Scarpato, SIlvia (2022) Innovative strategies in the identification of new natural lead compounds from marine organisms. [Tesi di dottorato]

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Marine environment represents one of the most interesting sources of inspiration for the development of new drugs. Marine organisms, for example porifera, tunicates, aquatic plants, can synthesize and/or accumulate an important plurality of secondary metabolites with peculiar and unusual molecular structures, implicated in defence mechanisms, in intra- and inter-species communication and in processes of adaptation to extreme conditions. In the spectrum of pharmacological activities boasted by natural products isolated from marine organisms, it is fundamental to mention the antibiotic, antifungal, antiviral and antineoplastic activities. In recent years, about 20 compounds from marine origin have been in phase I, II or III clinical trials, whereas some marine natural product-derived compounds are commercially available. Significant advances in the field of structural elucidation techniques, occurred in recent years, allow for the complete elucidation of sub milligram samples. In addition, the development of innovative techniques combining data obtained from sophisticated analytical techniques and efficient data analysis software, has allowed to stem the difficulty in identifying new natural compounds from complex extracts, facilitating the process of dereplication of the huge number of metabolites. Among these, molecular networking is an innovative computational technique, which is a valuable complement to traditional dereplication techniques. Molecular networking processes the massive amounts of data obtained from LC-MS/MS analysis of complex extracts, allowing the automated identification of the structural similarity between metabolites based on similarity between the fragmentation spectra of the compounds in the extract. The research work described in this PhD thesis fits into this scenario. A significant part of the research activity was focused on molecular networking, demonstrating its effectiveness both in the discovery of new natural products and in environmental analysis. In particular, the workflow that integrates the analysis of LC-MS/MS data of extracts of the widely studied sponge Stylissa caribica with molecular networking, in its feature-based molecular networking variant, allowed the rapid isolation of a new cyclic heptapeptide, stylissamide L. Moreover, the study with these methods of the extract of the sponge Clathria faviformis led to the isolation of a new phospholipid, favilipid A. The validity of the methodology was further verified with the analysis of extracts from the cyanobacterium Trichodesmium thiebautii, whose metabolome had shown traits in common with the metabolic profile of the marine sponge Smenospongia aurea. Thanks to Feature-Based Molecular Networking, it was possible to focus the analysis on halogenated compounds in the extract, leading to the isolation of 4 new polyketide compounds, included in the class of trichophycins (isotrichophycin C and trichophycins G-I). The cytotoxicity of the newly isolated compounds, along with the previously identified trichophycins was evaluated against the mouse neuroblastoma cell line N2A, as part of a broader structure-activity relationship (SAR) study, aimed at determining the features that modulate bioactivity. In the context of environmental monitoring of cyanobacteria, the validity of a pipeline that integrates the investigation of the territory thanks to specific satellites, allowing the rapid identification of potentially toxic cyanobacterial blooms, and the subsequent chemical analysis of collected environmental samples, based on molecular networking, was verified in the analysis of an unusual cyanobacteria bloom that took place in Lake Avernus at the turn of the lockdown period from COVID-19 in Italy. The presence in the extracts of cyanotoxins, particularly microcystins, was verified by genome amplification analysis and by in-depth analysis of molecular networking clusters containing nodes recognized as known toxins by in-silico tools, provided by the freely accessible website GNPS. In addition, the cytotoxic activity of the chloroform extracts was confirmed. Another important part of the research activity was focused on the detailed structural elucidation of new natural products isolated from the extracts of the seagrass Zostera marina. The structure of the new cyclic diarylheptanoids, zosteraphenol A and zosteraphenol B was completely elucidated. They both displayed broad and unresolved signals in 1D and 2D NMR spectra even at low temperature, which were shown to be related to a conformational equilibrium between rotamers with opposite axial chirality with the support of quantum mechanical calculations. Further analysis of extracts from Z. marina led to the discovery of two unique diarylheptanoid dimers, zosterabisphenone A and B, each composed of two different cyclic diarylheptanoids. They showed the same conformational equilibrium as zosteraphenols A and B, and their structure elucidation required extensive use of DFT calculations. A possible biosynthesis of zosterabisphenone A was proposed. In cytotoxicity assays performed for these compounds, zosterabisphenone B exhibited time-dependent and concentration-dependent cytotoxic activity towards HCT116 cells. Finally, the new phenolic acid, 7″,8″-didehydrosalviolanic acid B was isolated and characterized as part of a study investigating seasonal variations of phenolic compounds in Z. marina. Its absolute configuration was assigned by comparison of the ECD spectrum of methyl (3,4-dihydroxyphenyl)lactate produced by degradation of the isolated compound, and methyl (3,4-dihydroxyphenyl) lactate produced by degradation of rosmarinic acid, whose absolute configuration is known from previous studies.

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