Development of MKI-3: A Potent and Selective MASTL Inhibitor with Improved Efficacy for Cancer Treatment

Microtubule-associated serine/threonine kinase-like (MASTL) is a primary regulator of mitosis and a potential cancer target. We report the discovery and optimization of MASTL kinase inhibitor-3 (MKI-3), a potent and selective MASTL inhibitor with nanomolar cellular activity and enhanced metabolic stability, suitable for <i>in vivo</i> assessment. Structure-activity relationship studies identified MKI-3, a quinazoline-based scaffold that enhances MASTL binding, suppresses endosulfine alpha (ENSA) phosphorylation, and activates protein phosphatase 2A. MKI-3 inhibited MASTL with a half-maximal inhibitory concentration of 5.72 nM and exhibited strong selectivity over other AGC kinases. In breast cancer cell lines (MCF7, MDA-MB-231, BT549, and 4T1), MKI-3 exhibited robust antiproliferative effects at nanomolar concentrations. Mechanistically, MKI-3 disrupted the MASTL-ENSA-Aurora A signaling axis, inducing chromosomal instability, mitotic catastrophe, and apoptosis. MKI-3 significantly reduced tumor growth in treatment-refractory 4T1 triple-negative breast cancer without toxicity and outperformed MKI-2 supporting MASTL-targeted therapy.