Discovery of [1,2,4]triazolo[1,5-a]pyrimidine-based disruptors of the Skp2-Cks1 protein-protein interaction through integrated computational and experimental analysis.

The Skp2-Cks1 protein-protein interaction is essential for SCFSkp2/Cks1-mediated recognition and ubiquitination of cell-cycle regulators, making this interface an attractive target for anticancer drug discovery. In this study, we identified new small-molecule Skp2-Cks1 disruptors from a focused [1,2,4]triazolo[1,5-a]pyrimidine library using an integrated computational and experimental workflow. A scaffold-based set of 40 compounds was filtered by diversity, purchasability, and drug-likeness, followed by docking into the Skp2-Cks1 interfacial pocket. Five hits (C1-C5) were prioritized and further evaluated by 1000 ns molecular dynamics simulations, MM/PBSA calculations, free-energy landscape analysis, and in silico ADMET profiling. Docking and simulation results supported stable occupancy of the Skp2-Cks1 hotspot by the selected compounds, with ligand-dependent effects on interfacial stability and conformational sampling. Among the series, C3 showed the clearest computational signature of protein-protein interaction weakening, while C4, C3, and C5 showed the most favorable predicted binding to the Skp2-Cks1 pocket; however, favorable ligand binding was not always accompanied by equivalent weakening of the protein-protein interaction. Free-energy landscape analysis indicated that ligand binding reshaped the conformational ensemble of Skp2-Cks1 toward more defined low-energy states. Experimental validation using an in vitro HTRF assay confirmed dose-dependent inhibition of the Skp2-Cks1 interaction. C3 emerged as the most potent inhibitor (IC50 = 1.88 ± 0.16 μM), outperforming the reference inhibitor NSC689857 (IC50 = 2.89 ± 0.35 μM), while C5 also showed strong activity (IC50 = 5.76 ± 0.68 μM). Overall, this work identifies [1,2,4]triazolo[1,5-a]pyrimidines as promising Skp2-Cks1 inhibitors and establishes C3 as a lead scaffold for further optimization.