In Silico Screening for CDK5 Inhibitors Leads to SAR Analysis of Flavonoids, Three Diverse Inhibitor Scaffolds and Fisetin Demonstrating Potent In Vitro Glioblastoma Effects

There is an urgent need for more effective treatment protocols for glioblastoma (GBM), the most common malignant brain tumour in adults. Kinase inhibitors have demonstrated significant potential, with cyclin-dependant kinase 5 (CDK5) recognised as a promising target. Here, we present an in silico led approach towards the discovery of CDK5 inhibitors with anti-GBM potential. Active/decoy benchmarking computations considered the available CDK5–ligand complex crystal structures, to design and optimize a virtual screening protocol that involved 3D pharmacophore pre-filtering of compounds and Glide docking calculations. Screening of the ZINC15 biogenic and Analyticon Discovery natural product databases led to the identification of six low micromolar CDK5/p35 flavonoid inhibitors, with fisetin (IC 50 = 0.90 µM) and apigenin (IC 50 = 1.19 µM) validated as the most potent. Additionally, three diverse chemical scaffolds with xanthone, aristolactam and β-carboline cores were identified as hit compounds for further optimization. Structure activity relationship (SAR) analysis of sixteen flavonoids tested in the CDK5/p35 in vitro binding assays unravelled structural features governing the observed potencies, while selectivity screening against five homologous kinases revealed cirsiliol as the most selective flavonoid for CDK5 (IC 50 = 4.73 µM). Meanwhile, fisetin demonstrated the most potent effects on cell viability (IC 50 s ∼ 20-40 µM at 72 h) against three GBM cell lines (U87-MG, T98G and U251-MG), in a generally time- and concentration-dependent manner.