Investigating the anti-TNBC potential of new quinazoline-based PAK4 inhibitors: design, synthesis and biological evaluation.

A series of quinazoline-based PAK4-selective inhibitors were designed via structure-based modification of leads Czh-226 and compound 12i using a heterocyclic tail strategy. Kinase and cell viability assays identified two close analogues, 11j and 11k, differing only by the position of one nitrogen atom. Both showed low nanomolar PAK4 inhibition, high selectivity over PAK1, and low-micromolar antiproliferative activity against three TNBC cell lines, with 11j consistently more potent. Molecular docking and dynamics simulations indicated that 11j formed more stable interactions with the PAK4 catalytic pocket. Mechanistically, 11j inhibited PAK4 and downstream signaling pathways, reduced MAPK activation, up-regulated p53, induced G0/G1 arrest, promoted apoptosis, and raised ROS levels, thereby suppressing TNBC cell proliferation, migration, and invasion. Notably, 11j down-regulated c-Src phosphorylation and sensitized TNBC cells to Gefitinib, an EGFR-TKI. Co-treatment with a non-toxic concentration of 11j markedly lowered the IC50 of Gefitinib across three TNBC lines, and Gefitinib reciprocally enhanced 11j's anti-TNBC effects. In an MDA-MB-231 orthotopic xenograft model, 11j alone at 25 and 50 mg/kg/d significantly and dose-dependently inhibited tumor growth (TGI = 33.70% and 53.17%, respectively), while the combination of 11j (15 mg/kg/d) with Gefitinib (10 mg/kg/d) produced enhanced efficacy (TGI = 60.39%) and reduced lung metastasis without overt toxicity. Collectively, these results validate 11j as a promising lead for TNBC therapy, especially in combination with EGFR-targeted agents.