Novel Bioactive Molecules with Biopharmaceutical Potential from the Marine Environment (ERI Open Day October 10 th, 2012) Professor Alan Dobson, Environmental Research Institute and Microbiology Department, 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 1 PhD 2 PhD

Novel Marine Bio-actives for Health Marine-origin Bioactives in the Market Pharmaceuticals UpJohn: Anti-cancer drug Cytosar-U® $1.36 billion in sponge PharmaMar: Anti-cancer drug Yondelis® €45 million in sea squirt Parkedale: Anti- herpes and anti- shingles drug Vidarabine €27 million in 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 Products Distribution of new natural products by phyla From Blunt et al., NPR 2009

Marine Sponges Porifera –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 damicornis Cliona 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 Sponges Deep Sea 2010 and 2011 Biodiscovery Cruises on Celtic Explorer Kilkieran Bay, Galway Haliclona simulans Lough Hyne, West Cork Marine Nature Reserve UCC Research Laboratories

Culture dependent approach Sponges Culturing Marine 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; R f ≈ 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 Culturing Marine isolates Bioactive compounds Sporeformers Bacillus 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 kDa A B De novo sequencing ALPHALYSE, Denmark

Bacillus species Lytic effect of putative lantibiotic Whole genome sequencing –Gene cluster identified –Further characterisation

Streptomyces SM2 Example 3 Streptomyces SM2 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 antibiotic Qualitative Assay – Disk Diffusion Quantitative Assay

Analysis of active fraction HPLC 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 Metagenomics Screen for activity Isolate sponge DNA Construct library Transform into E. coli Functional Screens Anti-bacterial Anti-fungal Quorum sensing inhibitors Calcineurin inhibition PHA production Antibiotic resistance Biosurfactant Protease Lipase Esterase Phosphatase

Metagenomic Hits Lipase (tributyrin) Protease (skimmed milk) Phosphatase (X-phos) Biosurfactant (oil spray and emulsification) Olive oil Mineral oil Clones analysed by activity profile End-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)

Summary 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, MMA7 All 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 Lipases Proteases Phosphatases 16 17