Free energy perturbation and machine learning-assisted identification of potential MAP3K8 hit molecules: a comprehensive structure- and ligand-based studies.

MAP3K8, also known as COT or Tpl2, is a serine/threonine kinase that plays a pivotal role in regulating the MAPK signaling pathway and pro-inflammatory cytokine production, making it an attractive therapeutic target for inflammatory disorders and cancer. In this study, an integrated ligand- and structure-based virtual screening workflow was used to identify potential MAP3K8 inhibitors. A pharmacophore model derived from the co-crystal ligand bound to the COT kinase domain was used to screen the ChEMBL, PubChem, and ZINC databases, yielding 20,566 unique molecules. Sequential filtering through RMSD and binding-score thresholds, RDKit-based similarity searching, AutoDock Vina molecular docking, ADMET evaluation, and ChemMASTER-guided clustering reduced the dataset to four structurally diverse lead candidates designated as PM1, PM2, PM3, and PM4. All four molecules preserved the canonical hinge-binding pharmacophore of the reference ligand and exhibited binding energies between - 9.0 and - 10.2 kcal/mol, ranking more favourably than the co-crystal ligand with - 8.6 kcal/mol. The 200 ns molecular dynamics (MD) simulations confirmed the structural stability of the complexes, supported by favourable RMSD, RMSF, RoG, SASA, hydrogen-bond, PCA, and free energy landscape profiles. MM-GBSA calculations revealed that the binding free energies of PM2, PM3, and PM4 were - 42.12, - 38.46, and - 36.96 kcal/mol, respectively, outperformed the co-crystal ligand with - 34.36 kcal/mol, while FEP analysis identified PM3 with - 14.59 kcal/mol as the most thermodynamically favourable test compound, followed by PM2 and PM4. The convergence of docking, dynamics, and free-energy results prioritized PM2, PM3, and PM4 as promising MAP3K8 hit candidates, which require further experimental validation and lead optimization.