Free energy perturbation-derived identification of natural compounds targeting Mycobacterium tuberculosis thymidylate kinase.

(Mtb), the cause of tuberculosis, remains a major global health threat. TB spreads via airborne transmission through sneezing and coughing, thereby compromising respiratory health. The emergence of drug-resistant strains necessitates the development of new anti-tubercular agents. Natural products from plants, microbes, and marine sources are promising for drug discovery because of their structural diversity and biological activities. Current research seeks natural thymidylate kinase inhibitors to prevent TB, using methods such as binding-site identification, similarity searches, interaction analyses, and 100-ns MD simulations. Binding free energy is evaluated with MM-GBSA, and FEP studies identify molecules based on binding properties. In silico methods to explore natural compounds as TMPK putative inhibitors include fingerprint searches, AutoDock Vina docking, ADMET predictions, GROMACS MD simulations, and energy calculations. Molecular docking revealed binding energies of -12.10, -11.90, and − 13.40 kcal/mol for CNP0015705, CNP0363196, and CNP0217487, respectively, which are better than those of the co-crystal ligand and Lifirafenib. Moreover, the MM-GBSA-based binding energy ranged from − 14.50 to -22.86 kcal/mol, indicating a strong association between the proposed molecules and the TMPK protein. A virtual library was screened against Mtb TMPK, with promising candidates showing strong binding. Selected compounds CNP0015705, CNP0363196, and CNP0217487 preferentially bind to TMPK, as shown by various in silico analyses. This approach demonstrates the potential of natural products in drug discovery, highlighting their role in developing new therapies targeting TMPK.