Background: Skp1 is a potential pharmacological target for cancer treatment. However, the few Skp1 inhibitors reported to date have exhibited limited structural diversity and target specificity. This highlights the need for new chemical scaffolds that could potentially be developed into potent and specific Skp1 inhibitors. Methods: We performed a large-scale, hierarchical, structure-based virtual screening (SBVS) integrated with molecular dynamics (MD) simulations against a hydrophobic and shallow P1 hotspot region within the F-box binding interface of Skp1, using a commercial compound library (~280,000 compounds). The top-ranked hits were then experimentally validated using in vitro biophysical and biochemical assays, followed by evaluation of cell viability. Results: The SBVS campaign yielded 28 potential hits. Using a thermal stability shift assay (TSA), we confirmed that several compounds directly bind to both full-length Skp1 and its truncated variant containing only the P1 hotspot (Skp1 1− 140 ), consistent with the SBVS strategy. Functional inhibition assessed by a fluorescence polarization (FP) assay showed that compounds #03, #05, #07, #09, #22, #24 and #28 effectively disrupted the Skp1:F-box peptide interaction in vitro. Furthermore, compounds #04, #12, #15, and #24 demonstrated low-to-moderate micromolar EC 50 values in cell viability assays across different cancer cell lines. Conclusion: Compounds #04, #12, and #24 represent interesting scaffolds and may serve as valuable starting points for future structure-activity relationship (SAR) studies aimed at developing a new class of selective Skp1 inhibitors. Keywords: virtual screening, Skp1 inhibitors, molecular docking, molecular dynamics simulation, binding free energy, hydrophobic hotspot
Hussain et al. (Sun,) studied this question.