Discovery of Novel Naphthylphenylketone and Naphthylphenylamine Derivatives as Cell Division Cycle 25B (CDC25B) Phosphatase Inhibitors: Design, Synthesis, Inhibition Mechanism, and in Vitro Efficacy against Melanoma Cell Lines.

CDC25 phosphatases play a critical role in the regulation of the cell cycle and thus represent attractive cancer therapeutic targets. We previously discovered the 4-(2-carboxybenzoyl)phthalic acid (NSC28620) as a new CDC25 inhibitor endowed with promising anticancer activity in breast, prostate, and leukemia cells. Herein, we report a structure-based optimization of NSC28620, leading to the identification of a series of novel naphthylphenylketone and naphthylphenylamine derivatives as CDC25B inhibitors. Compounds 7j, 7i, 6e, 7f, and 3 showed higher inhibitory activity than the initial lead, with Ki values in the low micromolar range. Kinetic analysis, intrinsic fluorescence studies, and induced fit docking simulations provided a mechanistic understanding of the activity of these derivatives. All compounds were tested in the highly aggressive human melanoma cell lines A2058 and A375. Compound 4a potently inhibited cell proliferation and colony formation, causing an increase of the G2/M phase and a reduction of the G0/G1 phase of the cell cycle in both cell lines.

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells.

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5-9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 µM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.