S292L mutation in Rv1258c efflux pump drives pyrazinamide efflux and a novel inhibitor designed for co-therapy to improve MDR-TB treatment outcomes.

Tuberculosis (TB) remains a major global health challenge, exacerbated by the rise of drug-resistant Mycobacterium tuberculosis (Mtb) strains that undermine first-line therapies. Resistance to pyrazinamide (PZA), an essential anti-TB drug, is often linked to mutations in the pncA gene and efflux pump genes such as Rv1258c. The Rv1258c efflux pump not only expels antibiotics, reducing intracellular drug concentrations, but also enhances bacterial survival under stress, making it a promising therapeutic target. This study investigated the effects of two critical Rv1258c mutations, V219A and S292L, on drug efflux function and inhibitor binding. Molecular dynamics simulations revealed that both mutations significantly altered the thermodynamic stability and binding energetics of the efflux pump. Comparative MM/GBSA analysis showed that the binding free energy (ΔG) of PZA with wild-type Rv1258c (-17.08 ± 1.66 kcal/mol) was moderately weakened in mutants S292L (-16.01 ± 0.82 kcal/mol) and V219A (-11.68 ± 3.31 kcal/mol), suggesting reduced drug affinity. Virtual screening further identified three potent inhibitors that met all ADMET criteria. MM/GBSA calculations indicated these inhibitors had substantially stronger binding affinities than PZA, with ΔG values of -46.49 ± 3.97 kcal/mol, -19.61 ± 4.58 kcal/mol, and -28.14 ± 3.61 kcal/mol for ZINC000000035064, ZINC000000040452, and ZINC000000040453, respectively. These findings suggest that targeting Rv1258c could effectively restore intracellular drug concentrations even in resistant strains. Overall, this study provides a strong foundation for the development and experimental validation of efflux pump inhibitors as a novel therapeutic strategy against multidrug-resistant TB.