Identification of a pyrazolo[3,4-b]pyridine chemotype for dual focal adhesion kinase 1/2 (FAK1/2) targeting in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) remains difficult to treat because of the lack of effective therapeutic targets and limited treatment options. Dual targeting of Focal adhesion kinase 1 (FAK1) and 2 (FAK2) has emerged as a promising strategy, yet current dual-targeted ligands are still limited in structural diversity and binding mode. In this study, a pyrazolo[3,4-b]pyridine scaffold was identified through virtual screening and optimized to generate 16 derivatives. Among them, compound 8a showed the strongest binding affinity for both FAK1 and FAK2, with KD values of 2.9 μM and 2.5 μM, respectively, and exhibited the best selectivity toward TNBC cells. Intracellular target engagement studies indicated that 8a protected specific peptides within the kinase domains of FAK1 and FAK2 in cells. Functionally, 8a induced G2/M-phase arrest and activated DNA damage-related signaling, accompanied by decreased phosphorylation of CDK1. The antiproliferative activity of 8a was markedly attenuated by FAK1/FAK2 knockdown but significantly enhanced in combined treatment with doxorubicin. In addition, 8a inhibited 4 T1 tumor growth in vivo without significant body weight loss. These findings identify pyrazolo[3,4-b]pyridine as a new chemotype for dual FAK1/FAK2 targeting and support 8a as a target-dependent lead compound for anti-TNBC development.