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
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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
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Axinella dissimilis
- 16S RNA gene analysis
- Pseudovibrio species
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Genetic diverse determined
Novel Pseudovibrio species characterised
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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
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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
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Lytic effect of putative lantibiotic
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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
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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