Marine Bioprospecting for Pharmaceuticals Shigeaki Harayama Department of Biological Sciences Faculty of Science Chuo University
Rise and decline of natural products in drug discovery ~ 1995 1995 2000 2005 2010 2010 ~ •HTS HTS (Robotization Robotization) •Combinatory Combinatory chemistry • Ultra-HTS: Ultra HTS: Natural product screening in big pharma closedz • No lead from chemical screening ? • Renaissance of natural products screening?
Why natural products not popular? • Product development speed slower than synthetic approaches • Low reproducibility: gene expression is strongly influenced by environmental factors, genes for secondary metabolites often unstable (deleted?) • Purification and identification of active compounds are required as extracts contain many molecules • Low productivity: chemical synthesis difficult for most of natural products, and no firm strategy for overproduction of active compounds • Difficult accesses to biodiversity due to the Convention on Biological Diversity
Combinatorial compounds Natural products Drugs Why combinatorial libraries failed to generate preclinical candidates? Chemical spaces explored explo ed by natural products and drugs overlap each other while that explored by combinatorial compounds is restricted Feher, Feher, M. & Schmidt, J. M. Property distributions: differences between drugs, natural products and molecules from combinatorial chemistry. J. Chem. Inf. Comput. Comput. Sci. 43: 218–227 218 227 (2003).
Is a return to natural products for screening warranted? • For the revival of natural product boom, all the drawbacks in natural product drug discovery programs should be improved • A way to improve traditional drug discovery process (phenotypic screening followed by bioassay-guided bioassay guided fractionation of natural product extracts) will be shown in the next slide
Necessary improvements In the shorter term • Access to better bioresources • Development at a faster rate: more sophisticated identification/dereplication identification/ dereplication of natural products In the longer term • Stable supplies of natural products in large quantities • Genetic studies to elucidate (i) mechanisms on the genetic instability of natural product biosynthesis; and (ii) regulatory circuits operating on secondary metabolite genes
Access to biodiversity: are marine bioresources better than terrestrial ones? The total number of bacterial species in marine environments (= sea surface) was estimated to be less than 10 6 species which is much smaller than that in terrestrial environment (higher than 10 9 species) 1. Hagström Hagstr et al. (2002) Use of 16S ribosomal DNA for delineation of marine bacterioplankton species. Appl. Environ. Microbiol. Microbiol. 68: 3628–3633 3628 3633 2. Curtis, T.P. et al. (2002) Estimating prokaryotic diversity and its limits. Proc. Natl. Acad. Sci. U. S. A. 99:10494–10499 99:10494 10499 3. Gans J, Wolinsky M, Dunbar J. (2005) Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science. 309: 1387-1390. 1387 1390.
Complex marine habitats support great biodiversity • Secondary metabolites seem to be abundant in certain niches of marine environments • For example, the ability of sponges to harbor a diverse assemblage of microorganisms producing diverse biologically active compounds is well documented • Some invertebrate-derived invertebrate derived natural products are structurally related to bacterial metabolites suggesting a microbial origin for some of these compounds
Screening of marine bacteria at Marine Biotechnology Institute (MBI) • More than 30,000 bacteria were isolated mainly from marine invertebrates • 16S rRNA gene sequences of the isolates were determined • About 5,000 taxonomically novel strains were identified • Several phenotype screenings were performed with the extracts of the 5,000 strains • More than 4,000 hits were identified • The 4,000 extracts were analyzed with LC/MS for dereplication • 250 compounds were isolated and their structures determined by NMR • 55 compounds were shown to be structurally novel • In a more recent work, a collection of Streptomyces from sponges yielded unprecedented number of new compounds
Conclusion Focusing of screening efforts on symbionts of marine invertebrates may be one of the best ways for the development of merchandisable natural products
Development of more sophisticated identification & dereplication methods for natural products • Prediction of the structure of secondary metabolites from the sequences of biosynthesis genes • Rapid identification of secondary metabolites by FT/MS
Polyketide synthesis by type I polyketide synthase (PKS) Polyketides are biosynthesized by the polymerization of acetyl and propionyl subunits in a similar process to fatty acid synthesis
Prediction of polyketide species by PKS phylogeny avermectin filipin pimaricin monensin niddamycin nystatin Knuc pteA pimS monA nanA nidA aveA nidA pikA gdmA nys polyene macrolide Polyether Macrolide polyene macrolide • Chemical structures of the secondary metabolites can be predicted, in some extent, from PKS protein sequences • If novel PKS protein sequences are found, these protein may synthesize a new metabolite
PCR amplification of various regions in PKS genes Type-I PKS genes KS AT (ER) (DH) (KR) ACP DNA isolation from each strain ↓ PCR amplification of each domain ↓ Cloning of amplified fragments n KS AT ACP TE Sequencing ↓ Blast search & Phylogenetic analysis ↓ Prediction of polyketide compounds
Comprehensive sequencing of the KS domains of pks genes in 16 strains in a single 1/16 run of a next generation sequencer (454) PCR amplification of pks from strain n (n = 1, 2..) Tag n Primer 1 Target sequence Primer 2 Tag n 454 adaptor A Tag n Primer 1 Target sequence Primer 2 Tag n 454 adaptor B Ligation Ligation Mixing of PCR products from strains 1, 2, … 454 sequencing
Coverage of pks and nrps sequences in Streptomyces greseus by the 454 sequencing Number of KS domain in pks pks (dc = 56) Number of A domain in nrps nrps (dc = 16) Expected Detected Expected Detected 26 26 31 22 Unknown 2-4 1-1 pks 2-3 3-2 dc = depth of coverage 4985 nrps 6716 586 3264
Rapid identification of secondary metabolites by FT/MS • Microorganisms harboring new pks genes may produce novel polyketide molecules. • Then, how can we detect and identify these polyketide compounds efficiently? The answer would be: the Fourier Transform Ion Cyclotron Resonance Mass ass Spectrometry (FT-ICR (FT ICR- MS) ・accurate accurate mass (m/z ( m/z) ・high high sensitivity ・high high throughput
Specific detection and identification of polyketide molecules by LC/FT-ICR LC/FT ICR-MS MS
Summary 1. Partial sequencing of pks genes allowed the prediction of their functional subfamilies 2. A collection of strains harboring novel pks genes may be a significant resource for the potential drug discovery 3. The expression of novel pks genes could readily be monitored by FTSICR-MS FTSICR MS
Overproduction of natural products • Many organisms produce interesting natural products in a tiny amount, or they are slow-growing slow growing • It is thus desirable to establish expression of their entire biosynthetic gene clusters in a more suitable host • European initiative on the subjects exits: ActinoGEN ActinoGEN aimed at developing novel genomics- based approaches for: Accessing new antibiotic biosynthetic pathways from diverse actinomycetes that have yet to be cultured Activating cryptic pathways from well- characterised actinomycetes Engineering novel hybrid antibiotics by combinatorial biosynthesis
Focused sampling of good biological resources (e.g. marine invertebrates) Isolation of microorganisms (bacteria) (New isolation/cultivation methods) pks/nrps gene sequencing Expression confirmation by FTICR/MS] High quality bacterial libraries possessing novel pks/nrps genes Distribution to industries Hits Overexpression of genes of intrests Commercialization Metagenomic libraries (Gene capture methods) Expression of genes of intrests in E. coli or other hosts
Conclusion Grapes hanging from a low vine have already been harvested New idea and new tools will be required to harvest grapes hanging from high vine