Albiani, Federica Identification of natural products for the development of immunoceuticals and candidate drugs. [Tesi di dottorato]

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Abstract

Natural Products (NPs) have always represented a huge source of molecules with relevant potential in pharmacological field. In particular, Marine Natural Products (MNPs) are characterized by an enormous structural diversity and potential bioactivity. Many natural products displayed chemo-protective functions, drug-like benefits to skin health, and immunomodulant potential both alone or in association with other treatments. In this doctoral thesis, I evaluated the activity of MNPs as modulator of the innate immune system with the aim of identifying novel candidates with either defensive properties to use as cosmeceuticals (anti-oxidant and skin protection) or anticancer activity to use in immunotherapy. Conventional chemotherapeutic drugs are cytotoxic substances affecting vitality of normal cells together with tumor cell. In contrast, immunotherapeutic drugs offer a new strategy to fight cancer by stimulating the immune system to recognize and eliminate the malignant cells. Selection of the molecules was based on the ability to bind and activate Dectin-1 and Triggering Receptor Expressed on Myeloid cells 2 (TREM2), two membrane receptors occurring on cells of the innate immune system. Dectin-1 is a pattern recognition receptor (PRR) belonging to the C-type lectin receptors (CLRs) and is predominantly expressed on macrophages and Dendritic cells (DCs), while TREM2 is a transmembrane protein of the immunoglobulin family that is involved in modulation of several physiological and pathological processes, which are involved in several types of diseases, especially neurodegenerative ones. The research activities of this PhD thesis regard eight case studies: Cacospongia mollior and Haliclona vansoesti (sponges), Ophiura ophiura (echinoderm),Cyclope neritea and Haminoea orbygnana (molluscs), Clavelina lepadiformis (tunicate), Schizotrix (cyanobacterium), Alexandrium andersonii (dinoflagellate) and Colpomoenia sp. (red alga). The marine extracts were submitted to an advanced biological screening platform which includes innovative approaches of fractionation, with the application of orthogonal chromatographic phases allowing us to both validate the biological response and identify the bioactive products. Moreover, marine extracts were submitted to MS-based deconvolution to find molecules with a specific functional group on the skeleton, that could be correlated to the interaction with a specific immune system receptor. These strategies allowed to isolate new and known active small metabolites of different chemotypes. Between the isolated compounds, the alkaloid Lepadin A resulted to be a good Immunogenic Cell Death (ICD) inducer, whereas the terpene Molliorin F, glycolipids cerebrosides and halisphingosines were found to be cytotoxic, at low concentrations, on human melanoma epithelial cells. Concerning the identification of cosmeceuticals, the research activities were focused on the investigation of three microalgae species- Tetraselmis chuii, Cyclotella cryptica and Isocrysis galbana-already known to be reach of immunomodulant lipids, on which were evaluated the anti-oxidant activity (AOA) and anti-oxidant capacity (AOC). AOA and AOC were assessed through four different assays (DPPH, ABTS, ORAC and FRAP). Also in this case, the extract was submitted to bioassay-guided screening platform, allowing to observe even more promising results for the fractions compared to the extracts. All microalgal extracts exhibited a good scavenging ability in the ABTS assay, but the most promising outcome was obtained with the green microalga T. chuii. After fractionation, fraction B of C. cryptica showed the best results in DPPH and ORAC assays. Fractions B of C. cryptica and I. galbana, fractions A and C of T. chuii showed a removal of the ABTS radical higher than 90%. Looking for conventional anti-oxidant molecules in each species: the highest phenolic content was found in fractions B and E of I. galbana and fraction E of T. chuii; whereas fraction B of I. galbana was the richest in carotenoids and the highest vitamin content was found in the fraction E of T. chuii. Therefore, the highest antioxidant property of the microalgae did not correlate with conventional antioxidant molecules. So, fraction B of C. cryptica yielded the best results as candidate for developments of new natural antioxidants, thus suggesting the presence of different molecules with promising potential as scavenger ingredients. Part of my PhD thesis project was also developed at the company Pharmamar S.p.a (Madrid, Spain), a pharmaceutical company, leader in drug discovery, focused on the exploring of new anti-tumour agents from marine biodiversity. During this period, six different marine extracts, with potential cytotoxic activity, were analyzed and fractionated, through different chromatographic techniques, such as flash chromatography and HPLC, and spectroscopic techniques such as NMR, HRESI-LC-MSMS, to identify sixteen different pure compounds. In compliance with the privacy company policy, only six chemical structures, of the sixteen isolated, are reported in the thesis.

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