The conformational landscape of dengue NS2B-NS3 protease is modulated by ligand stereochemistry: An integrated molecular dynamics, Markov state modeling, and explainable AI study

The dengue virus NS2B-NS3 protease is a validated antiviral target whose pronounced conformational plasticity presents a major challenge for structure-based inhibitor design. Although stereochemistry is a fundamental determinant of molecular recognition, how ligand chirality modulates the dynamic and kinetic states of the protease remains poorly understood. Here, we investigated whether stereochemical optimization can reshape the conformational landscape of NS2B-NS3 using a series of diketopiperazine (DKPZ)-based ligands and their D-enantiomeric derivatives. An integrated computational framework combining molecular docking, 200-ns molecular dynamics simulations, Markov state modeling (MSM), principal component analysis (PCA), free-energy landscape (FEL) mapping, dynamic residue network (DRN) analysis, MM/PBSA, and explainable artificial intelligence (XAI) was employed to resolve ligand-induced changes across structural, energetic, kinetic, and communication levels. While all ligands maintained stable engagement with the protease binding pocket, D-enantiomeric derivatives produced markedly distinct conformational signatures. In particular, d-DKPZ promoted compact, energetically favorable conformational ensembles and persistent metastable states, accompanied by reduced flexibility and more organized residue-to-residue communication. MSM further revealed that stereochemical optimization altered state populations and transition dynamics, favoring kinetically persistent conformations rather than highly fluctuating ensembles. DRN and SHAP-based XAI independently converged on key catalytic-pocket and stabilizing residues, providing an interpretable link between ligand stereochemistry, local interactions, and global conformational behavior. Comparative analysis of the parent compounds and their D-enantiomeric analogues reveal how stereochemical changes influence binding energetics, conformational dynamics, and residue communication in dengue NS2B-NS3 protease. These findings establish stereochemical optimization as a promising strategy for guiding the design and prioritization of candidate dengue protease inhibitors.