Molecular dynamics insights into allosteric and orthosteric co-targeting of Mycobacterium tuberculosis thymidylate kinase: A structure-based approach for bitopic ligand design

• This study explored allosteric regulation of Mycobacterium tuberculosis thymidylate kinase (TmpK), a key enzyme in nucleotide metabolism. • 4-Hydroxyestrone (4HY) exhibited strong binding at both orthosteric (−11.5 kcal/mol) and allosteric (−7.1 kcal/mol) sites during virtual screening. • Molecular Dynamics Simulations revealed that single-site binding-maintained stability, whereas dual-site occupancy disrupted allosteric communication and induced conformational changes. • Root mean square fluctuation analysis indicated increased flexibility in the Ser150-Glu170 loop, highlighting its probable role in allosteric regulation. • Findings support allosteric site targeting and bitopic ligands as a strategy for developing new treatments for multidrug-resistant tuberculosis. Allosteric regulation is a fundamental mechanism in enzyme function and drug design, enabling conformational changes upon ligand binding at sites distinct from the active site. In this study, we employed computational approaches to identify and characterize the allosteric site of Mycobacterium tuberculosis thymidylate kinase (TmpK), a crucial enzyme for bacterial nucleotide metabolism. The 3D crystal structure of TmpK (PDB ID: 1W2G ) was analyzed for potential allosteric pocket residues using the PASSer server. Virtual screening and molecular docking studies revealed 4-Hydroxyestrone (4HY) as high-affinity binding moiety at both the orthosteric and allosteric sites, with docking scores of −11.5 kcal/mol and −7.1 kcal/mol, respectively. Molecular dynamics (MD) simulations also demonstrated that ligand binding at a single site to conserve structural stability, whereas simultaneous occupancy of both sites induced significant conformational perturbations, disrupting allosteric network. Pocket volume analysis also further indicated that ligand binding at one site dynamically modulated the cavity size of the other, driven by the mobility of the Ser150-Glu170 loop. Additionally, root mean square fluctuation (RMSF) analysis also highlighted increased flexibility in this region, reinforcing its role in allosteric regulation. These findings provide mechanistic insights into TmpK allostery and establish a computational framework for rational drug design. Furthermore, the study underscores the potential of allosteric inhibitors and bitopic ligands, which simultaneously target orthosteric and allosteric sites, as a promising strategy to combat multidrug-resistant tuberculosis, a critical challenge in global TB Elimination efforts.