Novel active site -targeted fluorescent probes for real-time monitoring of CDK4/6 in tumor cells and tissues.

Breast cancer is the leading female malignancy, and CDK4/6, key members the cyclin-dependent kinases (CDK) family, have become validated therapeutic targets. However, small-molecule tools that can track these kinases in living cells and tissues remain scarce. Therefore, guided by the co-crystal structure of the CDK4/6 inhibitor Palbociclib, we carried out two rounds of optimization to develop a mini-library of fluorescent probes (Q1-Q9) and subsequently screened them for selective labeling capability. Docking and biochemical assays confirmed that the new ligands retain the binding mode of Palbociclib. Among them, Q2 exhibited good affinity for CDK4 and CDK6 (IC = 151 ± 43 nM and 193 ± 70 nM, respectively) and provided exceptional, higher-resolution images of the kinases in live cells, outperforming antibody staining. The probe may serve as a cost-effective, stable and user-friendly alternative to antibody staining, and a potential visualization tool of drug preliminary screening by showing the changed fluorescent signals when incubated with active compounds. Importantly, Q2 retained bioactivity, inducing cell-cycle arrest and suppressing tumor-cell growth, mirroring the activity of the parent inhibitor Palbociclib. Thus, Q2 is a ready-to-use chemical tool for real-time visualization of CDK4/6 dynamics and holds promise for improving the diagnosis and treatment of breast cancer.