Novel Bioactive Molecules with Biopharmaceutical Potential from the Marine Environment (ERI Open Day October 10th, 2012) Professor Alan Dobson, Environmental Research Institute and Microbiology Department, University College Cork Beaufort Marine Biodiscovery Work Programme • • • • • • 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 Implementation of Marine Biodiscovery Programme QUB 2 PD, 4 PhD Initial Capacity Building • Beaufort Marine Research Award: – funding of €7.2m over 7 years PI, 2 PD, 4 PhD NUIG – to NUIG, UCC and QUB. DCU 2 PhD UCD 1 PhD • MI IRCSET funding – to DCU and UCD 2 PD, 4 PhD UCC Novel Marine Bio-actives for Health Marine bioactives market = $1.9bn by 2012 Pharmaceuticals, nutraceuticals, cosmetics, agri products Driven by life sciences research – turning to marine for novelty 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 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 Suberites carnosus • Important source of new bioactive metabolites Axinella dissimilis Suberites ficus Cliona celata Sponge samples for antimicrobial screens • 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 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 Example 1 Sponges Culturing Marine isolates Pseudovibrio sp. W19 Bioactive compounds 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 • Characterisation of bioactive compound region extracted; Rf ≈ 0.72 MALDI-TOF mass spec analysis Extraction Purification of Ad2 bioactive compound by preparative TLC Antimicrobial activity of crude extracts of W19 vs S. aureus, MRSA, and VISA Ad2 bioactive compound (MW 234) Preliminary NMR scaling up Culture dependent approach Example 2 Sporeformers Bacillus sp. Sponges Culturing Marine isolates Bioactive compounds 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 kDa A 32 B 14 7.6 3.4 1 2 3 1 2 3 HPLC purified compound De novo sequencing L. monocytogenes overlay ALPHALYSE, Denmark Bacillus species • Lytic effect of putative lantibiotic • Whole genome sequencing – Gene cluster identified – Further characterisation Example 3 Streptomyces SM2 Strong bioactivity: Bacillus subtilis Staphylococcus aureus MRSA VISA VRE Clostridium difficile Kennedy, et al., Mar. Biotech. 2009, Extraction of antibiotic Fermentation • 14 days • 1.6 L grown Extraction • Amberlite XAD-16 resin • Eluted with acetone, methanol, and ethyl acetate • Concentrated by evaporation Qualitative Assay – Disk Diffusion Purification • silica gel flash chromatography – Active fractions pooled – Crude extract MIC (B. subtilis) • ~0.45mg/ml – Most active fraction MIC • ~0.015mg/ml 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 Isolate sponge DNA Transform into E. coli Functional Screens Construct library Screen for activity 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 Mineral oil 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 • Diverse collection of sponges (33) • Culturable microbes 17 16 • • 1,500 bacterial and 85 fungal isolates >700 bacterial strains from deep sea sponges Bioactivities Microbial Ecology Genomics 3 lead antimicrobials SM2, SM8, MMA7 All have novel structures and good antibacterial, anti-fungal activities (mg.ml range) Biocatalysts 16s rRNA analysis of >1,400 bacteria from 9 coastal >350 bacteria from 5 deep sea sponges Sequenced genomes of Streptomyces strains SM2, SM8, Bacillus subtilis MMA7 Lipases Proteases Phosphatases