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

Nocardiaceae Mycobacteriaceae

Tsukamurellaceae Dietziaceae

Corynebacteriaceae Segniliparaceae

Actinosynnemataceae Pseudonocardiaceae

Streptomycetaceae Actinospicaceae

Catenulisporaceae Micromonosporaceae

Nakamurellaceae Cryptosporangiaceae

Sporichthyaceae Geodermatophilaceae

Acidothermaceae Frankiaceae

Kineosporiaceae Dermacoccaceae Intrasporangiaceae

Dermatophilaceae Yaniellaceae

Micrococcaceae Brevibacteriaceae

Dermabacteraceae Jonesiaceae

Rarobacteraceae Sanguibacteraceae

Beutenbergiaceae Microbacteriaceae

Promicromonosporaceae Bogoriellaceae

Cellulomonadaceae Streptosporangiaceae

Thermomonosporaceae Nocardiopsaceae

Nocardioidaceae Propionibacteriaceae Actinopolysporaceae

Actinomycetaceae Glycomycetaceae Bifidobacteriaceae

Nitriliruptoraceae Acidimicrobiaceae

Coriobacteriaceae Solirubrobacteraceae

Conexibacteraceae Patulibacteraceae

Thermoleophilaceae Rubrobacteraceae

92 99

97

89

79

50

84

68

62

98

93

98

82

64

68

68

91

97

55

52 68

63

0.02

Bifidobacteriales

Acidimicrobiales Coriobacteriales

Rubrobacterales

Solirubrobacterales

Thermoleophilales

Nitriliruptorales/Euzebyales

Actinomycetales

= Actinomycetes

Corynebacterineae

Pseudonocardineae

Streptomycineae

Catenulisporineae

Micromonosporineae

Frankineae

Kineosporiineae

Micrococcineae

Streptosporangineae

Propionibacterineae

Actinopolysporineae Actinomycineae

Glycomycineae

The Class Actinobacteria

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

The case for Actinobacteria: why prioritize for Drug Discovery?

• 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.

“Arguably the richest source of small molecule

diversity on the planet”

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?

Extract Analysis Organisms

Novel Compound Bioactivity

Dereplication: differentiation

of phenotypically ambiguous

strains (and chemicals) to

facilitate efficient screening

and minimizing cost and time

in sorting large collections.

H I T

Compound Characterization

Compound Evaluation

(ADMET Screening)

L E A D Dereplication filters

Drug Target Screen

Chemistry Positive

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

-18C to 121C

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

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

• Limits to Growth:

• Extreme Earth Environments:

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

Bifidobacteriales

Nitriliruptorales

Corynebacterineae

Frankinease

Micrococcinease

Streptomycineae

Actinopolysporinease

Microbacteriaccae

Micrococcaceae

Streptomycetaceae

Actinopolysporaceae

Nitriliruptoraceae

Acidimicrobiales Actidimicrobineae Acidimicrobiaceae

Coriobacteriales

Rubrobacteriales

Thermoleophilales

Solirubrobacterales

“Rubrobacterineae” Rubrobacteraceae

Thermoleophilaceae

Conexibacteraceae

Patulibacteraceae

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

Hyper-arid deserts

[Atacama]

Polar ecosystems

[Terra Nova Bay,

Antarctica]

Deep-seas

[Mariana Trench]

Our Bioprospecting Grounds

Recent Deep-Sea Actinomycete Hit Strains Isolated by our Group

Depth (m) Organism Compound

>200 Verrucosispora Abyssomicins

atrop-

Abyssomicin C

Enediyene

Proximicins

2004

2007

2005

2008

>2000 Bathyal Streptomyces

Streptomyces

Caboxamycin

Benzoxacystol

2009

2011

>4000 Abyssal Streptomyces Albidopyrone 2007

>6000 Hadal Micromonospora

Dermacoccus

Dipeptide

Dermacozines

2009

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

Micrococcus lylae DSM 20315T (X80750)

Streptomyces cheonanensis JCM 14549T (AY822606)

Streptomyces carpaticus NRRL B-16359T (DQ442494)

Streptomyces thermolineatus DSM 41451T (Z68097)

Streptomyces ribosidificus NBRC 13796T (AB184487)

Streptomyces albus subsp. albus DSM 40313T (AJ621602)

Streptomyces rimosus subsp. rimosus JCM 4667T (AB045883)

Streptomyces sporocinereus NBRC 100766T (AB249933)

Streptomyces morookaensis NBRC 13416T (AB184878)

Streptomyces mashuensis DSM 40221T (X79323)

Streptomyces variegatus LMG 20315T (AJ781371)

Streptomyces rutgersensis subsp. rutgersensis DSM40077T (Z76688)

Streptomyces koyangensis NBRC 100598T (AY079156)

Streptomyces coelicolor DSM 40233T (Z76678) 83

Streptomyces coeruleofuscus NRRL-ISP 5144T (AJ399473)

Streptomyces speibonae ATCC BAA-411T (AF452714)

Streptomyces ambofaciens ATCC 23877T (M27245)

Streptomyces caelestis NRRL 2418T (X80824) 95

67

83

Streptomyces somaliensis DSM 40738T (AJ007403)

Streptomyces mexicanus JCM 12681T (AF441168)

Streptomyces thermodiastaticus DSM 40573T (AB018096) 83

Streptomyces acidiscabies ATCC 49003T (D63865)

Streptomyces galbus DSM 40089T (X79852)

Streptomyces mirabilis DSM 40553T (AY999730)

Streptomyces griseoplanus AS 4.1868T (AY999894)

Streptomyces griseus subsp. griseus NBRC 15744T (AB184699)

Streptomyces rubiginosohelvolus NBRC 12912T (AB184240)

Streptomyces mediolani NBRC 15427T (AB184674)

Streptomyces tanashiensis IFO 12919T (AY999856)

strain NTK935

64

Streptomyces globisporus NRRL B-2872T (EF178686)

100

68

85

96

99

0.1

Biological activity

First benzoxazine inhibitor of

glycogen synthase kinase 3β

(IC50 1.35μM).

Alzheimer’s; Type 2 diabetes

J. Nachtigall et al. (2011) J. Antibiotics, in press.

The Genus Dermacoccus

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

Dermacoccus profondi MT2.2T (AY894329)

Dermacoccus bathari MT2.1T (AY894328)

Dermacoccus abyssi MT1.1T (AY894323)**

Dermacoccus nishinomiyaensis DSM 20448T (X87757)

Dermacozines ex. Dermacoccus abyssi: Mariana Trench 10,898m

N.M. Abdel-Mageed et al. (2010) Org. Biomol. Chem. 8: 2352.

Biological activity of A and D

Anti cancer and radical scavenging

Anti-trypanosomal

NOVEL NEW

N

N

CONH2

HNO O

N

N

CONH2

OO O

NH

N

CONH2

O

O

COOCH3

NH

N

CONH2 CONH2

O

N

N

CONH2

O

OH

N

N

CONH2

O

O

OH

OH

N

N

O

HO

NH

N

CONH2

CONH2

Dermacozine A Dermacozine B Dermacozine C Dermacozine D

Dermacozine E Dermacozine F Dermacozine G Dermacozine H

The Atacama Desert

B. Gomez-Silva (2010) Astrobio, Santiago

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)

Yungay – the Hyper-arid Desert core Gypsum crust Nitrate

Rock desert Salar

Anti-cancer macrolides

Several novel NPs

Novel aromatic polyketide

Polyene antibiotic

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

Anti-MRSA ansamycins

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

O

CH3

O

R1

OCH3

OH

H3C

R2

R3

CH3

A: R1 =

R2 = OCH3

R3 = OH

B: R1 =

R2 = H

R3 = OH

C: R1 =

R2 = H

R3 = Hatacamycin A-C

Atacamycins : New Macrolactones produced by Atacama Streptomyces strain C38

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

Fatty acid synthesis

RNA synthesis

DNA synthesis

Cell envelop

Cell wall 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

Ammosamides (2009) Streptomyces sp. DSS Pyrroloquinoline

alkaloids Cell cycle modulators

Arenamides (2009) Salinispora arenicola MS Depsipeptides NFB inhibitors

Caboxamycin (2009) Streptomyces sp. DSS Benzoxazole Antibacterial

Antitumour

Dermacozines (2009) Dermacoccus abyssi DSS Phenazines Antioxidant

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

Lucentamycins (2007) Nocardiopsis

lucentensis MS

Non-ribosomal

peptides Antitumour

Piperazimycins (2007) Streptomyces sp. MS Non-ribosomal

peptides Antitumour

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)

[atrop-abyssomicin C] (2007) Verrucosispora maris MS Polycyclic polyketide Antibacterial

Chandrananimycins (2003) Actinomadora sp. MS Phenoxazin Antitumour

Salinosporamide A (2003) Salinispora tropica MS Polyketide/non-

ribosomal peptide Antitumor

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

(B, C, D, E, G, H, I)

2004 – 2010

Complete chemical synthesis: discovery of

atrop-abyssomicin C

2005, 2006

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-

deoxychorismate synthase at Cys-263

2007

Patent filing: Germany, European Union, USA 2003

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

Novartis, Sanofi-Aventis 2004-2008

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

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

A natural product from a region of underexploited chemical space

?? 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:

Koki Horikoshi [Tokyo]

Doug Masson [Southampton]

Gjert Knutsen [Bergen]

Juan Asenjo [Santiago]

Luis Caceres V. [Antofagasta]

Microbiology/Molecular Biology:

Mike Goodfellow [Newcastle]

Jem Stach [Kent/Newcastle]

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