Integrative subtractive genomics and molecular dynamics-based approach for drug repurposing against female genital...

Female genital tuberculosis (FGTB) often remains untreated because many patients show no clear symptoms or present with vague clinical symptoms. Poor reproductive outcomes and prolonged treatment are usually the results of this delay in detection. Current TB therapies are costly and toxic, especially for strains of the disease that are resistant to several drugs. These drawbacks highlight the urgent need for safer and more effective treatment options. Computational drug discovery approaches targeting uterine TB remain limited, and essential pathogen-specific proteins non-homologous to humans are underexplored. This work identified GMP synthase as an important and specific target in Mycobacterium tuberculosis using subtractive genomics and drug repurposing. To improve specificity, targets that are similar to commensal and human proteins were discarded. 2619 FDA-approved drugs were virtually screened, and probenecid was found to be a probable choice. Quantum chemical analysis confirmed the robust binding and stable interactions of probenecid with the target. Molecular dynamics simulations and RMSD- Rg -based- Free Energy Landscape (FEL), along with PCA- based- Free Energy Landscape (FEL), confirmed the stability of the protein-ligand complex, and molecular docking identified favorable binding patterns. Probenecid inhibited M. tuberculosis H37Rv at a minimum inhibitory concentration (MIC) of 50 µg/mL, according to in vitro studies, while isoniazid and rifampicin had MICs of 0.20 µg/mL and 0.25 µg/mL, respectively. However, Probenecid's well-established safety profile and pharmacokinetic characteristics indicate that localized administration via a liposomal vaginal formulation may achieve therapeutic concentrations at the infection site while lowering systemic exposure, even though it is less effective than first-line medications. Future study will focus on mucosal safety, local pharmacokinetics, and formulation development. Overall, our findings highlight the usefulness of combining subtractive genomics, computational modeling, and experimental validation in tuberculosis drug discovery and indicate the possibility of repurposing probenecid for FGTB.