His Pro Ser Gln Met Ala Trp
85 90 95

<210s, SEQ ID NO 3 O
&211s LENGTH: 109
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 30

Met Lieu. Ile Phe Glu Gly Lys Glu Ile Glu Thir Asp Thr Glu Gly Tyr
1. 5 1O 15

Lieu Lys Glu Ser Ser Glin Trp Ser Glu Pro Lieu Ala Val Val Ile Ala
2O 25 3O

Glu Asn. Glu Gly Ile Ser Lieu. Ser Pro Glu. His Trp Glu Val Val Arg
35 4O 45

Phe Val Arg Asp Phe Tyr Lieu. Glu Phe Asn Thr Ser Pro Ala Ile Arg
SO 55 6O
US 2010/0028334 A1 Feb. 4, 2010
62

- Continued
Met Lieu Val Lys Ala Met Ala Asn Llys Phe Gly Glu Glu Lys Gly Asn
65 70 7s 8O

Ser Arg Tyr Lieu. Tyr Arg Lieu. Phe Pro Lys Gly Pro Ala Lys Glin Ala
85 90 95

Thir Lys Ile Ala Gly Lieu Pro Llys Pro Wall Lys Cys Ile
1OO 105

<210s, SEQ ID NO 31
&211s LENGTH: 251
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 31

Met Val Asp Llys Ser Glin Glu Thir Thr His Phe Gly Phe Glin Thr Val
1. 5 1O 15

Ala Lys Glu Glin Lys Ala Asp Met Val Ala His Val Phe His Ser Val
2O 25 3O

Ala Ser Lys Tyr Asp Wal Met Asn Asp Lieu Met Ser Phe Gly Ile His
35 4O 45

Arg Lieu. Trp Lys Arg Phe Thir Ile Asp Cys Ser Gly Val Arg Arg Gly
SO 55 6O

Glin Thr Val Lieu. Asp Lieu Ala Gly Gly. Thr Gly Asp Lieu. Thir Ala Lys
65 70 7s 8O

Phe Ser Arg Lieu Val Gly Glu. Thr Gly Llys Val Val Lieu. Ala Asp Ile
85 90 95

Asn Glu Ser Met Pro Llys Met Gly Arg Glu Lys Lieu. Arg Asn. Ile Gly
1OO 105 11 O

Val Ile Gly Asn Val Glu Tyr Val Glin Ala Asn Ala Glu Ala Lieu Pro
115 12 O 125

Phe Pro Asp Asn Thr Phe Asp Cys Ile Thr Ile Ser Phe Gly Lieu. Arg
13 O 135 14 O

Asn Val Thir Asp Lys Asp Lys Ala Lieu. Arg Ser Met Tyr Arg Val Lieu
145 150 155 160

Llys Pro Gly Gly Arg Lieu. Lieu Val Lieu. Glu Phe Ser Llys Pro Ile Ile
1.65 17O 17s

Glu Pro Leu Ser Lys Ala Tyr Asp Ala Tyr Ser Phe His Val Leu Pro
18O 185 19 O

Arg Ile Gly Ser Lieu Val Ala Asn Asp Ala Asp Ser Tyr Arg Tyr Lieu
195 2OO 2O5

Ala Glu Ser Ile Arg Met His Pro Asp Glin Asp Thir Lieu Lys Ala Met
21 O 215 22O

Met Glin Asp Ala Gly Phe Glu Ser Val Asp Tyr Tyr Asn Lieu. Thir Ala
225 23 O 235 24 O

Gly Val Val Ala Lieu. His Arg Gly Tyr Llys Phe
245 250

<210s, SEQ ID NO 32
&211s LENGTH: 392
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 32

Met Ser Val Ile Ile Val Gly Gly Gly Met Ala Gly Ala Thr Lieu Ala
1. 5 1O 15
US 2010/0028334 A1 Feb. 4, 2010
63

- Continued
Lieu Ala Ile Ser Arg Lieu. Ser His Gly Ala Lieu Pro Val His Lieu. Ile
2O 25 3O

Glu Ala Thir Ala Pro Glu Ser His Ala His Pro Gly Phe Asp Gly Arg
35 4O 45

Ala Ile Ala Lieu Ala Ala Gly Thr Cys Glin Glin Lieu Ala Arg Ile Gly
SO 55 6O

Val Trp Glin Ser Leu Ala Asp Cys Ala Thr Ala Ile Thr Thr Val His
65 70 7s 8O

Val Ser Asp Arg Gly. His Ala Gly Phe Val Thir Lieu Ala Ala Glu Asp
85 90 95

Tyr Glin Lieu Ala Ala Lieu. Gly Glin Val Val Glu Lieu. His Asn Val Gly
1OO 105 11 O

Glin Arg Lieu. Phe Ala Lieu. Lieu. Arg Lys Ala Pro Gly Val Thir Lieu. His
115 12 O 125

Cys Pro Asp Arg Val Ala Asn. Wall Ala Arg Thr Glin Ser His Val Glu
13 O 135 14 O

Val Thir Lieu. Glu Ser Gly Glu Thir Lieu. Thr Gly Arg Val Lieu Val Ala
145 150 155 160

Ala Asp Gly Thr His Ser Ala Lieu Ala Thr Ala Cys Gly Val Asp Trip
1.65 17O 17s

Glin Glin Glu Pro Tyr Glu Gln Lieu Ala Val Ile Ala Asn. Wall Ala Thr
18O 185 19 O

Ser Val Ala His Glu Gly Arg Ala Phe Glu Arg Phe Thr Glin His Gly
195 2OO 2O5

Pro Lieu Ala Met Lieu Pro Met Ser Asp Gly Arg Cys Ser Lieu Val Trp
21 O 215 22O

Cys His Pro Lieu. Glu Arg Arg Glu Glu Val Lieu. Ser Trp Ser Asp Glu
225 23 O 235 24 O

Llys Phe Cys Arg Glu Lieu. Glin Ser Ala Phe Gly Trp Arg Lieu. Gly Lys
245 250 255

Ile Thr His Ala Gly Lys Arg Ser Ala Tyr Pro Lieu Ala Lieu. Thir His
26 O 265 27 O

Ala Ala Arg Ser Ile Thr His Arg Thr Val Lieu Val Gly Asn Ala Ala
27s 28O 285

Gln Thr Lieu. His Pro Ile Ala Gly Glin Gly Phe Asn Lieu. Gly Met Arg
29 O 295 3 OO

Asp Wal Met Ser Lieu Ala Glu Thir Lieu. Thr Glin Ala Glin Glu Arg Gly
3. OS 310 315 32O

Glu Asp Met Gly Asp Tyr Gly Val Lieu. Cys Arg Tyr Glin Glin Arg Arg
3.25 330 335

Glin Ser Asp Arg Glu Ala Thir Ile Gly Val Thr Asp Ser Lieu Val His
34 O 345 35. O

Lieu. Phe Ala Asn Arg Trp Ala Pro Lieu Val Val Gly Arg Asn. Ile Gly
355 360 365

Lieu Met Thr Met Glu Lieu Phe Thr Pro Ala Arg Asp Val Leu Ala Glin
37 O 375 38O

Arg Thr Lieu. Gly Trp Val Ala Arg

385 390

<210s, SEQ ID NO 33
&211s LENGTH: 513
212. TYPE: PRT
US 2010/0028334 A1 Feb. 4, 2010
64

- Continued
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 33

Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile
1. 5 1O 15

Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val
2O 25 3O

Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met
35 4O 45

Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu.
SO 55 6O

Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala
65 70 7s 8O

Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu
85 90 95

Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly
1OO 105 11 O

Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala
115 12 O 125

Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin
13 O 135 14 O

Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly
145 150 155 160

Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr
1.65 17O 17s

Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys
18O 185 19 O

Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val
195 2OO 2O5

Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val
21 O 215 22O

Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr
225 23 O 235 24 O

Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala
245 250 255

Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin
26 O 265 27 O

Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly
27s 28O 285

Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val
29 O 295 3 OO

Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys
3. OS 310 315 32O

Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp
3.25 330 335

Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin
34 O 345 35. O

Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val
355 360 365

Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys
37 O 375 38O
US 2010/0028334 A1 Feb. 4, 2010
65

- Continued
Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr
385 390 395 4 OO

Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala
4 OS 41O 415

Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys
42O 425 43 O

Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu.
435 44 O 445

Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile
450 45.5 460

Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala
465 470 47s 48O

Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile
485 490 495

Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser
SOO 505 51O

Trp

<210s, SEQ ID NO 34
&211s LENGTH: 392
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

< 4 OO > SEQUENCE: 34

Met Ser Val Ile Ile Val Gly Gly Gly Met Ala Gly Ala Thr Lieu Ala
1. 5 1O 15

Lieu Ala Ile Ser Arg Lieu. Ser His Gly Ala Lieu Pro Val His Lieu. Ile
2O 25 3O

Glu Ala Thir Ala Pro Glu Ser His Ala His Pro Gly Phe Asp Gly Arg
35 4O 45

Ala Ile Ala Lieu Ala Ala Gly Thr Cys Glin Glin Lieu Ala Arg Ile Gly
SO 55 6O

Val Trp Glin Ser Leu Ala Asp Cys Ala Thr Ala Ile Thr Thr Val His
65 70 7s 8O

Val Ser Asp Arg Gly. His Ala Gly Phe Val Thir Lieu Ala Ala Glu Asp
85 90 95

Tyr Glin Lieu Ala Ala Lieu. Gly Glin Val Val Glu Lieu. His Asn Val Gly
1OO 105 11 O

Glin Arg Lieu. Phe Ala Lieu. Lieu. Arg Lys Ala Pro Gly Val Thir Lieu. His
115 12 O 125

Cys Pro Asp Arg Val Ala Asn. Wall Ala Arg Thr Glin Ser His Val Glu
13 O 135 14 O

Val Thir Lieu. Glu Ser Gly Glu Thir Lieu. Thr Gly Arg Val Lieu Val Ala
145 150 155 160

Ala Asp Gly Thr His Ser Ala Lieu Ala Thr Ala Cys Gly Val Asp Trip
1.65 17O 17s

Glin Glin Glu Pro Tyr Glu Gln Lieu Ala Val Ile Ala Asn. Wall Ala Thr
18O 185 19 O

Ser Val Ala His Glu Gly Arg Ala Phe Glu Arg Phe Thr Glin His Gly
195 2OO 2O5

Pro Lieu Ala Met Lieu Pro Met Ser Asp Gly Arg Cys Ser Lieu Val Trp
21 O 215 22O
US 2010/0028334 A1 Feb. 4, 2010
66

- Continued
Cys His Pro Lieu. Glu Arg Arg Glu Glu Val Lieu. Ser Trp Ser Asp Glu
225 23 O 235 24 O

Llys Phe Cys Arg Glu Lieu. Glin Ser Ala Phe Gly Trp Arg Lieu. Gly Lys
245 250 255

Ile Thr His Ala Gly Lys Arg Ser Ala Tyr Pro Lieu Ala Lieu. Thir His
26 O 265 27 O

Ala Ala Arg Ser Ile Thr His Arg Thr Val Lieu Val Gly Asn Ala Ala
27s 28O 285

Gln Thr Lieu. His Pro Ile Ala Gly Glin Gly Phe Asn Lieu. Gly Met Arg
29 O 295 3 OO

Asp Wal Met Ser Lieu Ala Glu Thir Lieu. Thr Glin Ala Glin Glu Arg Gly
3. OS 310 315 32O

Glu Asp Met Gly Asp Tyr Gly Val Lieu. Cys Arg Tyr Glin Glin Arg Arg
3.25 330 335

Glin Ser Asp Arg Glu Ala Thir Ile Gly Val Thr Asp Ser Lieu Val His
34 O 345 35. O

Lieu. Phe Ala Asn Arg Trp Ala Pro Lieu Val Val Gly Arg Asn. Ile Gly
355 360 365

Lieu Met Thr Met Glu Lieu Phe Thr Pro Ala Arg Asp Val Leu Ala Glin
37 O 375 38O

Arg Thr Lieu. Gly Trp Val Ala Arg

385 390

<210s, SEQ ID NO 35
&211s LENGTH: 316
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 35

Met His Cys Lys Gly Ala Cys Met Llys Pro Lieu. Lieu. Asp Wall Lieu Met
1. 5 1O 15

Ile Lieu. Asp Ala Lieu. Glu Lys Glu Gly Ser Phe Ala Ala Ala Ser Ala
2O 25 3O

Lys Lieu. Tyr Lys Thr Pro Ser Ala Leu Ser Tyr Thr Val His Llys Lieu.
35 4O 45

Glu Ser Asp Lieu. Asn. Ile Glin Lieu. Lieu. Asp Arg Ser Gly His Arg Ala
SO 55 6O

Llys Phe Thr Arg Thr Gly Llys Met Lieu. Lieu. Glu Lys Gly Arg Glu Val
65 70 7s 8O

Lieu. His Thr Val Arg Glu Lieu. Glu Lys Glin Ala Ile Llys Lieu. His Glu
85 90 95

Gly Trp Glu Asn Glu Lieu Val Ile Gly Val Asp Asp Thr Phe Pro Phe
1OO 105 11 O

Ser Lieu. Leu Ala Pro Leu. Ile Glu Ala Phe Tyr Gln His His Ser Val
115 12 O 125

Thir Arg Lieu Lys Phe Ile Asn Gly Val Lieu. Gly Gly Ser Trp Asp Ala
13 O 135 14 O

Lieu. Thr Glin Gly Arg Ala Asp Ile Ile Val Gly Ala Met His Glu Pro
145 150 155 160

Pro Ser Ser Ser Glu Phe Gly Phe Ser Arg Lieu. Gly Asp Leu Glu Gln
1.65 17O 17s

Wall Phe Ala Wall Ala Pro His His Pro Leu Ala Lieu. Glu Glu Glu Pro
18O 185 19 O
US 2010/0028334 A1 Feb. 4, 2010
67

- Continued

Lieu. Asn Arg Arg Ile Ile Lys Arg Tyr Arg Ala Ile Val Val Gly Asp
195 2OO 2O5

Thir Ala Glin Ala Gly Ala Ser Thr Ala Ser Glin Lieu. Lieu. Asp Glu Glin
21 O 215 22O

Glu Ala Ile Thr Val Phe Asp Phe Llys Thir Lys Lieu. Glu Lieu. Glin Ile
225 23 O 235 24 O

Ser Gly Lieu. Gly Cys Gly Tyr Lieu Pro Arg Tyr Lieu Ala Glin Arg Phe
245 250 255

Lieu. Asp Ser Gly Ala Lieu. Ile Glu Lys Llys Val Val Ala Glin Thr Lieu.
26 O 265 27 O

Phe Glu Pro Val Trp Ile Gly Trp Asn Glu Gln Thr Ala Gly Lieu Ala
27s 28O 285

Ser Gly Trp Trp Arg Asp Glu Ile Lieu Ala Asn. Ser Ala Ile Ala Gly
29 O 295 3 OO

Val Tyr Ala Lys Ser Asp Asp Gly Lys Ser Ala Ile
3. OS 310 315

<210s, SEQ ID NO 36
&211s LENGTH: 382
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 36

Met Ala Ala Ser Thr Phe Phe Ile Pro Ser Val Asn Val Ile Gly Ala
1. 5 1O 15

Asp Ser Lieu. Thir Asp Ala Met Asn Met Met Ala Asp Tyr Gly Phe Thr
2O 25 3O

Arg Thr Lieu. Ile Val Thr Asp Asn Met Lieu. Thir Lys Lieu. Gly Met Ala
35 4O 45

Gly Asp Val Glin Lys Ala Lieu. Glu Glu Arg Asn. Ile Phe Ser Val Ile
SO 55 6O

Tyr Asp Gly Thr Glin Pro Asn Pro Thr Thr Glu Asn Val Ala Ala Gly
65 70 7s 8O

Lieu Lys Lieu. Lieu Lys Glu Asn. Asn. Cys Asp Ser Val Ile Ser Lieu. Gly
85 90 95

Gly Gly Ser Pro His Asp Cys Ala Lys Gly Ile Ala Lieu Val Ala Ala
1OO 105 11 O

Asn Gly Gly Asp Ile Arg Asp Tyr Glu Gly Val Asp Arg Ser Ala Lys
115 12 O 125

Pro Gln Leu Pro Met Ile Ala Ile Asn. Thir Thr Ala Gly Thr Ala Ser
13 O 135 14 O

Glu Met Thr Arg Phe Cys Ile Ile Thr Asp Glu Ala Arg His Ile Llys
145 150 155 160

Met Ala Ile Val Asp Llys His Val Thr Pro Lieu Lleu Ser Val Asn Asp
1.65 17O 17s

Ser Ser Leu Met Ile Gly Met Pro Llys Ser Lieu. Thir Ala Ala Thr Gly
18O 185 19 O

Met Asp Ala Lieu. Thir His Ala Ile Glu Ala Tyr Val Ser Ile Ala Ala
195 2OO 2O5

Thr Pro Ile Thir Asp Ala Cys Ala Lieu Lys Ala Val Thr Met Ile Ala
21 O 215 22O

Glu Asn Lieu Pro Lieu Ala Val Glu Asp Gly Ser Asn Ala Lys Ala Arg
US 2010/0028334 A1 Feb. 4, 2010
68

- Continued
225 23 O 235 24 O

Glu Ala Met Ala Tyr Ala Glin Phe Lieu Ala Gly Met Ala Phe Asn. Asn
245 250 255

Ala Ser Lieu. Gly Tyr Val His Ala Met Ala His Glin Lieu. Gly Gly Phe
26 O 265 27 O

Tyr Asn Lieu Pro His Gly Val Cys Asn Ala Val Lieu Lleu Pro His Val
27s 28O 285

Glin Val Phe Asin Ser Llys Val Ala Ala Ala Arg Lieu. Arg Asp Cys Ala
29 O 295 3 OO

Ala Ala Met Gly Val Asn Val Thr Gly Lys Asn Asp Ala Glu Gly Ala
3. OS 310 315 32O

Glu Ala Cys Ile Asn Ala Ile Arg Glu Lieu Ala Lys Llys Val Asp Ile
3.25 330 335

Pro Ala Gly Lieu. Arg Asp Lieu. Asn. Wall Lys Glu Glu Asp Phe Ala Val
34 O 345 35. O

Lieu Ala Thr Asn Ala Lieu Lys Asp Ala Cys Gly Phe Thr Asn Pro Ile
355 360 365

Glin Ala Thr His Glu Glu Ile Val Ala Ile Tyr Ala Ala Arg
37 O 375 38O

<210s, SEQ ID NO 37
&211s LENGTH: 571
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 37

Met Asin Ser Leu Glin Ile Leu Ser Phe Val Gly Phe Thr Lieu. Leu Val
1. 5 1O 15

Ala Val Ile Thr Trp Trp Llys Val Arg Llys Thr Asp Thr Gly Ser Glin
2O 25 3O

Glin Gly Tyr Phe Lieu Ala Gly Arg Ser Lieu Lys Ala Pro Val Ile Ala
35 4O 45

Ala Ser Lieu Met Lieu. Thir Asn Lieu. Ser Thr Glu Gln Lieu Val Gly Lieu
SO 55 6O

Ser Gly Glin Ala Tyr Lys Ser Gly Met Ser Val Met Gly Trp Glu Val
65 70 7s 8O

Thir Ser Ala Val Thr Lieu. Ile Phe Leu Ala Lieu. Ile Phe Leu Pro Arg
85 90 95

Tyr Lieu Lys Arg Gly Ile Ala Thir Ile Pro Asp Phe Lieu. Glu Glu Arg
1OO 105 11 O

Tyr Asp Llys Thir Thr Arg Ile Ile Ile Asp Phe Cys Phe Lieu. Ile Ala
115 12 O 125

Thr Gly Val Cys Phe Leu Pro Ile Val Lieu. Tyr Ser Gly Ala Leu Ala
13 O 135 14 O

Lieu. Asn Ser Leu Phe His Val Gly Glu Ser Leu Glin Ile Ser His Gly
145 150 155 160

Ala Ala Ile Trp Lieu. Lieu Val Ile Lieu. Lieu. Gly Lieu Ala Gly Ile Lieu
1.65 17O 17s

Tyr Ala Val Ile Gly Gly Lieu. Arg Ala Met Ala Val Ala Asp Ser Ile
18O 185 19 O

Asn Gly Ile Gly Lieu Val Ile Gly Gly Lieu Met Val Pro Val Phe Gly
195 2OO 2O5
US 2010/0028334 A1 Feb. 4, 2010
69

- Continued
Lieu. Ile Ala Met Gly Lys Gly Ser Phe Met Glin Gly Ile Glu Gln Leu
21 O 215 22O

Thir Thr Val His Ala Glu Lys Lieu. Asn Ser Ile Gly Gly Pro Thr Asp
225 23 O 235 24 O

Pro Leu Pro Ile Gly Ala Ala Phe Thr Gly Lieu. Ile Leu Val Asn Thr
245 250 255

Phe Tyr Trp Cys Thr Asn Glin Gly Ile Val Glin Arg Thr Lieu. Ala Ser
26 O 265 27 O

Llys Ser Lieu Ala Glu Gly Glin Lys Gly Ala Lieu. Lieu. Thir Ala Val Lieu.
27s 28O 285

Llys Met Lieu. Asp Pro Lieu Val Lieu Val Lieu Pro Gly Lieu. Ile Ala Phe
29 O 295 3 OO

His Lieu. Tyr Glin Asp Lieu Pro Lys Ala Asp Met Ala Tyr Pro Thir Lieu.
3. OS 310 315 32O

Val Asn Asn Val Lieu Pro Val Pro Met Val Gly Phe Phe Gly Ala Val
3.25 330 335

Lieu. Phe Gly Ala Val Ile Ser Thr Phe Asin Gly Phe Lieu. Asn Ser Ala
34 O 345 35. O

Ser Thr Lieu Phe Ser Met Gly Ile Tyr Arg Arg Ile Ile Asin Glin Asn
355 360 365

Ala Glu Pro Glin Gln Leu Val Thr Val Gly Arg Llys Phe Gly Phe Phe
37 O 375 38O

Ile Ala Ile Val Ser Val Lieu Val Ala Pro Trp Ile Ala Asn Ala Pro
385 390 395 4 OO

Glin Gly Lieu. Tyr Ser Trp Met Lys Glin Lieu. Asn Gly Ile Tyr Asn. Wall
4 OS 41O 415

Pro Leu Val Thir Ile Ile Ile Met Gly Phe Phe Phe Pro Arg Ile Pro
42O 425 43 O

Ala Lieu Ala Ala Lys Val Ala Met Gly Ile Gly Ile Ile Ser Tyr Ile
435 44 O 445

Thir Ile Asn Tyr Lieu Val Llys Phe Asp Phe His Phe Lieu. Tyr Val Lieu.
450 45.5 460

Ala Cys Thr Phe Cys Ile Asn Val Val Val Met Leu Val Ile Gly Phe
465 470 47s 48O

Ile Llys Pro Arg Ala Thr Pro Phe Thr Phe Lys Asp Ala Phe Ala Val
485 490 495

Asp Met Llys Pro Trp Lys Asn. Wall Lys Ile Ala Ser Ile Gly Ile Lieu
SOO 505 51O

Phe Ala Met Ile Gly Val Tyr Ala Gly Leu Ala Glu Phe Gly Gly Tyr
515 52O 525

Gly Thr Arg Trp Leu Ala Met Ile Ser Tyr Phe Ile Ala Ala Val Val
53 O 535 54 O

Ile Val Tyr Lieu. Ile Phe Asp Ser Trp Arg His Arg His Asp Pro Ala
5.45 550 555 560

Val Thr Phe Thr Pro Asp Gly Lys Asp Ser Leu
565 st O

<210s, SEQ ID NO 38
&211s LENGTH: 3 OO
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 38

US 2010/0028334 A1 Feb. 4, 2010
70

- Continued

Met Thr Met Ile Arg Val Ala Cys Val Gly Ile Thr Val Met Asp Arg
1. 5 1O 15

Ile Tyr Tyr Val Glu Gly Lieu Pro Thr Glu Ser Gly Lys Tyr Val Ala
2O 25 3O

Arg Asn Tyr Thr Glu Val Gly Gly Gly Pro Ala Ala Thr Ala Ala Val
35 4O 45

Ala Ala Ala Arg Lieu. Gly Ala Glin Val Asp Phe Ile Gly Arg Val Gly
SO 55 6O

Asp Asp Asp Thr Gly Asn. Ser Lieu. Lieu Ala Glu Lieu. Glu Ser Trp Gly
65 70 7s 8O

Val Asn. Thir Arg Tyr Thr Lys Arg Tyr Asn Glin Ala Lys Ser Ser Glin
85 90 95

Ser Ala Ile Met Val Asp Thir Lys Gly Glu Arg Ile Ile Ile Asn Tyr
1OO 105 11 O

Pro Ser Pro Asp Lieu Lleu Pro Asp Ala Glu Trp Lieu. Glu Glu Ile Asp
115 12 O 125

Phe Ser Glin Trp Asp Val Val Lieu Ala Asp Val Arg Trp His Asp Gly
13 O 135 14 O

Ala Lys Lys Ala Phe Thr Lieu Ala Arg Glin Ala Gly Wal Met Thr Val
145 150 155 160

Lieu. Asp Gly Asp Ile Thr Pro Glin Asp Ile Ser Glu Lieu Val Ala Lieu.
1.65 17O 17s

Ser Asp His Ala Ala Phe Ser Glu Pro Gly Lieu Ala Arg Lieu. Thr Gly
18O 185 19 O

Val Lys Glu Met Ala Ser Ala Lieu Lys Glin Ala Glin Thr Lieu. Thir Asn
195 2OO 2O5

Gly His Val Tyr Val Thr Glin Gly Ser Ala Gly Cys Asp Trp Leu Glu
21 O 215 22O

Asn Gly Gly Arg Gln His Glin Pro Ala Phe Llys Val Asp Val Val Asp
225 23 O 235 24 O

Thir Thr Gly Ala Gly Asp Val Phe His Gly Ala Lieu Ala Val Ala Lieu.
245 250 255

Ala Thir Ser Gly Asp Lieu Ala Glu Ser Val Arg Phe Ala Ser Gly Val
26 O 265 27 O

Ala Ala Lieu Lys Cys Thr Arg Pro Gly Gly Arg Ala Gly Ile Pro Asp
27s 28O 285

Cys Asp Gln Thr Arg Ser Phe Leu Ser Leu Phe Val
29 O 295 3 OO

<210s, SEQ ID NO 39
&211s LENGTH: 412
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 39

Met Met Tyr Gly Val Tyr Arg Ala Met Lys Lieu Pro Ile Tyr Lieu. Asp
1. 5 1O 15

Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met
2O 25 3O

Glin Phe Met Thr Met Asp Gly Thr Phe Gly ASn Pro Ala Ser Arg Ser
35 4O 45

His Arg Phe Gly Trp Glin Ala Glu Glu Ala Val Asp Ile Ala Arg Asn
US 2010/0028334 A1 Feb. 4, 2010
71

- Continued
SO 55 6O

Glin Ile Ala Asp Lieu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr
65 70 7s 8O

Ser Gly Ala Thr Glu Ser Asp Asn Lieu Ala Ile Lys Gly Ala Ala Asn
85 90 95

Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu. His
1OO 105 11 O

Lys Ala Val Lieu. Asp Thr Cys Arg Glin Lieu. Glu Arg Glu Gly Phe Glu
115 12 O 125

Val Thr Tyr Lieu Ala Pro Glin Arg Asn Gly Ile Ile Asp Lieu Lys Glu
13 O 135 14 O

Lieu. Glu Ala Ala Met Arg Asp Asp Thir Ile Lieu Val Ser Ile Met His
145 150 155 160

Val Asn. Asn. Glu Ile Gly Val Val Glin Asp Ile Ala Ala Ile Gly Glu
1.65 17O 17s

Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Glin Ser
18O 185 19 O

Val Gly Lys Lieu Pro Ile Asp Lieu. Ser Glin Lieu Lys Val Asp Lieu Met
195 2OO 2O5

Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu
21 O 215 22O

Tyr Val Arg Arg Llys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly
225 23 O 235 24 O

Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Lieu Pro Val His Glin
245 250 255

Ile Val Gly Met Gly Glu Ala Tyr Arg Ile Ala Lys Glu Glu Met Ala
26 O 265 27 O

Thr Glu Met Glu Arg Lieu. Arg Gly Lieu. Arg Asn Arg Lieu. Trp Asn Gly
27s 28O 285

Ile Lys Asp Ile Glu Glu Val Tyr Lieu. Asn Gly Asp Lieu. Glu. His Gly
29 O 295 3 OO

Ala Pro Asn. Ile Lieu. Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser
3. OS 310 315 32O

Lieu. Ile Met Ala Lieu Lys Asp Lieu Ala Val Ser Ser Gly Ser Ala Cys
3.25 330 335

Thir Ser Ala Ser Lieu. Glu Pro Ser Tyr Val Lieu. Arg Ala Lieu. Gly Lieu.
34 O 345 35. O

Asn Asp Glu Lieu Ala His Ser Ser Ile Arg Phe Ser Lieu. Gly Arg Phe
355 360 365

Thir Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Lieu Val Arg Llys Ser
37 O 375 38O

Ile Gly Arg Lieu. Arg Asp Lieu. Ser Pro Lieu. Trp Glu Met Tyr Lys Glin
385 390 395 4 OO

Gly Val Asp Lieu. Asn. Ser Ile Glu Trp Ala His His
4 OS 41O

<210s, SEQ ID NO 4 O
&211s LENGTH: 345
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 O

US 2010/0028334 A1 Feb. 4, 2010
72

- Continued
Met His Gly Asn Ser Glu Met Gln Lys Ile Asn Gln Thr Ser Ala Met
1. 5 1O 15

Pro Glu Lys Thr Asp Val His Trp Ser Gly Arg Phe Ser Val Ala Pro
2O 25 3O

Met Lieu. Asp Trp Thr Asp Arg His Cys Arg Tyr Phe Lieu. Arg Lieu. Lieu.
35 4O 45

Ser Arg Asn Thr Lieu. Leu Tyr Thr Glu Met Val Thr Thr Gly Ala Ile
SO 55 6O

Ile His Gly Lys Gly Asp Tyr Lieu Ala Tyr Ser Glu Glu Glu. His Pro
65 70 7s 8O

Val Ala Lieu. Glin Lieu. Gly Gly Ser Asp Pro Ala Ala Lieu Ala Glin Cys
85 90 95

Ala Lys Lieu Ala Glu Ala Arg Gly Tyr Asp Glu Ile Asn Lieu. Asn Val
1OO 105 11 O

Gly Cys Pro Ser Asp Arg Val Glin Asn Gly Met Phe Gly Ala Cys Lieu.
115 12 O 125

Met Gly Asn Ala Glin Lieu Val Ala Asp Cys Val Lys Ala Met Arg Asp
13 O 135 14 O

Val Val Ser Ile Pro Val Thr Val Lys Thr Arg Ile Gly Ile Asp Asp
145 150 155 160

Gln Asp Ser Tyr Glu Phe Lieu. Cys Asp Phe Ile Asn Thr Val Ser Gly
1.65 17O 17s

Lys Gly Glu. Cys Glu Met Phe Ile Ile His Ala Arg Lys Ala Trp Lieu.
18O 185 19 O

Ser Gly Lieu. Ser Pro Llys Glu Asn Arg Glu Ile Pro Pro Lieu. Asp Tyr
195 2OO 2O5

Pro Arg Val Tyr Gln Leu Lys Arg Asp Phe Pro His Lieu. Thr Met Ser
21 O 215 22O

Ile Asin Gly Gly Ile Llys Ser Lieu. Glu Glu Ala Lys Ala His Lieu. Glin
225 23 O 235 24 O

His Met Asp Gly Val Met Val Gly Arg Glu Ala Tyr Glin Asn Pro Gly
245 250 255

Ile Lieu Ala Ala Val Asp Arg Glu Ile Phe Gly Ser Ser Asp Thr Asp
26 O 265 27 O

Ala Asp Pro Val Ala Val Val Arg Ala Met Tyr Pro Tyr Ile Glu Arg
27s 28O 285

Glu Lieu Ser Glin Gly Thr Tyr Lieu. Gly His Ile Thr Arg His Met Leu
29 O 295 3 OO

Gly Lieu. Phe Glin Gly Ile Pro Gly Ala Arg Glin Trp Arg Arg Tyr Lieu.
3. OS 310 315 32O

Ser Glu Asn Ala His Lys Ala Gly Ala Asp Ile Asn Val Lieu. Glu. His
3.25 330 335

Ala Lieu Lys Lieu Val Ala Asp Lys Arg

34 O 345

<210s, SEQ ID NO 41
&211s LENGTH: 217
212. TYPE: PRT
<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 41

Met Lieu. Cys Wall Lys Asn. Wal Ser Lieu. Arg Lieu Pro Glu Ser Arg Lieu.
1. 5 1O 15
US 2010/0028334 A1 Feb. 4, 2010
73

- Continued

Lell Thir Asn Wall Asn Phe Thr Val Asp Gly Asp Ile Wall Thir Lieu.
25

Met Gly Pro Ser Gly Gly Lys Ser Thir Luell Phe Ser Trp Met Ile
35 4O 45

Gly Ala Luell Ala Glu Glin Phe Ser Thir Gly Glu Lell Trp Luell Asn
SO 55 6O

Glu Glin Arg Ile Asp Ile Leul Pro Thir Ala Glin Arg Glin Ile Gly Ile
65 70 8O

Lell Phe Glin Asp Ala Lieu. Luell Phe Asp Glin Phe Ser Wall Gly Glin Asn
85 90 95

Lieu. Luell Luell Ala Leul Pro Ala Thir Lieu. Gly Asn Ala Arg Arg Asn
1OO 105 11 O

Ala Wall Asn Asp Ala Lieu. Glu Arg Ser Gly Lieu. Glu Gly Ala Phe His
115 12 O 125

Glin Asp Pro Ala Thr Lell Ser Gly Gly Glin Arg Ala Arg Wall Ala Lieu
13 O 135 14 O

Lell Arg Ala Lieu. Luell Ala Glin Pro Ala Lieu. Lieu. Lieu Asp Glu Pro
145 150 155 160

Phe Ser Arg Lieu. Asp Val Ala Lieu Arg Asp Asn. Phe Arg Glin Trp Val
1.65 17O 17s

Phe Ser Glu Wall Arg Ala Lell Ala Ile Pro Wal Wall Glin Wall Thir His
18O 185 19 O

Asp Lieu. Glin Asp Val Pro Ala Asp Ser Ser Val Lieu. Asp Met Ala Glin
195 2O5

Trp Ser Glu Asn Tyr Asn Lys Luell Arg

21 O 215

SEQ ID NO 42
LENGTH: 5
TYPE PRT
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide
<4 OOs, SEQUENCE: 42
Gly Gly Gly Gly Cys
1. 5

1. A composition comprising an antimicrobial agent and an
enhancer to the antimicrobial agent, wherein the enhancer to
the antimicrobial agent is an inhibitor of a gene product that
by inactivating the gene product potentiates the effectiveness
of the antimicrobial agent.
2. The composition of claim 1, wherein the antimicrobial
agent is an antimicrobial peptide.
3. The composition of claim 2, wherein the antimicrobial
peptide is a lipopeptide.
4. The composition of claim3, wherein the lipopeptide is a
cyclic lipopeptide.
5. The composition of claim 4, wherein the cyclic lipopep
tide is a polymyxin class of antibiotic or derivative thereof.
6. The composition of claim 5, wherein the polymyxin is
selected from the group of polymyxin A, B1, B2, D1, D2, E1
and/or E2, F, G, M, P S and/or T
7. The composition of claim 5, wherein the polymyxin is
selected from polymyxin B1, polymyxin B2, and a mixture of
polymyxin B1 and polymyxin B2.
8. The composition of claim 5, wherein the polymyxin is
selected from colistin A, colistin B, and a mixture of colistin
A and colistin B.
9. The composition of claim 5, wherein the polymyxin is in
the form of a colistin salt.
10. The composition of claim 9, wherein the colistin salt is
a methane Sulphonate and/or Sulfate salt.
11. The composition of claim 1, wherein the gene product
is selected from a group consisting of agaA, atp A, atpC, atpB.
atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB,
iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L.
pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA,
visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/or ynjD or
homologues, variants or fragments thereof.
US 2010/0028334 A1
74
12. The composition of claim 1, wherein the inhibitor is
selected from a group consisting of mefloquine, Venturicidin
A, diary guinoline, betaine aldehyde chloride, acivein, psico
furaine, buthionine Sulfoximine, diaminopemelic acid,
4-phospho-D-erythronhydroxamic acid, motexafin gado
linium and/or Xycitrin or modified versions or analogues
thereof.
13. The composition of claim 1, wherein the gene product
is atp A, atpF or atpH or homologues, variants or fragments
thereof, and the inhibitor is mefloquine and/or venturicidin A
and/or diarycuinoline or modified versions or analogues
thereof.
14. The composition of claim 1, wherein the gene product
is betB or homologues or variants thereof, and the inhibitor is
betaine aldehyde chloride or modified versions or analogues
thereof.
15. The composition of claim 1, wherein the gene product
is guaA or guaB or homologues or variants thereof, and the
inhibitor is acivin and/or psicofluranine or modified versions
or analogues thereof.
16. The composition of claim 1, wherein the gene product
is LipA or homologues or variants thereof, and the inhibitoris
buthionine Sulfoximine or modified versions or analogues
thereof.
17. The composition of claim 1, wherein the gene product
is LySA or homologues or variants thereof, and the inhibitoris
diaminopimelic acid or modified versions or analogues
thereof.
18. The composition of claim 1, wherein the gene product
is rpiA or homologues or variants thereof, and the inhibitor is
4-phospho-D-erythronhydrixamic acid or modified versions
or analogues thereof.
19. The composition of claim 1, wherein the gene product
is trx A or homologues or variants thereof, and the inhibitor is
motexafin gadolinium and/or Xycitrin acid or modified ver
sions or analogues thereof.
20. The composition of claim 1, wherein the inhibitor
comprises a small molecule, nucleic acid, nucleic acid ana
logue, peptide, ribosome, antibody, and variants and frag
ments thereof.
21. (canceled)
22. (canceled)
23. The composition of claim 1, wherein the microorgan
ism is selected from the group consisting of a bacterium, a
gram-positive bacterium, a gram-negative bacterium, a multi
drug resistant microorganism.
24. (canceled)
25. (canceled)
Feb. 4, 2010
26. (canceled)
27. The composition of claim 23, wherein the multi-drug
resistant microorganism is resistant to at least one member of
the polymyxin class of antibiotics or derivatives or analogues
thereof.
28. (canceled)
29. (canceled)
30. (canceled)
31. The composition of claim 1 further comprising a phar
maceutically acceptable carrier.
32. (canceled)
33. The composition of claim 1, where the amount of the
antimicrobial agents is at least 25% less than the same anti
microbial agent in an isogenic cell except for the addition of
the enhancer of antimicrobial agent without reduction of anti
microbial effect.
34. (canceled)
35. A method of treatment and/or prophylaxis of an infec
tion caused by an microorganism comprising steps of admin
istering to a subject in need thereof an effective amount of the
composition according to claim 1.
36. The method of claim 35, wherein the subject is mam
malian, avian, amphibian or a plant.
37. The method of claim 36, wherein the mammalian is
human.
38.-41. (canceled)
42. The method of claim 35, wherein the infection is
selected from the group consisting of bacterial wound infec
tions, mucosal infections, enteric infections, septic condi
tions, infectious in airways, cerebrospinal fluid, blood, eyes
and skin.
43.-51. (canceled)
52. A method for identifying gene products, wherein inac
tivation of the gene products potentates antimicrobial agent
activity, the method comprising the steps of
(a) mutating one or more genes in a cell,
(b) contacting the cell with the antimicrobial agent,
(c) incubating the cell for a sufficient amount of time to
allow for growth, and;
(d) assessing the number of cells, wherein the number of
cells is compared to steps (a)-(c) performed on a non
mutated cell, wherein the decrease in numbers of cells
identifies a gene product that when inactivated potenti
ates antimicrobial peptide activity.
53. The method of claim 52, wherein the cell is bacterium.
54-58. (canceled)