Novel Bioactive Molecules with BiopharmaceuticalNovel Bioactive Molecules with BiopharmaceuticalPotential from the Marine Environment Potential from the Marine Environment

(ERI Open Day October 10(ERI Open Day October 10thth, 2012), 2012)

Professor Alan Dobson,Professor Alan Dobson,Environmental Research Institute and Microbiology Department,Environmental Research Institute and Microbiology Department,

University College Cork University College Cork

• Mapping and genetic characterisation of Ireland’s marine biodiversity

• Sampling, extraction and identification of biochemical components

• Screening and culturing process for isolation of bioactive compounds

• Application of research results into generation of new biomaterials, compounds and agents

• Integrated data management system

• Educational, outreach and technology transfer

Beaufort Marine Biodiscovery Work Programme

Initial Capacity Building

• Beaufort Marine Research Award:

– funding of €7.2m over 7 years

– to NUIG, UCC and QUB.

• MI IRCSET funding

– to DCU and UCD

Implementation of Marine Biodiscovery Programme

UCC

DCU

QUB

NUIG

UCD

PI, 2 PD, 4 PhD

2 PD, 4 PhD

2 PD, 4 PhD

1 PhD

2 PhD

Novel Marine Bio-actives for Health Novel Marine Bio-actives for Health

Marine-origin Bioactives in the Market

Pharmaceuticals• UpJohn: Anti-cancer drug Cytosar-U® $1.36 billion in 2009

- sponge• PharmaMar: Anti-cancer drug Yondelis® €45 million in

2009 - sea squirt• Parkedale: Anti- herpes and anti- shingles drug Vidarabine

€27 million in 2008 - sponge• Azurpharma: Anti-neuropathic pain drug Prialt® (IE) $16.5

million for 2008/9 – snail

Marine bioactives market = $1.9bn by 2012 Pharmaceuticals, nutraceuticals, cosmetics, agri products

Driven by life sciences research – turning to marine for novelty

Marine Natural ProductsMarine Natural Products• Distribution of new natural products by phyla

From Blunt et al., NPR 2009

Marine SpongesMarine SpongesPoriferaPorifera

– Simple animals• No nervous system• No internal organs

– Sessile filter feeders• 1000 L/kg/hr

– Chemical defence system• Important source of new bioactive metabolites

Suberites carnosus

Axinella dissimilis Cliona celataSuberites ficus

• Galway Bay Haliclona simulans

• Lough Hyne Marine Nature Reserve 16 sponge species (33 samples)

Amphilectus fucorum, Axinella dissimilis, Axinella damicornisCliona celata, Dysidea fragilis, Eurypon major, Leucosolenia sp.

Pachymatisma johnstonia, Polymastia boletiformis, Raspailia hispida, Raspailia ramosa, Rhaphidostyla kitchingi, Suberites carnosus,

Suberites ficus, Stelligera rigida, Tethya citrina

Sponge samples for antimicrobial screens

Sources of SpongesSources of Sponges

Deep Sea2010 and 2011

Biodiscovery Cruises on Celtic Explorer

Kilkieran Bay, Galway

Haliclona simulans

Lough Hyne, West Cork

Marine Nature ReserveUCC Research

Laboratories

Culture dependent approach

Sponges

CulturingMarine isolates

Bioactive compounds

Pseudovibrio sp.W19

Example 1

Pseudovibrio species

• Axinella dissimilis

- 16S RNA gene analysis- Pseudovibrio species

• Genetic diverse determined• Novel Pseudovibrio species characterised

• Majority of strains anti-microbial activity- Gram negative bacteria (e.g. E. coli, S. typhimurium, Y. enterocolitica)

- Gram positive bacteria (e.g. S. aureus, C. difficile, L. monocytogenes)

(other)

Pseudovibrio species

Extraction

Antimicrobial activity of crude extracts of W19 vs

S. aureus, MRSA, and VISA

region extracted; Rf ≈ 0.72

Purification of Ad2 bioactive compound by preparative TLC

Preliminary NMR

scaling up

MALDI-TOF mass spec analysis

Ad2 bioactive compound (MW 234)

• Characterisation of bioactive compound

Culture dependent approach

Sponges

CulturingMarine isolates

Bioactive compounds

SporeformersBacillus sp.

Example 2

Bacillus species

• Majority of strains antimicrobial activity

– Range of Gram positive bacteria– Gram negative; only Enterobacter species

Characterisation of B. subtilis MMA7 • Broad spectrum of antimicrobial activity

– Listeria monocytogenes– Clostridium difficile

L. monocytogenes overlay

HPLC purified compound

1 2 3 1 2 3

De novo sequencingALPHALYSE, Denmark

Bacillus species

• Lytic effect of putative lantibiotic

• Whole genome sequencing– Gene cluster identified– Further characterisation

Example 3 Streptomyces Streptomyces SM2SM2

Strong bioactivity:

Bacillus subtilis

Staphylococcus aureus

MRSA

VISA

VRE

Clostridium difficile

Kennedy, et al., Mar.Biotech. 2009,

Fermentation• 14 days

• 1.6 L grown

Extraction• Amberlite XAD-16 resin

• Eluted with acetone, methanol, and ethyl acetate

• Concentrated by evaporation

Purification• silica gel flash chromatography

– Active fractions pooled– Crude extract MIC (B. subtilis)

• ~0.45mg/ml– Most active fraction MIC

• ~0.015mg/ml

Extraction of antibioticExtraction of antibiotic

Qualitative Assay – Disk Diffusion

Quantitative Assay

Analysis of active fractionAnalysis of active fractionHPLC

Single major UV active peak

UV Spectrum

Mass Spectrum of major peak

MS and NMR data indicates major product is a novel compound.Structure currently being evaluated.

Functional MetagenomicsFunctional Metagenomics

Screen for activity

Isolate sponge DNA

Construct library

Transform into E. coli

Functional Screens

Anti-bacterialAnti-fungal

Quorum sensing inhibitors

Calcineurin inhibitionPHA production

Antibiotic resistanceBiosurfactant

ProteaseLipase

EsterasePhosphatase

Metagenomic HitsMetagenomic Hits

Lipase

(tributyrin)Protease

(skimmed milk)Phosphatase

(X-phos)Biosurfactant(oil spray and emulsification)

Olive oil

Mineral oil

Clones analysed by activity profileEnd-sequenced to determine phylogenetic origin

Selected clones fully sequenced

New funding opportunities

EU FP7• Excellent international partners• Large consortium

Granted• MicroB3• PharmaSEA• MaCuMBA

National postdoctoral fellowship granted• IRCSET EMPOWER

Marine Biotechnology Centre (Dobson)Biomerit Research Centre (O’Gara)

SummarySummary

O

HN

OO

OH

O

OO

O

O

HO

H

• Culturable microbes• 1,500 bacterial and 85 fungal isolates• >700 bacterial strains from deep sea

sponges

• Diverse collection of sponges (33)

Microbial Ecology

16s rRNA analysis of>1,400 bacteria from 9 coastal >350 bacteria from 5 deep sea

sponges

Bioactivities

3 lead antimicrobials SM2, SM8, MMA7All have novel structures and good anti-

bacterial, anti-fungal activities (g.ml range)

Genomics

Sequenced genomes of Streptomyces strains SM2, SM8,

Bacillus subtilis MMA7

Biocatalysts

LipasesProteases

Phosphatases

1617