| Summary: | Chemical compounds synthetized by living organisms have historically contributed to improved human health. Ancient populations made use of local natural products through herbal medicine, which today are to be found as bioactive molecules contained in marketed drugs. Previously, natural products’ major contribution to pharmacology was provided by terrestrial plant and microbe compounds, mainly due to the ease of access to these resources by humans. Marine natural products have gained interest for pharmacology since the beginning of marine exploration for research purposes and currently represent the most promising habitat for the description of new organisms and bioactive molecules. The “Deep-Sea”, constituting over 95% of all Oceans and Seas, represents the predominant marine ecosystem and the widest environment existing on Earth. Currently, only about 0.1% of this environment has been explored but several studies recorded an extraordinary biodiversity and species richness, comparable only to tropical rainforests or shallow-water coral reefs. Marine natural product drug discovery aims at isolating new molecules synthetized by marine organisms with the potential to cure human diseases. Previous research has been mainly focused on the discovery of anti-cancer and antimicrobial molecules, while testing for widespread chronic conditions associated to bone and cartilage degeneration was rarely performed. This thesis describes the investigation of compounds produced by deep-sea fungi and invertebrates to influence human stem cell differentiation and modulate inflammation aiming at the discovery of small molecules to aid regenerative medicine and treat musculoskeletal diseases. To implement a drug discovery program, we initially developed an automated high throughput screening platform based on miniaturized cellular assays using mammalian cells. Primary human Mesenchymal Stem Cells (hMSCs) were used to detect pro-osteogenic, pro-chondrogenic, proliferative and cytotoxic activity of marine compounds. An immortalized hepatocyte cell line (HepG2) was used to develop a cytotoxicity assay, while immortalized macrophages (THP1) were used to test marine libraries for associated anti-inflammatory activity. This methodology was validated by testing twelve pure compounds isolated from the X marine derived fungus Penicillium antarcticum and the screening results identified a derivative of itaconic acid able to inhibit hMSCs osteogenic and chondrogenic differentiation as well as inhibiting the inflammation of stimulated THP1 cells. This data identified a potential drug candidate to treat diseases such as atherosclerosis, where undesired stem cell differentiation and local inflammation are responsible for disease development. To expand the drug discovery program and harvest environmental resources for bioactive small molecule biosynthesis, the diversity of fungi associated to deep- sea sediments and invertebrates forming cold-water coral gardens offthe coast of Ireland was explored. Samples obtained from the deep-sea at the Porcupine Bank and Whittard Canyon locations were processed in the laboratory to isolate fungi in pure culture and identify them through taxonomy. Phylogenetic analysis was used to identify possible new fungal species, while database research using the Python-based tool FUNGuild was used to describe the community ecological role. Several isolated species were previously observed in the North Atlantic while other fungi were described for the first time in this environment and one strain is presented as a new species in the genus Arachnomyces. Microbial communities associated with the two habitats were composed of fungal species with distinct trophic preferences to support the theory of an active selection of fungi populating different habitats in the marine environment. A selection of fungi isolated from the deep-sea were cultured on rice medium for secondary metabolite chemical extraction. Fungi were stimulated with epigenetic modifiers for the biosynthesis of a wide pattern of secondary metabolites and a library of 160 crude fungal extracts was prepared. Fungal extracts’ bioactivity was tested on the previously developed platform: the screening for compounds influencing hMSCs differentiation did not provide any reliable bioactive hit, while the screening for extracts downregulating inflammation in activated macrophages showed eight species synthetizing anti-inflammatory compounds. Along with the fungal library, we tested 320 extracts from deep-sea invertebrates for their influence on hMSCs differentiation. Several extracts showed potential to induce differentiation toward osteogenic and chondrogenic lineages, with extracts from sea-pens able to induce reliable differentiation in all three hMSC donors tested. Corals and sponges forming coral gardens in the deep-sea proved XI to be valuable resources with the potential to influence stem cell differentiation while their associated fungi produced anti-inflammatory compounds relevant for chronic disease drug discovery. In this study we performed the first high throughput screening ofmarine natural products for stem cell-based drug discovery. We validated a novel screening platform to detect compounds influencing hMSC differentiation and macrophage inflammation as well as highlighted the potential of marine fungi and invertebrates to synthetise compounds relevant for regenerative medicine drug discovery. Moreover, the first description of fungal biodiversity associated to a deep-sea coral garden was provided in this research framework blending applied and basic studies to benefit the field of marine biotechnology 2022-09-27
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