Design, Synthesis, and Computational Insights into PKMYT1 Inhibitors for the Treatment of Breast Cancer

<b>Background</b>: Membrane-associated tyrosine-threonine protein kinase 1 (PKMYT1), which is identified as a synthetic lethal partner of <i>CCNE1</i>, emerged as a promising therapeutic target in oncology. <b>Methods</b>: A series of novel PKMYT1 inhibitors were designed by employing a pharmacophore fusion strategy. The underlying mechanisms were investigated by means of pharmacological experiments and molecular simulations. <b>Results</b>: Compound MY-14 demonstrated optimal kinase inhibition (IC₅₀ = 0.002 μM) and significant anti-proliferative efficacy against <i>CCNE1</i>-amplified cells (IC_50-HCC1569 = 1.06 μM and IC_50-OVCAR3 = 0.80 μM). Furthermore, MY-14 induced concentration-dependent apoptosis, inhibited colony formation, and effectively arrested cell-cycle progression at the S-phase through synthetic lethality. Molecular dynamics simulations, Hirshfeld surface analysis, dynamic cross-correlation matrix (DCCM), and MM/GBSA calculations elucidated the molecular mechanism underlying MY-14's interaction with PKMYT1. <b>Conclusions</b>: MY-14 emerged as a promising compound for the development of a novel PKMYT1 inhibitor.