New Drugs from Marine BacteriaNew Drugs from Marine Bacteria

Sergey B. Zotchev, NTNU/Biosergen ASSergey B. Zotchev, NTNU/Biosergen AS

August 7, 2009August 7, 2009

OutlineOutline

• Bioprospecting for new drugs in the Trondheim fjord

• Biosynthetic engineering at Biosergen AS

Bioprospecting for antibiotics: whatBioprospecting for antibiotics: what’’s important? s important?

• Where to look – search for unique ecological niches;

• How to look – establishment of cultivation techniques;

• How to reveal the potential – choice of production media and conditions;

• How to assess activity – assay development;

• How to assess novelty – development of analytical techniques;

• How to reveal mechanism of action – use of molecular biology;

• How to evaluate usefulness – in vitro and in vivo studies.

Trondheim fjordTrondheim fjord

Sampling sponges in the Trondheim fjordSampling sponges in the Trondheim fjord

Total: 960 isolatesTotal: 960 isolates

Actinobacteria from sponges Actinobacteria from sponges

Sponges:Sponges:

Geodia barrettiGeodia barrettiIsops phlegraeiIsops phlegraeiMycale linguaMycale linguaPhakellia ventilabrumPhakellia ventilabrumAntho dichotomaAntho dichotoma

DepthDepth: 60: 60--121 m121 m

MicromonosporaMicromonospora

RhodococcusRhodococcusPseudonocardiaPseudonocardia

ActinoalloteichusActinoalloteichus

StreptomycesStreptomyces

NonomuraeaNonomuraeaNocardiopsisNocardiopsis

StreptosporangiumStreptosporangium

IsoptericolaIsoptericolaPromicromonosporaPromicromonospora

TSI 123-18

TSI 128-18

TSI 124-18

TSI 129-2

TSI 118-17

TSI 122-5

TSI 125-23

M icromonospora matsumotoense IM SNU 22...

TSI 125-1

TSI 115-7

TSI 121-5

TSI 117-18

TSI 117-17

M icromonospora sp. lupac 09

TSI 117-5

M icromonospora carbonacea DSM 43815

TSI 114-4

M icromonospora auratinigra TT1-11

TSI 121-19

TSI 129-13

Pseudonocardia petroleophila IM SNU 22...

TSI 115-15

TSI 116-13

Rhodococcus opacus B-4

TSI 124-19

Actinoalloteichus spitiensis M TCC 6194

TSI 127-17

TSI 115-14

TSI 129-23

TSI 116-3

TSI 124-17

Streptosporangium sp. 14363

TSI 129-5

TSI 124-2

TSI 115-20

Streptosporangium amethystogenes DSM ...

TSI 129-12

Nonomuraea kuesteri GW 14-1925T

TSI 116-8

Nocardiopsis sp. YIM 80031

TSI 112-9

TSI 112-24

TSI 119-9

Streptomyces ramulosus NRRL B-2714

TSI 113-5

TSI 120-18

TSI 117-4

Streptomycetaceae bacterium CNR530

TSI 112-12

TSI 116-4

TSI 113-17

Streptomyces sp. Fiji-204

TSI 115-17

TSI 112-13

TSI 112-10

TSI 112-23

TSI 115-22

TSI 105-24

Isoptericola sp. TUT1258

Promicromonospora sp. YIM C653

TSI 106-17

TSI 98-01

TSI 105-21

Bifidobacterium bifidum DSM 20456T

1 0 0

7 6

9 8

1 0 0

6 6

1 0 0

9 9

1 0 0

9 9

8 7

1 0 0

6 4

9 8

1 5

1 6

2 7

9 0

1 0 0

9 2

5 3

1 0 0

1 0 0

1 0 0

1 0 0

8 6

5 9

5 2

9 8

3 0

6 1

3 9

7 3

1 0 0

1 0 0

1 0 0

1 0 0

9 9

3 4

4 1

6 8

6 5

5 1 9 8

9 1

1 0 0

4 5

4 6

5 3

5 2

8 2

7 7

3 7

4 8

1 0 0

6 3

0 .0 2

Streptomyces Streptomyces (marine)(marine)

NewNewAntiAnti--bacterialbacterial??ActinoalloteichusActinoalloteichus sp.sp.

NewNewAntiAnti--bacterialbacterialPolyketidePolyketideStreptomycesStreptomyces sp.sp.

NewNewAntiAnti--bacterialbacterialThiopeptideThiopeptideNocardiopsisNocardiopsis sp. sp.

NewNewAntiAnti--fungalfungalPolyene macrolidePolyene macrolideStreptomycesStreptomyces sp.sp.

NewNewCytotoxicCytotoxicMacrolactamMacrolactamStreptomycesStreptomyces sp.sp.

ValinomycinValinomycinAntiAnti--bacterialbacterialCyclodepsipeptideCyclodepsipeptideStreptomycesStreptomyces sp.sp.

BEBE--1410614106CytotoxicCytotoxicMacrolactamMacrolactamStreptomycesStreptomyces sp.sp.

IodininIodininCytotoxicCytotoxicPhenazinePhenazineStreptosporangiumStreptosporangium sp.sp.

Actinomycin DActinomycin DAntiAnti--bacterial, bacterial, cytotoxiccytotoxicPolypeptidePolypeptideStreptomycesStreptomyces sp.sp.

NewNewAntiAnti--bacterialbacterial??ActinomaduraActinomadura sp. sp.

PyrrolomycinsPyrrolomycinsAntiAnti--bacterialbacterialPolyketidePolyketide--pyrrolepyrroleStreptomycesStreptomyces sp.sp.

Candicidin DCandicidin DAntiAnti--fungalfungalPolyene macrolidePolyene macrolideStreptomycesStreptomyces sp.sp.

IdentificationIdentificationBiological Biological activityactivityChemical classChemical classProducing organismProducing organism

Compounds identified: some examplesCompounds identified: some examples

Structures of the new molecules fromStructures of the new molecules frommarine bacteriamarine bacteria

TPTP--11611161

Antibacterial Antibacterial thiopeptidethiopeptide

MLML--449449

Cytotoxic Cytotoxic macrolactammacrolactam

N

O

OHOH

Hidden genome treasuresHidden genome treasures

AntiAnti--bacterial activitybacterial activity(halogenated polyketide)(halogenated polyketide)

Geodia barrettiGeodia barretti

Actinoalloteichus sp.Actinoalloteichus sp.

Genome sequencingGenome sequencing

Nocardicin (monocyclic Nocardicin (monocyclic ββ--lactam, antilactam, anti--bacterial)bacterial)Halogenated polyketide 1 (antiHalogenated polyketide 1 (anti--bacterial)bacterial)Halogenated polyketide 2 (antiHalogenated polyketide 2 (anti--bacterial?)bacterial?)Aromatic poyketide 1 (antiAromatic poyketide 1 (anti--cancer?)cancer?)Aromatic polyketide 2 (antiAromatic polyketide 2 (anti--cancer/anticancer/anti--bacterial?)bacterial?)Glycosylated macrolide (antiGlycosylated macrolide (anti--bacterial?)bacterial?)Glycosylated aromatic polyketide (antiGlycosylated aromatic polyketide (anti--cancer?)cancer?)Polyene macrolide (antiPolyene macrolide (anti--fungal)fungal)Enediyne (antiEnediyne (anti--cancer)cancer)Lantibiotic (antiLantibiotic (anti--bacterial)bacterial)Isoprenoid (?)Isoprenoid (?)Hybrid polyketide/peptide (?)Hybrid polyketide/peptide (?)

What do we have so far?What do we have so far?

• > 10.000 actinomycete isolates10.000 actinomycete isolates (neuston layer, sediments and sponges)

•• Technology establishedTechnology established: from cultivation to identification/isolation of active compounds

• Identified >100 anti>100 anti--bacterial and >30 antibacterial and >30 anti--fungal fungal ””hitshits”” (currently under characterization)

•• 29 anti29 anti--cancer cancer ””hitshits”” (currently under characterization)

• Identified at least 6 new antibiotics – 4 anti-bacterial, 1 anti-fungal, 1 cytotoxic

• New opportunities for drug discovery through draft genome sequencing

• Unique antibiotic biosynthesis gene clusters identifed/cloned

Nystatin: an antiNystatin: an anti--fungal polyene fungal polyene macrolidemacrolide

Elizabeth Hazen & Rachel F. BrownElizabeth Hazen & Rachel F. Brown

Streptomyces nourseiStreptomyces noursei

O

OOH

CH3

OH OHO OH

OH

OH

OH

O

COOHCH3

CH3

O

OH NH2

OHCH3

OH

Nystatin A1

Toxic. Used for treatment of superficial fungal infections onlyToxic. Used for treatment of superficial fungal infections only

Nystatin biosynthesis in Nystatin biosynthesis in Streptomyces nourseiStreptomyces noursei

Fjærvik & Zotchev, 2005

Engineered biosynthesis of a heptaene nystatinEngineered biosynthesis of a heptaene nystatin analogueanalogue

O

OOH

CH3

OH OHO OH

OH

OH

OH

O

COOHCH3

CH3

O

OH NH2

OHCH3

OH

28

29

Nystatin

Bruheim et al., 2004

DH

ModuleModule 55

KRER

MIC50 = 0.45 µg/ml

DH

ModuleModule 55

KRER

MIC50 = 0.075 µg/ml

O

OOH

CH3

OH OHO OH

OH

OH

OH

O

COOHCH3

CH3

O

OH NH2

OHCH3

OH

28

29

S44HP

Activity increasedActivity increased ca 6ca 6--foldfold

• Established – December 2004

•• ShareholdersShareholders –– SINTEF Venture AS (Norway), Verdane Capital AS (Norway), Karolinska Development AB (Sweden), OBS Østersjøstiftelsen (Sweden)

• Mission – development of a safer and more efficient drug against systemic fungal infections

• Technology – manipulation of the nystatin biosynthetic genes + optional chemical modifications

• Partners – SINTEF, NTNU, Gause Institute of New Antibiotics (Russia), Biovian (Finland), Visionar (Sweden)

How do we make new nystatin analogues?How do we make new nystatin analogues?

•• Molecular design according to current knowledge on structureMolecular design according to current knowledge on structure--activity activity relationship;relationship;

•• Genetic design in accordance with nystatin biosynthetic pathway;Genetic design in accordance with nystatin biosynthetic pathway;

•• Genetic manipulation of the bacterium, verification of the mutatGenetic manipulation of the bacterium, verification of the mutation;ion;

•• Fermentation of the genetically manipulated bacterium, identificFermentation of the genetically manipulated bacterium, identification of ation of desired product, and its purification;desired product, and its purification;

•• Chemical modifications of genetically engineered analogues.Chemical modifications of genetically engineered analogues.

Engineered antibioticEngineered antibiotic analoguesanaloguesO

OOH

CH3

OH OHO OH

OH

OH

OH

O

COOHCH3

CH3

O

OH NH2

OHCH3

OH

28

29

S44HP

GeneticsGenetics ChemistryChemistry

15 analogues15 analogues 49 analogues & salts49 analogues & salts

in vitroin vitro & & in vivoin vivo evaluation,evaluation,analysis of SAR dataanalysis of SAR data

6 pre6 pre--CD candidatesCD candidates

Candidate drugCandidate drug

PrePre--CD study packageCD study package

Now hereNow here

Bioprospecting + Biosergen AS? Bioprospecting + Biosergen AS?

• Large collection of actinobacteria, many belonging to rare species

• Large pool of unique antibiotic biosynthesis genes: at least 15 gene clusters for biosynthesis of potentially novel antibiotics identified through genome sequencing

• Two novel gene clusters for biosynthesis of cytotoxic antibiotics cloned, sequenced and annotated (one forms a basis for a new anti-cancer project at Biosergen)

• Possibility to expand project portfolio: biosynthetic engineering of new anti-cancer and anti-bacterial antibiotics

•• NTNUNTNU: S.E.F. Borgos, A. Nedal, E. Fjærvik, P. Bruheim, H. Bredholt, G. Johnsen, H. Jørgensen, S. Hakvåg, K. Engelhardt, E. Ian, S. Valla, A.R. Strøm

•• SINTEFSINTEF: H. Sletta, T. Brautaset, G. Klinkenberg, K. Degnes, K. Josefsen, K. Zahlsen, R. Aune, T.E. Ellingsen

•• Biosergen ASBiosergen AS: I. Bakke, O. Sekurova, O. Volokhan

•• University of BergenUniversity of Bergen: L. Herfindal, H.T. Rapp, S.O. Døskeland

•• Gause InstituteGause Institute (Russia)(Russia): L. Terekhova, E. Olsufyeva, M. Preobrazhenskaya

•• ChemDiv, IncChemDiv, Inc. . (USA/Russia)(USA/Russia): D. Tyrsin, V. Kazey

•• Biofocus DPIBiofocus DPI (Switzerland)(Switzerland): M. Kemmler

AcknowledgementsAcknowledgements

Financial supportFinancial support: Research Council of Norway, Sinvent AS, Biosergen AS, NTNU, UiB