Protein phosphorylation/dephosphorylation regulates crucial cel- lular processes, such as cell growth, differentiation, migration, survival, and apoptosis. In particular, cell division cycle 25 (Cdc25) enzymes are dual phosphatases, playing a pivotal role in the regulation of cell cycle progression. Because of its altered expression in different tumors, Cdc25 could be considered as a promising target for cancer therapy. In a previous work from my research group it has been demonstrated that the compound NSC28620 acts as a reversible inhibitor of Cdc25 (Ki 5.3 lM) and affects the cell viability of some cancer cell lines at 200 lM. In order to identify more potent inhibitors of Cdc25, a lead opti- mization program on NSC28620 was undertaken and a group of thirty-one derivatives was considered. The properties exhibited by the novel molecules were investigated through kinetic measure- ments of the phosphatase activity sustained by the recombinant form of the catalytic domain of Cdc25B in the absence or in the presence of these inhibitors. Interestingly, some derivatives are endowed with a stronger inhibition power compared to the lead NSC28620 and the most active one (cpd 7j) had a Ki value of 0.8 lM. The inhibition mechanism of the new compounds was evaluated through an inspection of the corresponding Linewea- ver-Burk plots; this analysis suggested two main different types of reversible inhibition, i.e. non-competitive and un-competitive. Thanks to the presence of a unique tryptophan residue in Cdc25B, intrinsic fluorescence studies allowed an investigation on the protein region involved in the interaction with the various inhibitors. Finally, the cytotoxic effects of the most active com- pounds were evaluated in melanoma cell lines through MTT assay; at a low concentration, i.e. 2.5 lM, only one derivative (cpd 4a) significantly reduced the cell growth of melanoma cells in a time-dependent manner.
Optimization program of a novel inhibitor of the Cdc25B phosphatase activity: kinetic and structural studies of the inhibition mechanism and cytotoxic effects in melanoma cells
R Rullo;
2019
Abstract
Protein phosphorylation/dephosphorylation regulates crucial cel- lular processes, such as cell growth, differentiation, migration, survival, and apoptosis. In particular, cell division cycle 25 (Cdc25) enzymes are dual phosphatases, playing a pivotal role in the regulation of cell cycle progression. Because of its altered expression in different tumors, Cdc25 could be considered as a promising target for cancer therapy. In a previous work from my research group it has been demonstrated that the compound NSC28620 acts as a reversible inhibitor of Cdc25 (Ki 5.3 lM) and affects the cell viability of some cancer cell lines at 200 lM. In order to identify more potent inhibitors of Cdc25, a lead opti- mization program on NSC28620 was undertaken and a group of thirty-one derivatives was considered. The properties exhibited by the novel molecules were investigated through kinetic measure- ments of the phosphatase activity sustained by the recombinant form of the catalytic domain of Cdc25B in the absence or in the presence of these inhibitors. Interestingly, some derivatives are endowed with a stronger inhibition power compared to the lead NSC28620 and the most active one (cpd 7j) had a Ki value of 0.8 lM. The inhibition mechanism of the new compounds was evaluated through an inspection of the corresponding Linewea- ver-Burk plots; this analysis suggested two main different types of reversible inhibition, i.e. non-competitive and un-competitive. Thanks to the presence of a unique tryptophan residue in Cdc25B, intrinsic fluorescence studies allowed an investigation on the protein region involved in the interaction with the various inhibitors. Finally, the cytotoxic effects of the most active com- pounds were evaluated in melanoma cell lines through MTT assay; at a low concentration, i.e. 2.5 lM, only one derivative (cpd 4a) significantly reduced the cell growth of melanoma cells in a time-dependent manner.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
