From structural insight to molecule: Integrative molecular simulations identify candidate pyruvate kinase activators.

Pyruvate kinase (PKLR) is a key glycolytic enzyme that regulates red blood cell energy homeostasis. Although Mitapivat is the first approved allosteric activator for pyruvate kinase deficiency (PKD), its clinical utility is limited by metabolic and pharmacokinetic challenges, underscoring the need for improved analogues. This study aimed to identify novel Mitapivat-derived scaffolds with enhanced PKLR binding, stability, and drug-like properties using an integrated computational approach. A total of 190 structurally related derivatives were screened through molecular docking, pharmacophore modeling, frontier molecular orbital (HOMO-LUMO) analysis, 200 ns molecular dynamics (MD) simulations, MM/PBSA free energy calculations, ADMET profiling, and retrosynthetic feasibility assessment. Comparative analyses were performed against Mitapivat and phenylalanine, an endogenous PKLR inhibitor. Among the screened compounds, CHEMBL3729403 and CHEMBL3729860 emerged as the most promising candidates. CHEMBL3729403 exhibited the strongest docking affinity (-9.10 kcal/mol) and superior MD stability, with the lowest average RMSD (0.419 nm) and highest hydrogen-bond occupancy (1.039). MM/PBSA calculations revealed stronger binding free energies for CHEMBL3729403 (-26.39 ± 3.21 kcal/mol) and CHEMBL3729860 (-23.05 ± 3.59 kcal/mol) than Mitapivat (-21.35 ± 3.46 kcal/mol) and phenylalanine (-3.89 ± 3.91 kcal/mol). Pharmacophore analysis demonstrated conservation of the key interaction features required for PKLR activation, while HOMO-LUMO analysis revealed comparable electronic characteristics across the lead compounds, supporting their compatibility with ligand-protein interactions. ADMET profiling predicted favorable oral absorption (81.5% and 81.3%), good intestinal permeability, acceptable drug-likeness, and the absence of predicted mutagenic, tumorigenic, reproductive, or irritant liabilities for CHEMBL3729403 and CHEMBL3729860. Retrosynthetic analysis further supported their synthetic accessibility through feasible sulfonamide-coupling routes. Overall, CHEMBL3729403 and CHEMBL3729860 were identified as promising putative PKLR activators with improved predicted binding, stability, and pharmacokinetic properties compared with Mitapivat. These findings warrant further experimental validation to confirm their therapeutic potential in PKD and related metabolic disorders.