Dynamics‐Based Structural Insight Into the Covalent Inhibition Toward Dengue Virus NS2B‐NS3 Protease by Peptide‐Boronic Acid Inhibitors

ABSTRACT Dengue fever, an infectious disease that is caused by the Dengue virus (DENV), has become an increasingly global health concern. The nonstructural protein NS2B‐NS3 protease is a critical target for anti‐DENV drug development. However, the rational design of covalent inhibitors targeting DENV NS2B‐NS3 protease remains a challenging task. For a class of peptide‐boronic acid‐based NS2B‐NS3 protease inhibitors, the introduction of a carbon atom strikingly elevated the inhibitory activity; however, the mechanism for this significant discrepancy remains elusive. In this study, we combined multiple computational methods to investigate the binding modes of compounds 5 and 6 toward the DENV NS2B‐NS3 protease, and clarified the underlying mechanism. The introduction of a carbon atom enables a strong interaction with Asp81* and a favorable distance between the boronic acid warhead and Ser135, facilitating noncovalent binding and covalent attack on NS2B‐NS3 protease. Amino acid mutation and a series of dynamics simulations confirmed the pivotal role of Asp81* in stabilizing inhibitor‐target binding. This study underscores the dynamics‐based computation in the rational design of covalent NS2B‐NS3 protease inhibitors, and provides a guide for the further development of novel NS2B‐NS3 protease inhibitors.