NewNew DrugsDrugs fromfrom MarineMarine BacteriaBacteria Sergey B. Zotchev, NTNU/Biosergen AS August 7, 2009 OutlineOutline • Bioprospecting for new drugs in the Trondheim fjord • Biosynthetic engineering at Biosergen AS Bioprospecting for antibiotics: what’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 fjord Sampling sponges in the Trondheim fjord Total: 960 isolates Actinobacteria from sponges TSI 123-18 TSI 128-18 TSI 124-18 63 TSI 129-2 TSI 118-17 TSI 122-5 100 TSI 125-23 Micromonospora matsumotoense IMSNU 22... 48 TSI 125-1 53 TSI 115-7 TSI 121-5 Micromonospora 37 82 TSI 117-18 TSI 117-17 52 46 Micromonospora sp. lupac 09 77 45 TSI 117-5 Micromonospora carbonacea DSM 43815 100 TSI 114-4 Micromonospora auratinigra TT1-11 91 TSI 121-19 51 98 TSI 129-13 66 Pseudonocardia petroleophila IMSNU 22... 100 TSI 115-15 Pseudonocardia TSI 116-13 100 Rhodococcus opacus B-4 68 TSI 124-19 Rhodococcus 41 Actinoalloteichus spitiensis MTCC 6194 TSI 127-17 100 TSI 115-14 Actinoalloteichus 100 Actinoalloteichus 87 TSI 129-23 64 TSI 116-3 15 65 TSI 124-17 16 Streptosporangium sp. 14363 27 TSI 129-5 90 TSI 124-2 100 Streptosporangium TSI 115-20 Streptosporangium amethystogenes DSM ... 100 98 TSI 129-12 Sponges: Nonomuraea kuesteri GW 14-1925T 99 Nonomuraea 99 Nonomuraea TSI 116-8 Nocardiopsis sp. YIM 80031 100 TSI 112-9 Nocardiopsis 100 TSI 112-24 Geodia barretti 34 59 TSI 119-9 98 Streptomyces ramulosus NRRL B-2714 TSI 113-5 Streptomyces Isops phlegraei 100 TSI 120-18 100 TSI 117-4 Streptomycetaceae bacterium CNR530 Mycale lingua 73 100 TSI 112-12 86 TSI 116-4 39 30 TSI 113-17 Phakellia ventilabrum Streptomyces sp. Fiji-204 Streptomyces (marine) 61 TSI 115-17 TSI 112-13 52 Antho dichotoma TSI 112-10 Antho dichotoma 100 53 TSI 112-23 92 TSI 115-22 99 TSI 105-24 Isoptericola sp. TUT1258 Isoptericola Depth: 60-121 m 100 Promicromonospora sp. YIM C653 TSI 106-17 100 TSI 98-01 98 Promicromonospora 76 TSI 105-21 Bifidobacterium bifidum DSM 20456T 0.02 Compounds identified: some examples Biological Producing organism Chemical class Identification activity Anti-bacterial, Streptomyces sp. Polypeptide Actinomycin D cytotoxic Streptomyces sp. Polyene macrolide Anti-fungal Candicidin D Streptomyces sp. Polyketide-pyrrole Anti-bacterial Pyrrolomycins Streptomyces sp. Macrolactam Cytotoxic BE-14106 Streptomyces sp. Cyclodepsipeptide Anti-bacterial Valinomycin Streptosporangium sp. Phenazine Cytotoxic Iodinin Streptomyces sp. Polyketide Anti-bacterial New Streptomyces sp. Polyene macrolide Anti-fungal New Streptomyces sp. Macrolactam Cytotoxic New Nocardiopsis sp. Thiopeptide Anti-bacterial New Actinoalloteichus sp. ? Anti-bacterial New Actinomadura sp. ? Anti-bacterial New Structures of the new molecules from marine bacteria O N OH HO TP-1161 ML-449 Antibacterial Cytotoxic thiopeptide macrolactam Hidden genome treasures Geodia barretti Anti-bacterial activity Actinoalloteichus sp. (halogenated polyketide) Genome sequencing Nocardicin (monocyclic β-lactam, anti-bacterial) Halogenated polyketide 1 (anti-bacterial) Halogenated polyketide 2 (anti-bacterial?) Aromatic poyketide 1 (anti-cancer?) Aromatic polyketide 2 (anti-cancer/anti-bacterial?) Glycosylated macrolide (anti-bacterial?) Glycosylated aromatic polyketide (anti-cancer?) Polyene macrolide (anti-fungal) Enediyne (anti-cancer) Lantibiotic (anti-bacterial) Isoprenoid (?) Hybrid polyketide/peptide (?) WhatWhat dodo wewe havehave soso far?far? •> 10.000 actinomycete isolates (neuston layer, sediments and sponges) • Technology established: from cultivation to identification/isolation of active compounds • Identified >100 anti-bacterial and >30 anti-fungal ”hits” (currently under characterization) • 29 anti-cancer ”hits” (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 anti-fungal polyene macrolide Streptomyces noursei OH OH H3C O OH HO O OH OH OH OH O CH3 COOH CH3 CH O O 3 Elizabeth Hazen & Rachel F. Brown Nystatin A1 OH OH NH2 Toxic. Used for treatment of superficial fungal infections only Nystatin biosynthesis in Streptomyces noursei Fjærvik & Zotchev, 2005 Engineered biosynthesis of a heptaene nystatin analogue Module 5 Module 5 ER ER DH KR DH KR OH OH OH OH H3C O OH H3C O OH HO O OH OH OH OH O HO O OH OH OH OH O CH3 COOH CH3 COOH 29 29 CH3 28 CH3 28 CH CH O O 3 O O 3 Nystatin OH S44HP OH OH NH2 OH NH2 MIC50 =0.45µg/ml MIC50 = 0.075 µg/ml Activity increased ca 6-fold Bruheim et al., 2004 • Established – December 2004 • Shareholders – 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) HowHow dodo wewe makemake newnew nystatinnystatin analogues?analogues? • Molecular design according to current knowledge on structure-activity relationship; • Genetic design in accordance with nystatin biosynthetic pathway; • Genetic manipulation of the bacterium, verification of the mutation; • Fermentation of the genetically manipulated bacterium, identification of desired product, and its purification; • Chemical modifications of genetically engineered analogues. Engineered antibiotic analogues OH OH H3C O OH HO O OH OH OH OH O CH3 COOH 29 CH3 28 CH O O 3 S44HP OH OH NH2 Genetics Chemistry 15 analogues 49 analogues & salts in vitro & in vivo evaluation, analysis of SAR data 6 pre-CD candidates Pre-CD study package Candidate drug Now here 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 AcknowledgementsAcknowledgements • NTNU: 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 • SINTEF: H. Sletta, T. Brautaset, G. Klinkenberg, K. Degnes, K. Josefsen, K. Zahlsen, R. Aune, T.E. Ellingsen • Biosergen AS: I. Bakke, O. Sekurova, O. Volokhan • University of Bergen: L. Herfindal, H.T. Rapp, S.O. Døskeland • Gause Institute (Russia):(Russia) L. Terekhova, E. Olsufyeva, M. Preobrazhenskaya • ChemDiv, Inc. (USA/Russia):(USA/Russia) D. Tyrsin, V. Kazey • Biofocus DPI (Switzerland):(Switzerland) M. Kemmler Financial support: Research Council of Norway, Sinvent AS, Biosergen AS, NTNU, UiB.