Abstract
CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.
Highlights
CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy
CDC25 phosphatases are overexpressed in a variety of human cancers[9,41], are rate-limiting in tumorigenesis induced by Ras[10], and were recently proposed as target of choice in unresponsive, triple-negative breast cancer[22]
Taking advantage of knowledge gained in previous drug discovery programs[13,42], we initially defined a pharmacophore that is common to compounds belonging to three distinct classes of established CDC25 inhibitors (Fig. S1)
Summary
CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. The Cell Division Cycle 25 family encompasses three highly conserved members of dual specificity phosphatases that target Cyclin-Dependent Kinases (CDKs), acting as dose-dependent inducers of cell cycle transitions[1,2]. CDC25B initiates CDK1/CycB activation at centrosomes during the G2/M transition[4,5] and CDC25C causes full activation of CDK1 at mitotic entry[6]. Genetic studies showed that thermosensitive cdc[25] yeast mutants could be reversibly arrested in the cell cycle[7], providing the first demonstration of a regulatory role for CDC25.
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