Discovery and Pharmacological Characterization of a Dual-Acting 2-Amino-3-Cyanopyridine Inhibitor Targeting...

Microtubules are critical regulators of mitotic progression and cancer cell proliferation. Herein, we report a novel 2-amino-3-cyanopyridine-based scaffold that functions as a dual inhibitor of tubulin polymerization and cyclin G-associated kinase (GAK). Structure-activity relationship (SAR) optimization identified KMG-732 with nanomolar to submicromolar cytotoxicity across diverse cancer cell lines. Mechanistic investigations revealed that KMG-732 targets the colchicine-binding site, effectively disrupting microtubule assembly and triggering mitotic arrest followed by apoptosis. Beyond its antimitotic effects, KMG-732 significantly attenuates cancer cell migration and invasion. Importantly, KMG-732 displayed favorable pharmacokinetic properties and showed minimal P-glycoprotein-mediated efflux. KMG-732 demonstrated robust antitumor efficacy in both organoids and in vivo xenograft models, maintaining a superior safety profile with reduced systemic toxicity compared to colchicine. Kinome profiling and biochemical assays confirmed selective GAK inhibition, distinguishing KMG-732 from traditional tubulin inhibitors. Collectively, KMG-732 represents a promising dual-targeting antimitotic lead candidate with enhanced efficacy and therapeutic window for cancer treatment.