Structure-based discovery of diphenylpyrazines as disruptors of the Skp2-Cks1 protein-protein interaction: computational insights and biochemical validation.

The Skp2-Cks1 protein-protein interaction is essential for recognition of phosphorylated p27 and contributes to SCFSkp2-mediated ubiquitination, making this interface an attractive but challenging target for anticancer drug discovery. In this study, we applied an integrated in silico-in vitro workflow to identify novel Skp2-Cks1 disruptors from a focused 1,3-diphenylpyrazine library. A total of 131 compounds were prioritized through diversity, purchasability, and drug-likeness filtering, followed by docking against the p27-recognition pocket at the Skp2-Cks1 interface. Five hits (C1-C5) were selected for 1000 ns molecular dynamics simulations, MM/PBSA analysis, and free-energy landscape evaluation, with NSC681152 and NSC689857 used as reference inhibitors. Docking showed that all selected compounds occupied the targeted interfacial hotspot, while simulation-based analyses distinguished C3, C4, and C5 as the most promising disruption-oriented ligands. Free-energy landscape analysis further indicated that these compounds favored localized low-energy conformational states, with C3 showing the clearest energetic convergence. Experimental validation using a homogeneous time-resolved fluorescence assay confirmed potent inhibition of the Skp2-Cks1 interaction by C3, C4, and C5, with IC50 values of 2.96 ± 0.43, 4.32 ± 0.83, and 4.88 ± 0.59 µM, respectively. Notably, C3 exhibited activity comparable to the reference inhibitor NSC689857 (IC50 = 2.84 ± 0.29 µM) and was substantially more potent than NSC681152 (IC50 = 52.17 ± 3.81 µM). These findings validate the diphenylpyrazine scaffold as a promising chemotype for Skp2-Cks1 inhibition and identify C3 as a strong lead for further optimization.