Conformation-driven design, synthesis and biological evaluation of novel PAK1 inhibitors that downregulate PD-L1 for triple-negative breast cancer therapy.

The serine/threonine kinase PAK1 is increasingly recognized as a pivotal regulator of tumor progression and immune evasion in triple-negative breast cancer (TNBC). However, the development of PAK1 inhibitors with both high potency and favorable drug-like properties remains challenging. Herein, we report the design, synthesis, and biological evaluation of a series of novel PAK1 inhibitors guided by a conformation-driven optimization strategy. We identified ZMF-28, a potent PAK1 inhibitor with an IC50 of 0.02 μM, exhibiting high selectivity in the kinase profiling assay. Notably, ZMF-28 exhibited 10-fold higher cellular antiproliferative activity against 4T1 cells (IC50 = 0.22 μM) than the clinical candidate FRAX-486, and achieved significant tumor growth inhibition in vivo without overt toxicity, and demonstrated favorable pharmacokinetics. Mechanistically, ZMF-28 directly binds to PAK1 and blocks its kinase activity, thereby downregulating PD-L1 expression and reversing the immunosuppressive tumor microenvironment, representing a dual-action therapeutic strategy for TNBC. These findings establish ZMF-28 as a promising therapeutic candidate for TNBC and highlight the value of conformation-guided design in kinase inhibitor discovery.