Direct-to-biology high-throughput optimization enables rapid hit-to-lead discovery of potent CDK2 inhibitors.

Abstract

The growing demand for accelerated drug discovery has stimulated the development of Direct-to-Biology (D2B) strategies that integrate compound synthesis with immediate biological evaluation. Herein, we reported a D2B-enabled high-throughput optimization (HTO) platform that combined miniaturized parallel synthesis with fluorescence polarization (FP)-based in situ screening for the rapid discovery of cyclin-dependent kinase 2 (CDK2) inhibitors. Guided by structure-based design, a focused library of approximately 1800 triazole derivatives was efficiently generated in 96-well microplates through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) without purification. The resulting crude products were directly subjected to competitive FP assays, enabling rapid identification of active compounds through two rounds of biological screening. This workflow led to the discovery of LL-K2in10, which exhibited potent CDK2 inhibition with an IC50 value of 0.22 μM in the FP binding assay and 43.8 nM in the enzymatic assay. Structural characterization of the CDK2-ligand complex revealed multiple hydrogen-bonding interactions responsible for the high binding affinity and provided a structural basis for the observed structure-activity relationships. Subsequent cellular studies provided evidence consistent with CDK2 target engagement, reduced Rb phosphorylation, decreased cell viability, and altered cell-cycle progression in OVCAR3 cells. Collectively, this study established an efficient HTO platform for rapid hit-to-lead optimization and demonstrated its utility for accelerating the discovery of CDK2 inhibitors.

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