Inhibition of Mycobacterium tuberculosis UvrB by small molecules: Potent NER disruption and structural insights into...

The nucleotide excision repair (NER) pathway in Mycobacterium tuberculosis (Mtb) is important for DNA damage repair and bacterial survival under stress, yet specific inhibitors targeting its components remain scarce. Here, we targeted the UvrB protein, a central component of the Mtb UvrABC NER pathway, and identified novel small molecule inhibitors against its nucleotide binding domain (NBD). Using in silico structure-based screening involving the Maybridge library (∼54,000 compounds), Molecular dynamics (MD) simulations, and Biolayer interferometry (BLI), we identified four potent inhibitors: SPB08143, RJC04069, NRB00936, and DP00786 with IC50 values of 9.8 μM, 3.7 μM, 36.5 μM, and 37.68 μM, respectively, for disrupting the UvrB-DNA complex. Binding kinetics revealed a high affinity for SPB08143 (K_D = 0.31 μM), which is better than UvrB's affinity for its DNA substrate (K_D = 1.4 μM). Survival assays in Mycobacterium smegmatis demonstrated significant bactericidal activity, with SPB08143, RJC04069, and NRB00936 killing 85.3 %, 80.6 %, and 90 % of UV-treated cells, respectively, indicating effective NER inhibition. Small-angle X-ray scattering (SAXS) and Size exclusion chromatography (SEC) further revealed that apo Mtb UvrB adopts an open, extended dimeric conformation (Rg = 6.65 nm, Dmax = 17.4 nm), potentially facilitating DNA recognition. These inhibitors represent the first reported compounds targeting Mtb UvrB, and offer a novel strategy to inhibit Mtb DNA repair. Moreover, our findings provide structural and functional insights into UvrB inhibition by these compounds, with potential for development against drug-resistant Mtb strains.