Aliotta, Federica (2021) Biochemical studies of a novel class of Cdc25 inhibitors and biological analysis of the effect in melanoma cell lines. [Tesi di dottorato]

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Abstract

Cell division cycle 25 (Cdc25) phosphatases play a pivotal role in the regulation of cell cycle. Because of their altered expression in some tumors, Cdc25s are considered promising targets for cancer therapy. Previously my research group demonstrated that the compound NSC28620 acts as a reversible inhibitor of Cdc25s and affects the cell viability of some cancer cell lines at 200 µM. To identify more potent inhibitors of Cdc25s, thirty-one NSC28620 derivatives were characterized. From kinetic measurements of the phosphatase activity sustained by the recombinant form of Cdc25B emerged that some derivatives (7j, 7i, 6e, 7f, and 3) showed a higher inhibitory activity compared to the lead NSC28620, with the most potent inhibitor 7j (Ki, 0.8 μM). Intrinsic fluorescence measurements, performed to study the Cdc25B•inhibitor interaction, ranked the thirty-one derivatives in two main groups of inhibitors, i.e. noncompetitive, uncompetitive. Biological study revealed that only one derivative, compound 4a, exerted a specific toxic action in melanoma cells, at lower concentration (5-10 µM) respect to the lead compound. Cytofluorimetric analysis indicated that this molecule arrests the cells in G2/M phase and induces apoptosis. In particular, fluorimetric measurements of caspase-3 and -9 activity and western blotting analysis of sub-cellular localization of cytochrome c suggest that 4a activates an apoptotic program, mainly mitochondria and caspase-mediated. Furthermore, 4a reduces the protein levels of all three forms of Cdc25 and affects the expression of typical proliferation and apoptotic markers, such as pAkt and p53.In the complex, the discovery and production of more potent bioactive molecules can contribute to the advancement of research on new therapeutic strategies against one of the most deadly tumors such as melanoma.

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