Study on the binding mechanism between SARS-CoV-2 3CL protease and chiral isomers of its inhibitor pyridyl-urea diyne ester

3CL protease (3CLpro) of SARS-CoV-2 is a pivotal enzyme required in coronavirus replication and transcription. Its highly conserved structure and the absence of homologous proteins in the host make it an ideal target for broad-spectrum anti-coronavirus drug development. In this work, we systematically investigated and compared the binding modes and dynamic properties of the four stereoisomers of a pyridyl-urea diyne ester (PyDU) molecule with two chiral centers within the 3CLpro active site. Through molecular docking, MD simulations, MM/GBSA binding free-energy calculations, and DCCM analysis, all four stereoisomers were stabilized primarily by hydrophobic packing. Among them, the (R,S) isomer exhibited the best overall performance, including docking score, binding free-energy components, and key residue interactions. The (R,S) and (S,R) isomers also enhanced the cooperative motions around the binding pocket, while the (R,S) isomer further modulated the flexibility of domain III, which may influence 3CLpro dimerization. Conversely, the (S,S) isomer exhibited the weakest affinity due to insufficient hydrophobic contact. By innovatively integrating chirality, binding energy and protein dynamical features, we revealed the dual role of chirality in modulating affinity and dynamic responses, which provides a theoretical basis for the chiral-guided design of coronavirus inhibitors.