Structure‐Based Discovery of Fumiquinazolines as Skp2–Cks1 Protein–Protein Interaction Disruptors: Mechanistic Computational Insights and Experimental Validation

Abstract

The S‐phase kinase‐associated protein 2 (Skp2)–cyclin‐dependent kinase subunit 1 (Cks1) protein–protein interaction (PPI) plays a central role in recognition of phosphorylated p27 and therefore represents an attractive target for anticancer drug discovery. Herein, we sought to identify new small‐molecule disruptors of the Skp2–Cks1 interface from a focused fumiquinazoline‐scaffold library by combining virtual screening, molecular simulation, and experimental validation. Docking showed that all selected compounds occupied the Skp2–Cks1 interfacial hotspot. However, longer 1000‐ns MD simulations, comparative MM/PBSA calculations, and interface‐related metrics revealed distinct structural, energetic, and dynamic binding profiles among the selected compounds. Consistent with these analyses, comparative MM/PBSA evaluation of PPI stability showed that Fumiquinazoline D produced a positive , indicating weakening of the Skp2–Cks1 interface relative to the apo complex, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F generally yielded negative values consistent with interfacial stabilization. Free energy landscape analysis further supported a more weakly confined and dynamically disruptive binding mode for Fumiquinazoline‐ D. In vitro homogeneous time‐resolved fluorescence assays validated these predictions: Fumiquinazoline D inhibited the Skp2–Cks1 interaction with an IC 50 of 6.33 ± 0.44 µM, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F were substantially weaker. Although ADMET profiling identified substantial absorption and toxicity liabilities requiring future optimization, these findings identify Fumiquinazoline D as an early‐stage biochemical hit for disruption of the Skp2–Cks1 interaction and as a potential scaffold for further mechanistic validation and medicinal‐chemistry investigation.