Proposed Allosteric Inhibition of Cyclin-Dependent Kinase 4 by a Proline-Derived Acylsemicarbazide Compound with Antiproliferative Activity in Breast Cancer Cells.

Background: Cyclin-dependent kinase 4 (CDK4) is a key regulator of cell-cycle progression and an established therapeutic target for breast cancer. Although the unique architecture of its ATP-binding site has enabled the development of highly selective inhibitors, the emergence of acquired resistance highlights the need for alternative therapeutic strategies targeting protein conformational regulation. Methods: The conformational landscape of CDK4 was investigated in this study using accelerated molecular dynamics (aMD) simulations combined with Markov state model (MSM) analysis to identify cryptic conformational states and potential allosteric binding sites. Particular attention was given to the glycine-rich loop (G-loop), a critical structural element that shapes the ATP-binding pocket, and to the effects of compound 8i (1-(2-(3-chlorobenzoyl)hydrazine-1- carbonyl)-N-(pyridin-3-yl)pyrrolidine-2-carboxamide), previously synthesized in our laboratory, on CDK4 dynamics. Results: A distinct conformational transition was identified in which the G-loop shifted toward the N-terminus, resulting in the exposure of a previously unrecognized allosteric pocket adjacent to the catalytic site. Compound 8i interacted with Leu147 and was associated with stabilization of conformational states that favor exposure of the cryptic pocket. Conclusions: These observations suggest that ligand binding may modulate the conformational landscape of CDK4 and favor formation of a cryptic pocket with potential allosteric characteristics. The identified conformational mechanism provides new insights into the dynamic regulation of CDK4 and suggests that stabilization of transient allosteric states represents a promising strategy for the rational design of next-generation CDK4 inhibitors with the potential to overcome resistance in breast cancer therapy.