Targeting the CDK4-Cyclin D Complex: A New Generation of Selective Kinase Inhibitors for Cancer Therapy

Glioblastoma (GBM) is an aggressive malignancy with limited therapeutic options and poor survival. Cyclin-dependent kinase 4 (CDK4) is overexpressed in GBM and promotes tumor progression through canonical cell-cycle regulation and extranuclear functions related to metabolism, invasion, and tumor microenvironment modulation. Current ATP-competitive CDK4/6 inhibitors show limited selectivity, potentially causing off-target effects and resistance. We developed a strategy to identify novel CDK4 inhibitors targeting the CDK4-Cyclin D1 protein-protein interaction, a key determinant of CDK4 activation. Virtual screening of approximately 950,000 compounds, combining molecular docking and molecular dynamics simulations, identified candidate molecules that were subsequently evaluated using proximity ligation assays, in vitro kinase activity assays, phosphokinase profiling, and functional analyses in patient-derived GBM cell lines and additional cancer models. Diophenic emerged as the lead candidate, demonstrating consistent antiproliferative activity across multiple GBM and epithelial cancer models. The compound selectively disrupted the CDK4-Cyclin D1 interaction while sparing the closely related CDK6 complex, and inhibited CDK4 kinase activity in vitro. Functional studies showed that diophenic induced substantial cell death in GBM cells, significantly reduced invasiveness, and exhibited distinct effects on cell migration compared with CDK4/6 inhibitors. Furthermore, phosphokinase profiling revealed that diophenic modulates a more restricted signaling network than ATP-competitive inhibitors, supporting greater functional selectivity and reduced off-target activity. Aiming the CDK4-Cyclin D1 interface represents a promising alternative to ATP-competitive CDK4/6 inhibition, which target a highly conserved site across the protein kinase superfamily. This approach provides a proof of concept for developing next-generation CDK4-selective therapeutics. Further studies should evaluate its translational potential in GBM.