MTK_MCPA: A novel magnesium-competitive inhibitor of Mycobacterium tuberculosis thymidylate kinase.

Tuberculosis (TB) is one of the leading causes of mortality among infectious diseases. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains necessitates the development of novel therapeutic agents. The current study aimed to screen some novel natural inhibitors against TB. Here, we present the computational identification and characterization of N-mesityl-2-((2-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[d]pyrimidin-4-yl)thio)acetamide (MTK_MCPA) as a potential inhibitor of Mycobacterium tuberculosis thymidylate kinase (TMPKmt), an essential enzyme involved in DNA replication. Through structure-based virtual screening using DrugClip, MTK_MCPA was identified from a library of natural compounds and selected for its good binding affinity (Vina score -8.68 kcal/mol), favorable drug-like properties (MW 343.44 Da, LogP 3.05, QED 0.66), and no violations of Lipinski's Rule of Five. DFT computational analysis revealed a HOMO-LUMO energy gap of 0.1347 a.u (∼3.67 eV), with the electron density distributed across protein-ligand interactions. Molecular dynamics simulations over 500 ns demonstrated stable formation of the MTK_MCPA-TMPKmt complex and specific interactions involving hydrogen bonds with Tyr179, Lys13, Arg149, Arg95, and Asp9, along with hydrophobic contacts. Principal component and free energy landscape analyses confirmed that MTK_MCPA traps TMPKmt in multiple low-energy conformational states that prevent the magnesium coordination required for catalytic activity. Mechanistically, MTK_MCPA inhibits TMPKmt via a hybrid hydrophobic-electrostatic strategy and exhibits favorable drug-like properties, including the mesityl-thioacetamide region, which functions as a "hydrophobic clamp" that captures the LID in a semi-closed conformation, blocking Mg2+ binding. The average total binding free energy was (dG) of -55.32 kcal/mol, and a non-solvent contribution (dG(NS)) averaging -86.87 kcal/mol. These negative values indicate strong interactions, including van der Waals forces and hydrogen bonding. ADMET profiling confirmed highly favorable medicinal properties. These computational findings show that MTK_MCPA may serve as a promising lead compound for anti-TB drug development with potential efficacy against drug-resistant TB strains.