m Botulism causes flaccid paralysis of muscles.

m It is caused by a neurotoxin, generically called botulinum toxin

m produced by the bacterium Ú


 .
m Ú

  produces 7 distinct neurotoxins( A-G).
m Types A, B, E & F produce paralysis in humans.
m the toxin named 6
block neuromuscular transmissions by
blocking the release of acetylcholine from motor nerve endings.
m so the nerve cannot stimulate the muscle to contract, hence causes
paralyses.
m Botulinum neurotoxin is one of the most potent, lethal substance known.
m 1 ng/kg can be lethal to an individual.
m Estimated that about 1gm could potentially kill 1million people.
m So it is used, using & will be used as bioweapon.
m 



  
    
  
m À  
  is caused by eating foods that contain the botulinum
neurotoxin.
m a    is caused by neurotoxin produced from a wound that is
infected with the bacteria Ú


 .
m à    occurs when an infant consumes the spores of the
botulinum bacteria. The bacteria then grow in the intestines and release
the neurotoxin.





  
    
 

m Joesn't form endospores rather it produces neurotoxin.
m It is only to produce the neurotoxin during sporulation, which can only
happen in an aerobic environment.
m It is obligate anaerobe.
m It tolerate traces of O2 due to the enzyme called 3



(SOJ)
m îo commercial vaccine available or approved by the FJA.
m In US, an investigational 

   toxoid
vaccine(against A, B, C, J & E neurotoxins) can be used for laboratory
workers at high risk of expouser to botulinum toxin and by the military
for protection of troops against attack.
m Unfortunately, it takes several months to induce immunity.
Trivalent (A,B,E) Botulinum Antitoxin:
m derived from equine sources
m utilizing whole antibodies (Fab & Fc portions)
m This antitoxin is available from the local health department via the CJC in the USA.

Heptavalent (A,B,C,J,E,F,G) Botulinum Antitoxin:

m derived from "despeciated" equine IgG antibodies, which have had the Fc portion
cleaved off leaving the F(ab')2 portions.
m This is a less immunogenic antitoxin that is effective against all known strains of
botulism wher not contraindicated.
m This is available from the United States Army.
m Optimization of leads to be done based on the Lipinski rule of five.
m These optimized ligands are used to find its respective interactions
with the target protein.
ë
 , is the inventive process of finding new medications based on
the knowledge of the biological target.
ëA   

ë is a biopolymer such as a protein or nucleic acid whose activity can be
modified by an external stimulus
ë Biological targets are most commonly proteins such as enzymes, ion
channels, and receptors.
ë
ë most commonly an organic small molecule
ë which activates or inhibits the function of a biomolecule such as a protein
ë which in turn results in a therapeutic benefit to the patient.
ëJrug design really meant by drug design is ligand design
ëLigand is optimized using   
 
 
 for:
ë Binding affinity
ë Bioavailability
ë Metabolic half life
ë Lack of side effects
u In history new drugs discovered by  
 
u most famous such incident was the  
 
  
Alexander Fleming in 1928.
u discovered the first antibiotic when some bacterial cultures
he was working with became contaminated with
a bactericidal fungus.
u Of course, this type of chance discovery does not happen
very often.
u Older methods of developing new medications have several
flaws that make drug discovery an expensive business.
u As the 
  for new and more effective drugs has
increased, a new method of drug development
called rational drug design has begun to replace the old
methods.
m In this biologically active compounds are specifically
designed or chosen to work O   
is
the Rational Jrug Jesign
m It uses molecular design software, to create 3-J models of
drugs and their biological targets.
m For this reason, the process is also known as Computer-
Aided Jrug Jesign
u Ú
 s ca l cat  f r t sti i t ays:
u first iv lv s t s f c
iat rial lirary scr i.
u  v r si rati al r  si
t s ill scr  t lirary f r
c
 s.
u at is s cific  
t it ract it
t
r tar t f it r st.
u
s c 
t
 iv lv s t
actal  si f a c
  t
at ca it ract it

t
tar t.
u ir k l  f c

ical r s r ir  f r r it racti s c

ial

ak  f c
 .
u .

u
m First drug developed by Rational Jrug Jesign is Relenza.
m It is an antiviral drug interact with an influenza protein called
neuraminidase.
m Other rationally deisgned drugs are     of HIV
For selecting biomolecule as a drug target
m The target will 


 

m Target is 
i.e. capable of binding to small molecule & activity is
modulated by that small molecule.
m Once a suitable target has been identified, the target is
normally cloned and expressed.
m The expressed target is then used to establish a screening assay(High-
throughput screening (HTS)).
m Through this process one can  
active compounds,
antibodies or genes which modulate a particular bimolecular pathway.
m These results are used in:
m Starting points for drug design.
m Understanding the interaction or role of particular biochemical process in
biology.
m How selective the compounds are for the chosen target.
m Molecule which will interfere with only the chosen target
m  

  to avoid off-target toxicity

m
m Some compounds 
  
 

.

m At this time use 3


 3 ) to
improve certain features of the lead compound.
m increase  the chosen target
m 

 against unrelated targets
m  

! "
!  #$ properties of the molecule.
m ahile HTS is a commonly used method for novel drug discovery
ë Ideally the candidate drug compounds should be Dzdrug-likedz, it
should possess properties to
ë oral bioavailability
ë Adequate chemical & metabolic stability
ë Minimal toxic effects.
ë The rule describes 
  

   
for a drug's pharmacokinetics in the human body, including
their AJME
ë However, the rule does not predict if a compound is
pharmacologically active.
Lipinski's rule says that, in general, an orally active drug has no more than
one violation of the following criteria:
m îot more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or
more hydrogen atoms)
m îot more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms)
m A molecular weight under 500 daltons
m An octanol-water partition coefficient log  of less than 5

îote that all numbers are multiples of five, which is the origin of the rule's
name.
m Computer-assisted drug design uses computational chemistry to discover,
enhance, or study drugs and related biologically active molecules. The
most fundamental goal is to predict whether a given molecule will bind to
a target and if so how strongly
Applications:
m Find interesting lead molecules quickly
m Predicting properties and activities of untested molecules
m Propose compounds for synthesis
m Validate models of receptor binding sites
m Optimize pharmacokinetic properties of compounds
1. Ligand based drug design(or indirect drug design)
2. Structure based drug design( or direct drug design)
m ‰now the other molecules that bind to the
biological target of interest.
m By using these molecule a model of the biological
target may be built
m Require 3J-structure of the biological target obtained throuh
methodes such as:
m X-ray cyrastllography
m îMR spectroscopy
Current methods for structure-based drug design can be
divided roughly into two categories.
Pharmacophore
Π1) Dzfindingdz ligands for a given identification
receptor, which is usually referred as


 
ŒIn this case, a large number of potential ligand
Pharmacophore
molecules are screened to find those fitting the
modification
binding pocket of the receptor.
ŒThis method is usually referred as ligand-based
drug design.
ŒThe key advantage of database searching is that it List for the
saves synthetic effort to obtain new lead receptor
compounds. îo

Ye
s
Potential drug
 ë Another category of structure-based drug design
methods is about º  %  , which is usually
referred as 

 

 
ë In this case, ligand molecules are built up within the
constraints of the binding pocket by assembling
small pieces in a stepwise manner.
ë These pieces can be either individual atoms or
molecular fragments.
ë The key advantage of such a method is that novel
structures, not contained in any database, can be
suggested.
ë These techniques are raising much excitement to
the drug design community
Πin the field of molecular modeling,  "  is
a method which predicts the preferred
orientation of one molecule to a second
when bound to each other to form a

 

Œ º
% 
     
   

ΠJocking approaches
ΠOne approach uses a matching technique
that describes the  
  
  
 



ΠThe second approach  

 
 "  
in which the ligand-protein
pairwise interaction energies are calculated
ΠBoth approaches have significant advantages
as well as some limitations.
ë the receptorǯs molecular surface is described
in terms of its  





(measured in A0) and the ligandǯs
molecular surface is described in terms of its
matching surface description.
ë fast and robust,
ë
 
 
  & 
 
   
ë à "       

 
 
m It is more complicated process.
m In this approach, the protein and the ligand are
separated by some physical distance, and the
ligand finds its position into the proteinǯs active
site after a certain number of Dzmovesdz in its
conformational space.
m The moves incorporate rigid body transformations
such as translations and rotations, as well as
internal changes to the ligandǯs structure including
torsion angle rotations.
m To perform a docking, the first requirement 3J
STRUCTURE
m Usually the structure has been determined using a
biophysical technique such as x-ray crystallography, or
less often, îMR spectroscopy.
m This protein structure and a database of potential
ligands serve as inputs to a docking program.
m The success of a docking program depends on two
components: the search algorithm and the scoring
function.
m The search space in theory consists of all possible orientations
and conformations of the protein paired with the ligand.
m enumerating all possible distortions of each molecule and all
possible rotational and translational orientations of the ligand relative to
the protein at a given level of granularity.
m Most docking programs in use account for a flexible ligand, and several
attempt to model a flexible protein receptor.
m Flexible docking in addition considers all possible conformations of the
protein paired with all possible conformations of the ligand.
m Each "snapshot" of the pair is referred to as a  
.
m 3     used to predict the strength of the non-
covalent interaction (also referred to as binding affinity) between
two molecules after they have been docked.
ΠA binding interaction between a small molecule ligand and
an enzyme protein may result in activation or inhibition of the
enzyme.
Π. Jocking is most commonly used in the field of drug design most
drugs are small organic molecules, and docking may be applied
to:
Π
  docking combined with a scoring function can be
used to quickly screen large databases of potential drugs in silico to
identify molecules that are likely to bind to protein target of interest
Œ
 '  docking can be used to predict in where and in
which relative orientation a ligand binds to a protein (also referred to
as the binding mode or pose). This information may in turn be used to
design more potent and selective analogs.
Π6 

  Protein ligand docking can also be used to predict
pollutants that can be degraded by enzymes.
· Ú  
 

   

catalyzes the
Ú 
 ()

and poduce cytine
monopohsphare(CMP),(3-sn-phoshatidyl)-L-serine in Ú

 .
· CMP is helpful rna synthesis of Ú

 
· So CJP-diacylglycerol-serine o-phoshatidyltransferase is the lead
moleucle used for synthesis of rna of c.botulinum.
· By usinig Jocking program GOLJ, dock the protein with designed
ligand.
 m E Harikishan Reddy EH, Satpathy GRIdentification of potential targets
and lead molecules for designing inhibitory drugs against Ú 
 


  X.Bioinformatics  
http://users.comcen.com.au/~journals/leadprintabs2009.htm
m P. Aureli, L. Fenicia, B. Pasolini, M. Gianfrancesche, X. M. Mccroskey, C. L.
Hatheway. Two cases of type E infant botulism caused by neurotoxigenic
Clostridium botulinum in Italy. X. Infect. Jis. 1986, 154:207211.
http://www.ncbi.nlm.nih.gov/pubmed/3722863
m X. J. Hall, L. M. McCroskey, B. X. Pincomb, C. L. Hatheway, Isolation of an
organism resembling Clostridium baratii which produces a type F
botulinal toxin from an infant with botulism. X. Clin. Microbiol.1985,
21:654655.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC271744/pdf/jcm00117-
0202.pdf
m S. îotermans, A. H. Havellar, Removal and inactivation of botulinum
toxin during production of drinking water from surface water. Antonie van
Leeuwenhoek. 1980, 46:511514.
http://www.springerlink.com/index/lw73325nx4220745.pdf
m ‰roemer, R.T., Joughty, S.a., Robinson, A.X. and Richards, a.G.
Prediction of the three-dimensional structure of human interleukin-7 by
homology modeling. Protein Engineering. 1996, 9, 493498.
http://peds.oxfordjournals.org/cgi/content/abstract/9/6/493
m î. Eswar, M. A. Marti-Renom, B. aebb, M. S. Madhusudhan, J. Eramian,
M. Shen, U. Pieper, A.Sali. Comparative Protein Structure Modeling aith
MOJELLER. Current Protocols in Bioinformatics, Xohn ailey & Sons, Inc.,
Supplement. 2006, 15, 5.6.1-5.6.30.
http://salilab.org/pdf/Eswar_CurrentProtocolsinBioinformatics_2006.pdf
m Laskowski, R.A., MacArthur, M.a., Moss, J.S. and Thornton, X.M.,
PROCHEC‰: a program to check the stereochemical quality of protein
structures. Xournal of Applied Crystallography. 1993, 26, 283-291.
http://onlinelibrary.wiley.com/doi/10.1107/S0021889892009944/abstract