Identification of a 2-substituted Benzimidazole Derivative with Antimycobacterial Activity and Synergistic Interaction with Rifampicin

Infectious diseases remain a persistent global health burden, with bacterial infections accounting for a substantial disease burden. The increasing prevalence of antimicrobial resistance has emphasised the need for the development of novel antibacterial drugs. In the present study, the antibacterial activity of novel 2-substituted benzimidazole derivatives (NR-1 to NR-9) was evaluated against three bacteria, viz. M. smegmatis, B. subtilis and E. coli using estimation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Two (NR-4 and NR-5) exhibited inhibitory activity against M. smegmatis, while two (NR-5 and NR-7) were active against B. subtilis, with MICs ranging from 62.5 to 250 g/mL. Notably, NR-5 demonstrated antibacterial activity against both M. smegmatis and B. subtilis, with more efficacy against M. smegmatis (MICs: 125 g/mL vs 62.5 g/mL), bactericidal activity with an MBC/MIC ratio of 1 and low toxicity against Vero cells. Furthermore, NR-5 exhibited concentration-dependent killing kinetics and a synergistic interaction with Rifampicin (FICI: 0.498) against M. smegmatis. Molecular docking analysis predicted thymidylate kinase (Tmk) with a binding energy of -6.61 Kcal/mol as a plausible target of NR-5, and molecular dynamics simulation supported the stability of the Tmk-NR-5 complex over 100 ns of simulation. Thus, based on its potency, low-toxicity antimycobacterial activity and synergistic action with rifampicin, NR-5 is identified as a preliminary antimycobacterial lead against M. smegmatis.