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ESR1: Exploitation of new strains for Drug Discovery from deep sea sediments

Dr. Donatella de Pascale

Project details: Isolation and characterization of new bioactive compounds isolated from deep-sea sediment from the EUROFLEET2 Pharmadeep cruise.  Approach: 1) the cultivable fractions from the isolated sediments will be isolated and characterised, while 2) the uncultivable microbial fractions will be studied; 3) new and improved protocols of screening for antimicrobial and anti-biofilm activities will be set up, in particular Multi-Drug-Resistance (MDR) bacteria will be tested for discovering new compounds. This will be followed by 4) the identification of hyper-producer microorganisms of bioactive compounds. 5) The chemical profiling and compounds identification will be the final step.


ESR2: Drug discovery from marine microalgae Dr. Adrianna Ianora,, Dr. Giovanna Romano,

Project details: Selecting anticancer hits previously isolated from cultures of marine microalgae and screening for new anticancer compounds from deep-sea samples. 1) To improve the production of these compounds using variations in media composition and/or fermentation conditions. 2) Scaled-up production of bioactive species in bioreactors to maximize the production of targeted molecules. 3) Test newly isolated species for anti-cancer (A549 lung, HT-29 colon-rectal, Saos-2 osteosarcoma, MDA-MB-231 breast, PC-3 prostate, A2780 ovarian) activity. 4) Identify the mechanism of action and main pathways involved in cell signalling processes (autophagy, necrosis, apoptosis, inflammation, oxidative stress and other pathways). 5) Chemical characterization of compounds of interest.

ESR3: Novel anticancer compounds from Arctic marine microorganisms Prof. Jeanette Andersen,

Project details: Selecting antibacterial hits previously isolated from cultures of marine strains and screening for new antibacterials from deep-sea samples. The ESR will be included in the existing bioprospecting pipeline at Marbio and hence be involved in the following: extraction/fractionation, isolation, identification and biological characterization of antibacterial compounds. When bioactive extracts are identified, the bacterial strains will be sequenced and analyzed to identify putative secondary metabolism clusters at UCC. When the bioactive crude extracts are identified by preliminary bioactivity analysis, purification of the active compound(s) will commence. The process will involve identification of the suspected bioactive molecule(s) by HR-MS analysis. This will be followed by crude extract submission to mass-guided preparative HPLC for purification of the selected compound(s).  Structural elucidation of the isolated compound(s) will then be undertaken by combining results obtained from HR-MS analysis and various 1D and 2D NMR experiments. The purified compounds will be thoroughly tested in different bioassays to evaluate the potential as a candidate for follow-up studies.

ESR4: Complex marine natural products from extremophile bacteria Prof Marcel jaspars,

Project details: 1) Identification of lead compounds from 10 magnificent strains: dereplication of partition fractions using LC-MS. Elucidation of the 2D structure of lead compounds using spectroscopic methods (NMR and MS). Determination of the relative stereochemistry using the most appropriate approach for the structural class under study. 2) Synthesis of analogues of lead compounds. 3) Organisms obtained from extreme habitats (Pharmadeep) will be grown under a range of culture conditions to give rise to complex metabolic profiles representing a range of biosynthetic types. LC-MS based dereplication will be applied to identify the extracts most likely to contain novel chemical entities. Genome scanning may be applied in selected cases to identify organisms containing particular pathways of interest. Compounds will be isolated from promising extracts and dereplicated using molecular formula and basic 1D NMR data from the 1H and 13C NMR spectra. Library of fractions and pure compounds from newly isolated strains will be created.

 ESR5: Enhanced chemical diversity of antibiotic molecules from Extremophilic  

microorganisms monitored by metabolomics and chemical imaging Prof. Deniz Tasdemir,

Project details: New approaches to increase/detect chemical diversity will be implemented on newly isolated strains by ESR5: 1) OSMAC (One-Strain-MAny-Compounds), an effective method for mono-cultural microbial fermentation strategy seeking to identify the most suitable culture media and other conditions for optimal microbial growth, which also determines the type (and the amount) of secondary metabolite biosynthesis. 2) Co-culturing, i.e. the growth of a microbial species in the presence of another microbe(s) ideally from the same environment/same host species by creation of a competitive microbial ecosystem to induce a competitive chemical response between microorganisms. 3) Chemical imaging and direct detection/identification of compounds, particularly in live co-cultures by DESI-Imaging Mass Spectrometry (IMS) 4) Secondary metabolome analysis of both mono and co-cultures (by UPLC-QTOF-MS and NMR) as dereplication in combination to IMS. 5) Purification of novel metabolites by HPLC and other chromatographic methods. 6) Structure elucidation of new compounds by 1D and 2D NMR, MS and other spectroscopic techniques 7) Evaluation of antibiotic activity against large panel of strains.

 ESR6: Safety assessment of potential leads using zebrafish based assays Prof. Peter de Witte,

Project details: To bring a new drug to market is very costly and time consuming. Unfortunately, many drugs fail along this process due to safety concerns. To protect the patients, as well as improve the success rate of bringing new drugs to the market, the prediction of drug safety at an early stage must improve. For this purpose the zebrafish can play an important role. The embryonic and larvae stages, can be reared in the small μl-volumes in multi-well plate format, which facilitates high-throughput screening capacity of drugs/compounds and provides a rapid and cheap screening tool to screen hit molecules that can be further developed into drugs. Objectives: To perform safety assessment of the potential leads generated from MarPipe using zebrafish larvae. Approach: 1) Evaluate developmental toxicity (screening for lethality, edema, impaired circulation, change in heart rate, haemorrhaging, tissue necrosis, loss of posture, impaired motility), body curvature and swim bladder defects to determine the degree of toxicity. 2) Assess organ specific toxicity using transgenic lines where vital organs (i.e. liver, kidney, heart, brain/neurons) are highlighted with fluorescent markers. The effect of the compounds will be evaluated by screening procedures (microscope, IN CELL analyser) and by performing functional assays (e.g. quantification of heart beat, kidney filtration rate, locomotor and photomotor response).

ESR7: Genomic approaches for identification of novel biosynthetic pathways for bioactive marine bacterial strains Prof. Alan Dobson,

Project details: Identification of molecular targets of already identified lead compounds with antibacterial activity. Characterisation of new bacterial strains and description of a variety of metabolic pathways involved in the production of bioactive agents. Approach: Genomic libraries will be prepared in pCCERI for expression in Streptomyces, Pseudomonas and E. coli and clones containing novel secondary metabolism genes will be identified by hybridization and/or PCR. Positive clones will be introduced into Streptomyces strain M115 together with Pseudomonas putida and Pseudoalteromonas haloplanktis. Anti-bacterial screens targeting gram-negative clinical pathogens will be undertaken. Extracts will also be assayed for quorum quenching and quorum sensing inhibitory activity using specific indicator bacteria. Novel metabolites will be isolated and identified following purification and analysis by MS and NMR in the Aberdeen secondment.

ESR8: Streamlining the legal, policy and governance aspects & Innovation management and entrepreneurship of marine biodiscovery pipeline Dr. Thomas Vanagt,

Project details: Study the different legal and policy frameworks governing marine biodiscovery (from sample to product). Special attention will be given to hot topics such as the concept of registered collections and synthetic biology. Approaches: 1) Different legal and policy frameworks at international and EU level will be studied: Nagoya Protocol, EU Regulation 511, IPT, WIPO, etc. 2) Study the specifics of marine genetic resources in terms of governance, both within and beyond national jurisdictions; 3) Through interviews and questionnaires compile workable procedures to register a biological collection. Second objective- Innovation management and entrepreneurship aspects in relation to marine biodiscovery will be focused on case studies of successful and failed examples of marine biodiscovery ventures; 4) Improve business models for the different stages of the biodiscovery pipeline; 5) Develop guidelines for innovation management in biodiscovery SME’s to make them better prepared for change and competition; 6) Develop a methodology of what are the necessary steps to go from an idea/discovery to a venture, within a company or as a stand-alone. The specific challenge of this ESR is to align these two objectives and gain new insight from combining expertise in legal, policy and business.

ESR9: Scale-up of production, modification and determination of the preclinical safety of antimicrobial hits isolated from marine microorganisms Dr. Fernando Reyes,

Project details: After selection of one or two promising antimicrobial hits previously isolated from the magnificent 10 strains, a work plan to improve the production of these compounds will be developed. Scaled-up fermentations in the best conditions selected will allow the obtaining of gram amounts of the desired hits, which will then be used as a starting point for the generation of new and improved antimicrobials using biotransformation and/or semi-synthetic approaches. The biological profile of the compounds will be evaluated against an extended panel of microbial pathogens and completed with determination of the preclinical safety and toxicity tests that will permit the selection of the best candidates to be developed as potential leads.

ESR10: Cultivation and characterisation of novel marine microorganisms with high potential as sources of bioactive compounds Dr. Colin Ingham,

Project details: Generation of a strain collection of marine microorganisms to allow screening operations for bioactives. The use of novel cultivation techniques, particularly microcultivation and microprinting on culture chips using ceramic supports, which have been shown to target different groups of microorganisms compared to conventional techniques. This approach concentrates on using near natural cultivation conditions using substrates (e.g. sediments) provided by WP2 partners (from Pharmadeep deep-sea sediments) combined with culture chips, imaging techniques and micro-colony recovery. The aim will be to create an “intelligent” microbial strain collection with sufficient knowledge of microbe identity and likely chemistry that downstream screening has a higher chance of identifying novel compounds. Because of the need for scientific context, the ESR will work extensively with the academic partners (secondments).

ESR11: Drug-discovery of marine natural compounds from deep-sea sediments Dr. Rolando Lorenzetti   Angelo Fontana, Dr. Ilaria Re,

Project details: Identification of new lead compounds of biomedical interest is an iterative process that includes several steps of chromatographic fractionation and evaluation of the biological activity in collaboration with other partner of MarPipe. The ESR 11 will be focused on search of novel anticancer or immunomodulatory compounds, and he will work in the laboratory of Italbiotec at CNR-Institute of Biomolecular Chemistry (CNR-ICB) in Naples. Dry samples selected from marine invertebrates and protists will be extracted and fractionated according to the standard techniques developed by CNR-ICB.

ESR11 is expected to process at least 30 samples by solid-phase extraction on styrene-divinylbenzene resin. To this aim, hydroalcoholic suspension of the biological material will be loaded on pre-packed column and fractionated by an automated system that will allow highly reproducible partition of the chemical constituents in five fractions of different polarity. This material will be tested in phenotypic assay to evaluate the biological activity. The most promising 10 hits will be further fractionated by gel filtration on sephadex or reverse phase HPLC. These chromatographic steps will produce a number of samples ranging from 5 to 10 fractions that will be tested again for the anticancer or immunomodulatory properties. The active fractions will be processed again in order to obtain a homogeneous product (compound that does not show increase of activity after two consecutive chromatographic steps). Chemical consistency will be established by recording 1H-NMR and ESI+/ESI-MS spectra after every chromatographic step, whereas the homogeneous products will be fully characterized by 1D and 2D NMR experiments, CD analysis. Stereochemistry of the homogeneous compounds will be determined by chemical or computational methods. The aim is the unambiguous identification of the structure of the bioactive compounds and the definition of active/toxic concentration of the lead candidates.