BIOPROSP 2011 : Tromsø



Bioprospecting among the Actinobacteria

of Extreme Environments


Alan T. Bull
School of Biosciences,
University of Kent, Canterbury, UK
• Bioprospecting – the case for Natural Products

• The case for Actinobacteria

• The case for Systematics

• The case for Prospecting the Extremobiosphere

• Recent Case Studies from our group

• Lessons and Prospects

 The Class Actinobacteria
68 Nocardiaceae
Mycobacteriaceae
Tsukamurellaceae
Dietziaceae Corynebacterineae
68
Corynebacteriaceae
Segniliparaceae
Actinosynnemataceae Pseudonocardineae
68 Pseudonocardiaceae
Streptomycetaceae Streptomycineae
Actinospicaceae
Catenulisporineae
91 Catenulisporaceae
Micromonosporaceae Micromonosporineae
Nakamurellaceae
Cryptosporangiaceae
Sporichthyaceae
Geodermatophilaceae Frankineae
Acidothermaceae
98 Frankiaceae
Kineosporiaceae Kineosporiineae
52 Dermacoccaceae
68 Intrasporangiaceae
Dermatophilaceae Actinomycetales
98 Yaniellaceae = Actinomycetes
Micrococcaceae
63 Brevibacteriaceae
Dermabacteraceae
55 Jonesiaceae Micrococcineae
82 Rarobacteraceae
Sanguibacteraceae
Beutenbergiaceae
Microbacteriaceae
Promicromonosporaceae
Bogoriellaceae
Cellulomonadaceae
Streptosporangiaceae
93 Thermomonosporaceae Streptosporangineae
Nocardiopsaceae
Nocardioidaceae Propionibacterineae
97 64 Propionibacteriaceae
Actinopolysporaceae Actinopolysporineae
Actinomycetaceae Actinomycineae
84
62 Glycomycetaceae Glycomycineae
Bifidobacteriaceae Bifidobacteriales
79
Nitriliruptoraceae Nitriliruptorales/Euzebyales
50 Acidimicrobiaceae Acidimicrobiales
Coriobacteriaceae Coriobacteriales
92 Solirubrobacteraceae
99 Conexibacteraceae Solirubrobacterales
97
Patulibacteraceae
Thermoleophilaceae Thermoleophilales
89 Rubrobacterales
Rubrobacteraceae

0.02 A.T. Bull (2011) Extremophiles Handbook, Springer Tokyo pp 1203-1240

The case for Actinobacteria: why prioritize for
Drug Discovery?
“Arguably the richest source of small molecule
diversity on the planet”

• Size and diversity of the taxon

• Widespread global and environmental dispersal
• Rapid discovery of new taxonomic radiations
• Produce ca. 45% all microbial bioactive chemicals
(~9500)
• Possibly only ca. 10% of actinomycete bioactive
chemicals have been discovered to date.
• Large numbers of biosynthetic gene clusters from
whole genome sequencing.
 The Case for Systematics
Role of taxonomy in bioprospecting
• Enables classification and hence detection and identification
• Predicts metabolic potential – taxonomy as a roadmap to products
• Facilitates dereplication of isolates
• IP protection

But essential to develop

• Quicker, more reliable procedures for describing new taxa – current
methods are laborious and time-consuming
• Wider exploration of taxospace, e.g. deep lineage Actinobacteria
sub-classes
• More sophisticated screening for chemical diversity

How have evolutionary and environmental forces shaped the

distribution of bioactive natural products across microbial
diversity?
Drug Target Screen Extract Analysis Organisms

Chemistry Positive Novel Compound Bioactivity

Dereplication: differentiation
HIT of phenotypically ambiguous
strains (and chemicals) to
facilitate efficient screening
and minimizing cost and time
in sorting large collections.
Compound Characterization
Compound Evaluation
(ADMET Screening)

Dereplication filters
LEAD
 A Guide to Biodiscovery

Extreme Environments → Novel Organisms → Novel Natural Products

“Bacteria dwell in virtually every spot that can sustain

any sort of life. And we have underestimated their
global number because we, as members of a
Kingdom far more restricted in potential habitation,
never appreciated the full range of places that might
be searched.”

Stephen Jay Gould (1996) Planet of the bacteria, Washington Post Horizon.
 The Extremobiosphere
• Limits to Growth:

-18C to 121C
pH 0 to 13
P up to 110 MPa
Salinity up to 2.5M NaCl
-Radiation up to 60 Gy h-1
Nutrients down to nM concentrations
Moisture content down to aw 0.75

• Extreme Earth Environments:

Deep-seas (>200m) ca. 64% Earth surface

Deep biosphere (deep subterranean, deep submarine)
Cryosphere (permanent) ca. 11% Earth surface
Deserts ca. 15% Earth land surface
 Extremophilic/-tolerant Actinobacteria?
• Traditionally viewed as autochthonous members of soil and freshwaters
• Now found increasingly in all extreme environments, often as dominant
populations
Order Sub-order Family
Actinomycetales Corynebacterineae
Frankinease
Micrococcinease Microbacteriaccae
Micrococcaceae
Streptomycineae Streptomycetaceae
Actinopolysporinease Actinopolysporaceae
Bifidobacteriales
Nitriliruptorales Nitriliruptoraceae
Acidimicrobiales Actidimicrobineae Acidimicrobiaceae
Coriobacteriales
Rubrobacteriales “Rubrobacterineae” Rubrobacteraceae
Thermoleophilales Thermoleophilaceae
Conexibacteraceae
Patulibacteraceae
Solirubrobacterales Solirubrobacteraceae
A.T. Bull (2011) Extremophiles Handbook
Extremophilic Actinobacteria
Arthrobacter (pressure)
Catenulispora (acidity)
Kocuria (salinity, low temperature)
Nitriliruptor (salinity, alkalinity)
Rubrobacter (salinity, low temperature, high radiation, low moisture)
Thermoleophilum (high temperature)

Extremotolerant Actinobacteria
Dermacoccus (pressure)
Modestobacter (low moisture, low nutrient, high radiation)
Rhodococcus (low temperature, high radiation, pressure)
Streptomyces (low/high temperature, salinity, low moisture, pressure)

Note: some, not all, members of these genera contain extremophilic/

tolerant organisms.
 Extreme Environments  Novel Organisms  Novel Natural Products

Our Bioprospecting Grounds

Polar ecosystems Hyper-arid deserts

[Terra Nova Bay, [Atacama]
Antarctica]

Deep-seas
[Mariana Trench]
 Recent Deep-Sea Actinomycete Hit Strains
Isolated by our Group
Depth (m) Organism Compound
>200 Verrucosispora Abyssomicins 2004
atrop-
Abyssomicin C 2007
Enediyene 2005
Proximicins 2008
>2000 Bathyal Streptomyces Caboxamycin 2009
Streptomyces Benzoxacystol 2011
>4000 Abyssal Streptomyces Albidopyrone 2007
>6000 Hadal Micromonospora Dipeptide 2009
Dermacoccus Dermacozines 2010
Anti bacterial
Enzyme inhibitor
Anti cancer
Anti protozoal
Signalling (?)
Proximicins ex. Verrucosispora sp. MG-37: Raune
Fjord, Norway 250 m

H.-P. Fiedler et al. (2008) J. Antibiotics 61: 158.

Caboxamycin ex. Streptomyces sp. NTK 937
Canary Basin 3,814 m

C. Hohmann et al. (2009) J. Antibiotics, 62: 99

 Benzoxacystol ex. Streptomyces sp. NTK935
Canary Basin 3,814m
Streptomyces globisporus NRRL B-2872T (EF178686)
strain NTK935
100 Streptomyces tanashiensis IFO 12919T (AY999856)
Streptomyces mediolani NBRC 15427T (AB184674)
68 Streptomyces rubiginosohelvolus NBRC 12912T (AB184240)
Streptomyces griseus subsp. griseus NBRC 15744T (AB184699)
85 64
Streptomyces griseoplanus AS 4.1868T (AY999894)
96 Streptomyces mirabilis DSM 40553T (AY999730)
Streptomyces galbus DSM 40089T (X79852)
Streptomyces acidiscabies ATCC 49003T (D63865)
83 Streptomyces thermodiastaticus DSM 40573T (AB018096)
Streptomyces mexicanus JCM 12681T (AF441168)
Streptomyces somaliensis DSM 40738T (AJ007403)
95 Streptomyces caelestis NRRL 2418T (X80824)
67 Streptomyces ambofaciens ATCC 23877T (M27245)
Streptomyces speibonae ATCC BAA-411T (AF452714)
Streptomyces coeruleofuscus NRRL-ISP 5144T (AJ399473)
83 Streptomyces coelicolor DSM 40233T (Z76678)
Streptomyces koyangensis NBRC 100598T (AY079156)
83
Streptomyces rutgersensis subsp. rutgersensis DSM40077T (Z76688)
Streptomyces variegatus LMG 20315T (AJ781371) Biological activity
Streptomyces mashuensis DSM 40221T (X79323)
Streptomyces morookaensis NBRC 13416T (AB184878)
First benzoxazine inhibitor of
Streptomyces sporocinereus NBRC 100766T (AB249933) glycogen synthase kinase 3β
Streptomyces rimosus subsp. rimosus JCM 4667T (AB045883)
Streptomyces albus subsp. albus DSM 40313T (AJ621602) (IC50 1.35μM).
Streptomyces ribosidificus NBRC 13796T (AB184487)
Streptomyces thermolineatus DSM 41451T (Z68097)
Alzheimer’s; Type 2 diabetes
Streptomyces carpaticus NRRL B-16359T (DQ442494)
99 Streptomyces cheonanensis JCM 14549T (AY822606)
Micrococcus lylae DSM 20315T (X80750)
0.1
J. Nachtigall et al. (2011) J. Antibiotics, in press.
The Genus Dermacoccus
Dermacoccus profondi MT2.2T (AY894329)

Dermacoccus bathari MT2.1T (AY894328)

Dermacoccus abyssi MT1.1T (AY894323)**

Dermacoccus nishinomiyaensis DSM 20448T (X87757)

Pathom-aree et al. (2006) Int. J. System. Evol. Microbiol. 56: 2303

 Dermacozines
ex. Dermacoccus abyssi: Mariana Trench 10,898m
H O
O N O O O O
O
O
COOCH3

N N N
N

N N N
H
N
CONH2 CONH2 CONH2 H

CONH2 CONH2

Dermacozine A Dermacozine B Dermacozine C Dermacozine D

O
O
CONH2
O N
N N
OH N OH

N
N N
N H
O
CONH2 CONH2 HO
CONH2 OH

Dermacozine E Dermacozine F Dermacozine G Dermacozine H

Biological activity of A and D NOVEL NEW

Anti cancer and radical scavenging
Anti-trypanosomal N.M. Abdel-Mageed et al. (2010) Org. Biomol. Chem. 8: 2352.
The Atacama Desert
 Is there Microbial Life in the
Atacama Soils?

Oldest, driest on Earth

Extreme aridity for at least 10-15 My
Very low soil OM (0.02-0.04 mg C g-1)

B. Gomez-Silva (2010) Astrobio, Santiago

Yungay – the Hyper-arid Desert core
Gypsum crust Nitrate

Salar Rock desert

New Streptomyces clade isolated from soil of the Laguna de Chaxa,
Salar de Atacama, Chile

Anti-MRSA ansamycins
Anti-cancer macrolides

Several novel NPs

Novel aromatic polyketide

Polyene antibiotic
Chaxamycins : New Ansamycins produced by
Atacama Streptomyces strain C34

Bioactivities
Ansamycin 4: active against
Gram +ve and –ve bacteria
Clinical isolates of MRSA,
MIC 5 < 1μg ml-1 (rifamycin
0.002-0.008 μg ml-1)

Ansamycin 1 (methylated at
C28):
Inhibits intrinsic ATPase of
human chaperone Hsp90
(50% activity of 17-AA
Geldamycin)

M.E. Rateb et al. (2011) J. Org. Chem., submitted

 Atacamycins : New Macrolactones produced by
Atacama Streptomyces strain C38

OH A: R1 =
H3C R2 = OCH 3
OCH3 R3 = OH
R3
R1 O B: R1 =
R2 = H
R2 O R3 = OH

CH3 CH3
C: R1 =
R2 = H
atacamycin A-C R3 = H

Positive antitumor activity against 42 human cell lines but no

pronounced selectivity. Best activity vs. adeno carcinoma (IC50 5.9µM)
and breast carcinoma (IC50 2.7µM).

J Nachtigall et al. (2011), in preparation)

Novel species of Nonomurea isolated from the
Salar de Atacama
Reporter strain screening of antibiotic mode of
action : Atacama Nonomurea sp. JP10

DNA synthesis Fatty acid synthesis

Cell wall synthesis

Cell envelop RNA synthesis

 Misconceptions that obstruct natural product
search and discovery

1. Microbial systematics is simply stamp collecting.

2. The search for natural product is more stamp

collecting.

3. Natural products have become exhausted.

4. Synthetic compounds provide superior drug

candidates than natural products.

5. Isolation and characterization of natural products is

laborious and slow.
 Natural products from actinomycetes in the extremobiosphere
Compound/Discovered Organism Origin Chemistry Bioactivity
Barbumycin (2010) Streptomyces sp. MS Macrolactone Antitumor
Nocardiopsins (2010) Nocardiopsis sp. MS Macrolides Immuno-suppressive
TP-1161 (2010) Nocardiopsis sp. MSp Thiopeptide Anti-bacterial
J BUR-31 (2010) Streptomyces sp. MSp Thiocidin Cytotoxic
Albidopyrone (2009) Streptomyces sp. DSS -Pyrrone Insulin agonist
*
Pyrroloquinoline
Ammosamides (2009) Streptomyces sp. DSS Cell cycle modulators
alkaloids
Arenamides (2009) Salinispora arenicola MS Depsipeptides NFB inhibitors
Antibacterial
* Caboxamycin (2009) Streptomyces sp. DSS Benzoxazole
Antitumour
Antioxidant
* Dermacozines (2009) Dermacoccus abyssi DSS Phenazines
Antitumour
Echinomycin (2009) Streptomyces sp. MS Peptide Antibacterial
Indoxamycins (2009) Streptomycete MS Polyketide Antitumor
Marinosporolides (2009) “Marinispora” sp. MS Macrolides Anti-Candida
Splenocins (2009) Streptomyces sp. MS Lactones Anti-inflammatory
Marineosins (2008) Streptomyces sp. MS Spiroaminals Antitumour
Marinopyrroles (2008) Streptomyces sp. MS Halogenated pyrroles Antibacterial
Pacificanones (2008) Salinispora pacifica MS Polyketides Antitumour

* Proximicins (2008) Verrucosispora sp. MS Aminofuran Antitumour

Salinipyrrones (2008) Salinispora pacifica MS Polyketides Antitumour

MS, marine sediment; DSS, deep-sea sediment; MSp, marine sponge

 Compound/Discovered Organism Origin Chemistry Bioactivity
Arenicolides (2007) Salinispora arenicola MS Polyketides Antitumour
Nocardiopsis Non-ribosomal
Lucentamycins (2007) MS Antitumour
lucentensis peptides
Non-ribosomal
Piperazimycins (2007) Streptomyces sp. MS Antitumour
peptides
Saliniketals (2007) Salinispora arenicola MS Polyketides Antitumour
Daryamides (2006) Streptomyces sp. MS Polyketides Antitumour
Marinomycins (2006) “Marinispora” sp. MS Polyketides Antitumour
Streptokordin (2006) Streptomyces sp. DSS Methylpyridine Antitumor
Chinikomycins (2005) Streptomyces sp. MS Polyketide Antitumour

* Frigocyclinone (2005) Streptomyces griseus AS Angucyclinone Antitumour

* Gephyromycin (2005) Streptomyces griseus AS Angucyclinone Glutaminergic

* Lipocarbazoles (2005) Tsukamurella sp. DSS Carbazole Antioxidant
* Abyssomicins (2004)
Verrucosispora maris MS Polycyclic polyketide Antibacterial
[atrop-abyssomicin C] (2007)
Chandrananimycins (2003) Actinomadora sp. MS Phenoxazin Antitumour
Polyketide/non-
Salinosporamide A (2003) Salinispora tropica MS Antitumor
ribosomal peptide

MS, marine sediment; DSS, deep-sea sediment; AS, Antarctic soil

PCA analysis of Drugs and Natural Products

R.A. Bauer et al., Curr. Opin. Chem. Biol. 2010, 14: 308
Actinobacterial Taxospace

A.C. Ward & N. Bora. Curr. Opin. Microbiol. 2006, 9, 279.

 Abyssomicins: History of a Perfect Hit
“inspirational natural products” [K.C. Nicolaou]

Source: deep sea sediment 2001

Organism: new actinomycete (Verrucosispora maris) 2004

Product: new chemical entity 2001

Chemistry: family of spirotetronate polyketides 2004 – 2010
(B, C, D, E, G, H, I)
Complete chemical synthesis: discovery of 2005, 2006
atrop-abyssomicin C

Screen: nutrient reversion assay

Bioactivity: Gram +ve antibacterial including MRSA, VRSA 2004
2007
MIC abyssomicin C: MRSA 4μg ml-1; 20μM
MIC atrop-abyssomicin C: 15μM

Target: p-aminobenzoic acid synthesis, 1st-in-a-class compound 2004

Mode of action: co-valent binding to PabB subunit of 4-amino-4- 2007
deoxychorismate synthase at Cys-263
 A natural product from a region of underexploited chemical space

Patent filing: Germany, European Union, USA 2003

Pharma discussions: Actelion, AiCuris, Basilea, Cubist, Nereus,

2004-2008
Novartis, Sanofi-Aventis

Other organisms: Streptomyces incl. non-marine 2007-2010

New Targets: Mycobacterium tuberculosis H37Rv (bactericidal, 3.6μM) 2010

 ?? LESSONS ??

• Missing the stage from detecting a compound and its target to the
next step – checking the in vitro in in vivo assays and determining
the basic toxicity, i.e. Moving from hit to lead. ADMET and other
screens.

• Include medicinal chemistry

• MICs too high? cf. TB drugs (isoniazid 0.29μM)

• You need excellent personal contacts to a pharmaceutical company,

even when it’s a small one

• Pharma personnel often do not have a strong interest to develop a

compound coming from outside.
WHAT ROLE FOR ACADEMIC GROUPS IN NATURAL PRODUCT
DRUG PROSPECTING ?

• Search for new scaffolds in regions of unexplored chemical space

• Be circumspect in adhering too strictly to posited “Rules”

• Do not be discouraged by initially unpromising MICs or IC50s

 Strategies for Accessing Undiscovered Natural
Products
• Focus on novel organisms (innovative isolation, e.g.
diffusion chambers, phage indicators)
e.g. deep lineage actinobacteria
• Maximise gene expression (diversity of production
media and conditions)
• Research allelopathic phenomena and allelochemicals
• Metagenomics (analysis and reverse metagenomics of
environmental DNA)
• Whole genome sequencing (screen for biosynthetic
gene clusters)
_____________________________________________
• This is a multidisciplinary game - DON’T WORK ALONE!
 Thanks to:
Collecting Expeditions: Microbiology/Molecular Biology:
Koki Horikoshi [Tokyo] Mike Goodfellow [Newcastle]
Doug Masson [Southampton] Jem Stach [Kent/Newcastle]
Gjert Knutsen [Bergen]
Juan Asenjo [Santiago]
Luis Caceres V. [Antofagasta]

Chemistry:
Hans-Peter Fiedler [Tübingen ]
Roderich Süssmuth [Berlin]
Marcel Jaspars [Aberdeen]

ATB supported by: Natural Environment Research Council

UK; Biotechnology & Biosciences Research Council UK;
The Leverhulme Trust; Boehringer Ingelheim; The Royal
Society