Elucidating Binding Selectivity in Cyclin-Dependent Kinases (CDKs) 4, 6, and 9: Development of Highly Potent and Selective CDK4/9 Inhibitors

CDK4/6 inhibitors have proven effective in treating HR+/HER2- breast cancer, but their limited therapeutic indications and the emergence of acquired resistance are concerning. Recent studies suggest that hematological toxicity linked to CDK4/6 dual inhibitors may be due to strong CDK6 inhibition. To address these challenges, small molecules targeting different CDK subfamily members could provide clinical advantages in specific patient subgroups and expand CDK inhibitor indications beyond breast cancer. However, the high conservation of CDK's binding pocket makes designing molecules selectively targeting different CDKs challenging. In this study, we use in-depth in silico modeling and structural biology analysis of co-crystal data to analyze subtle variations in several CDKs' key binding pockets. Our findings indicate that a sequential difference of a αD-helix motif between CDK4 and CDK6 represents an ideal "sweet spot" that influences binding selectivity. To achieve this selectivity, we designed small molecules with a 1,4-trans-cyclohexanediamine sidechain that specifically promotes interactions with the αD-helix motif in CDK4 but not in CDK6. Interestingly, the same αD-helix motif in CDK9 presented an opportunity to develop a CDK4/9 inhibitor, potentially improving acquired resistance to CDK4/6 inhibitors. Our rational approach identified a series of analogs exhibiting distinct in vitro selectivity among CDK4, CDK6, and CDK9. The lead compound (29) was further evaluated for in vivo pharmacokinetics. This work revealed key molecular insights for developing next-generation CDK inhibitors with unique selectivity profiles. This approach could help overcome the challenges of developing selective CDK inhibitors and broaden their therapeutic indications beyond breast cancer.