Marin Marin bioprospekteringbioprospektering i Trondheimsfjordeni Trondheimsfjorden

Sergey B. Sergey B. ZotchevZotchevInstituttInstitutt for for bioteknologibioteknologi, NTNU, NTNU

BioprospectingBioprospecting

”Bioprospecting" was first defined in 1993 as "the exploration of biodiversity for commercially valuable

genetic resources and biochemicals."

Marine bioprospecting in Trondheim: history

• Started in October 2003 at NTNU & SINTEF (2 projects), funded by Norges Forskningsråd.

• 2003-2005: sample collection (sediments, sponges, water surface) and development of cultivation, screening and analytical techniques.

• 2006: identification of ”hits” in anti-bacterial, anti-fungal and anti-cancer screens.

• 2007-2008: identification of potential anti-bacterial and anti-cancer ”lead” compounds. Structure elucidation for 2 compounds.

• 2010: Initiation of genome-based bioprospecting.

TopTop 10 10 causescauses ofof deathdeath: : WHO data for 2010 WHO data for 2010

1,208,62910Road Traffic Accidents1,255,4779Diabetes Mellitus1,341,7688Tuberculosis1,387,1217Lung Cancers1,776,2706HIV/AIDS2,464,4255Diarrhoeal diseases3,278,1674Lung Disease3,463,2563Influenza & Pneumonia6,151,1542Stroke7,253,8241Coronary Heart Disease

> 12.200.000 peoiple died in 2010 of infectious diseases

Antibiotic: what is it?Antibiotic: what is it?

“… a chemical substance derived from microorganisms which has the capacity of inhibiting growth, and even destroying, other microorganisms in dilute solutions”

Selman A. Waksman (1888-1973)

The The goodgood old old daysdays......

......areare overover

Antibiotic resistance: a BIG problem!Antibiotic resistance: a BIG problem!

Why did antibiotic developmentWhy did antibiotic developmentby Big by Big PharmaPharma virtually stopped? virtually stopped?

•• ””LetLet’’ss gogo ””blockbusterblockbuster!!”” – concentrating on more profitable chronic disease drugs;

•• ””ForgetForget bioprospectingbioprospecting!!”” – abandonment of searchfor new antibiotics from natural sources – too costly, few novel compounds;

•• ””ResistanceResistance? ? WhatWhat resistanceresistance??”” – under-appreciation of the emerging resistance of pathogensto existing antibiotics.

Antibiotics: good or bad?Antibiotics: good or bad?

• Anti-infective drugs

• Immunosuppressants

• Antitumor agents

• Antiparasitic drugs

• Food preservatives

• Herbicides

• Side effects

• Emergence of

resistant

pathogens

Why new antibiotics?Why new antibiotics?

• Overcoming resistance in pathogens and cancer cells

• Reduction/elimination of side effects

• New properties

New antibiotics: how?New antibiotics: how?

•• Natural sourcesNatural sources

•• Chemical modificationsChemical modifications

•• Genetic engineering of Genetic engineering of producing organismsproducing organisms

MicrobialMicrobial bioprospectingbioprospecting: : searchsearchfor new for new antibioticsantibiotics

• Many new antibiotics can be found in nature: certain bacteria are known to be excellent antibiotic producers;

• At NTNU/SINTEF isolated bacteria from Trondheim fjord capable of producing antibiotics

Actinomycete bacteriaActinomycete bacteria

The The processprocessSampling of sediments, marine animals, and upperSampling of sediments, marine animals, and upper

water layer in the Trondheim fjordwater layer in the Trondheim fjord

Sample preSample pre--treatment and plating on thetreatment and plating on theselective agar mediaselective agar media

Selection of actinomycetes and preparation Selection of actinomycetes and preparation of master platesof master plates

Replication of master plates to Replication of master plates to ““productionproduction””agar mediaagar media

Preparation of extracts from production media, Preparation of extracts from production media, and testing for antibiotic activityand testing for antibiotic activity

WhatWhat’’ss nextnext??

• Selection of “hits”;• Up-scaling – production of active

compounds and their purification;• Structure determination by NMR;• Comprehensive in vitro activity &

toxicity testing;• Selection of “leads”;• IP protection.

BioprospectingBioprospecting: : wayway to marketto market

Sampling,isolation of

bacteria

Screening, ID,production,purification

Structureelucidation,

initial testing

IPprotection

Genetics

Chemistry

Pre-clinicaltesting

Clinicaltrials ApprovalApproval

New New spongesponge--associatedassociated bacteriabacteria from from thetheTrondheimsfjordTrondheimsfjord

SeaSea waterwater--dependentdependentactinomycetesactinomycetes from from spongessponges

Sea water (+) Sea water (-)

Establishment of screening Establishment of screening protocolsprotocols• Selection of test organisms (Gram-positive, Gram-negative

bacteria and fungi);• Development of production media;• Development of extraction protocols;• Assay development.

OverlayOverlaytechniquetechnique Glucose Glucose

consumptionconsumptiontechniquetechnique

““MicroDropMicroDrop””techniquetechnique

OverlayOverlaytechniquetechnique Glucose Glucose

consumptionconsumptiontechniquetechnique

““MicroDropMicroDrop””techniquetechnique

SampleSample fractionationfractionation and and characterisationcharacterisation

0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5 5.5-6.5 min

Bioassay

HPLC

LC-MS

0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5 5.5-6.5 min

Bioassay

HPLC

LC-MS

NewNewAntiAnti--bacterialbacterial??ActinoalloteichusActinoalloteichus sp.sp.

NewNewAntiAnti--bacterialbacterialPolyketidePolyketideStreptomycesStreptomyces sp.sp.

NewNewAntiAnti--bacterialbacterialThiopeptideThiopeptideNocardiopsisNocardiopsis sp. sp.

NewNewAntiAnti--fungalfungalPolyenePolyene macrolidemacrolideStreptomycesStreptomyces sp.sp.

NewNewCytotoxicCytotoxicMacrolactamMacrolactamStreptomycesStreptomyces sp.sp.

ValinomycinValinomycinAntiAnti--bacterialbacterialCyclodepsipeptideCyclodepsipeptideStreptomycesStreptomyces sp.sp.

BEBE--1410614106CytotoxicCytotoxicMacrolactamMacrolactamStreptomycesStreptomyces sp.sp.

IodininIodininCytotoxicCytotoxicPhenazinePhenazineStreptosporangiumStreptosporangium sp.sp.

ActinomycinActinomycin DDAntiAnti--bacterialbacterial, , cytotoxiccytotoxicPolypeptidePolypeptideStreptomycesStreptomyces sp.sp.

NewNewAntiAnti--bacterialbacterial??ActinomaduraActinomadura sp. sp.

PyrrolomycinsPyrrolomycinsAntiAnti--bacterialbacterialPolyketidePolyketide--pyrrolepyrroleStreptomycesStreptomyces sp.sp.

CandicidinCandicidin DDAntiAnti--fungalfungalPolyenePolyene macrolidemacrolideStreptomycesStreptomyces sp.sp.

IdentificationIdentificationBiologicalBiologicalactivityactivityChemical Chemical classclassProducingProducing organismorganism

WhatWhat have have wewe foundfound??

StructuresStructures ofof newnew moleculesmolecules from from thetheTrondheim fjord Trondheim fjord bacteriabacteria

TPTP--11611161

AntibacterialAntibacterialthiopeptidethiopeptide

MLML--449449

CytotoxicCytotoxicmacrolactammacrolactam

N

O

OHOH

HiddenHidden genomegenome treasurestreasures

2 x 2 x AntiAnti--bacterialbacterial activityactivity

GeodiaGeodia barrettibarretti

ActinoalloteichusActinoalloteichus sp.sp.

GenomeGenome sequencingsequencing

NocardicinNocardicin ((monocyclicmonocyclic --lactamlactam, , antianti--bacterialbacterial))HalogenatedHalogenated polyketidepolyketide 1 (1 (antianti--bacterialbacterial))HalogenatedHalogenated polyketidepolyketide 2 (2 (antianti--bacterialbacterial?)?)AromaticAromatic poyketidepoyketide 1 (anti1 (anti--cancer?)cancer?)AromaticAromatic polyketidepolyketide 2 (2 (antianti--cancer/anticancer/anti--bacterialbacterial?)?)GlycosylatedGlycosylated macrolidemacrolide ((antianti--bacterialbacterial?)?)GlycosylatedGlycosylated aromaticaromatic polyketidepolyketide (anti(anti--cancer?)cancer?)PolyenePolyene macrolidemacrolide ((antianti--fungalfungal))EnediyneEnediyne (anti(anti--cancer)cancer)LantibioticLantibiotic ((antianti--bacterialbacterial))IsoprenoidIsoprenoid (?)(?)Hybrid Hybrid polyketide/peptidepolyketide/peptide (?)(?)

Zotchev et al (2012) Curr Opin Biotechnol 23:941-947

GenomeGenome--basedbased bioprospectingbioprospecting

•• NTNUNTNU (Norway): H. Jørgensen, K. Engelhardt, O. Sekurova, E. Fjærvik, H. Bredholt, G. Johnsen, S. Hakvåg, E. Ian

•• SINTEFSINTEF ((NorwayNorway)): H. Sletta, G. Klinkenberg, K. F. Degnes, T.E. Ellingsen

•• University University ofof BergenBergen: L. Herfindal, H.T. Rapp, S.O. Døskeland

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

•• BioFocusBioFocus (Switzerland): M. Kemmler

AcknowledgementsAcknowledgements

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