Cipollone, Irene (2025) Studies of the interactions between target proteins and drugs by in silico, in vitro and ex vivo new generation approaches. [Tesi di dottorato]

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Abstract

The study of small molecule–protein interactions is of central importance, as it not only provides insights into the binding mode but can also shed light on the true mechanism of action of pharmacological agents. Over the past decades, a variety of techniques, including proteomics, have been developed to expand our understanding of these processes. Within this framework, my PhD project, co-funded by Dompé Farmaceutici S.p.A. (D.M. 352), has been carried out. During the first year, I focused on the covalent inhibition of the SARS-CoV-2 main pro-tease (3CLpro) by halicin, within a collaboration network including Elettra Sin-crotrone (Trieste), University of Cagliari and the EXSCALATE platform (Dompé). By applying biochemical and mass mapping strategies, in combination with X-Ray crystallography, Thermal Shift Assay and Molecular Dynamics ap-proaches, halicin was found covalently targeting not only the catalytic Cys145 but also Cys44, a residue crucial for the stabilization of the dimeric active form of 3CLpro. Through this finding, an inhibition process by halicin, relying also on its destabilizing effect upon Cys44 modification was proposed. In the last two years, the interest was turned to pulmonary fibrosis, one of the major long-term conse-quences of this viral infection, with a particular focus on c-Kit, a tyrosine kinase implicated in fibrotic progression. Using a Limited Proteolysis–Mass Spectrome-try strategy, in combination with Surface Plasmone Resonance, X-Ray crystallog-raphy and molecular dynamics (in collaboration with EXSCALATE, Dompé), the binding mode of four different c-Kit inhibitors was investigated. Since c-Kit exists in both hyperphosphorylated (active) and hypophosphorylated (inactive) states, phosphorylation-induced conformational changes were first examined, identify-ing regions that become more or less exposed depending on the kinase activation state. Structural alterations upon drug treatment were then assessed, demonstrat-ing that all inhibitors targeted the active site but with distinct preferences: one compound, Cpd2, bound selectively to the active form, two, Cpd3 and Cpd5, to the inactive form, while a fourth, Cpd7, was able to interact with both confor-mations with a comparable affinity. The research on fibrosis progressed also through functional studies aimed at identifying the target pathways in fibrotic fi-broblasts exposed preventively to three selected compounds. These experiments were carried out at the Plateforme Protéomique Necker in Paris in collaboration with Dr. Chiara Guerrera, employing both proteomic and phosphoproteomic anal-yses to characterize the compounds’ mechanisms of action. The results high-lighted diverse outcomes: one compound, Cpd79, completely prevented fibrotic progression, another, Cpd18, showed anti-fibrotic effects but was associated with side effects, while the third compound, Cpd108, failed to exert protective activity. In conclusion, this work provides an integrated perspective on small molecule–protein interactions, spanning from structural binding modes to functional cellular consequences. The findings emphasize the value of combining biochemical, bio-physical, computational and proteomic approaches to elucidate mechanisms of action, ultimately contributing to the rational development of more effective and selective therapeutics.

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