Abstract:

Objective: To screen synergist of clofazimine (CFZ), a core drug for multidrug-resistant tuberculosis treatment, and investigate the mechanism underlying its enhanced antitubercular activity in relation to the inhibition of Mycobacterium tuberculosis mycobacterial membrane protein large5-mycobacterial membrane protein small5 (MmpL5-MmpS5) efflux system. Methods: The Rv0678 mutant strain Rv0678-M1 (C305T) was selected as the screening strain. The microplate colorimetric method was employed to screen CFZ synergist. The checkerboard method was used to evaluate the combined effects of zuclopenthixol and CFZ against different Mycobacterium tuberculosis strains. Molecular docking was performed to predict interactions between zuclopenthixol and MmpL5 or MmpL5-MmpS5 system. Surface plasmon resonance was utilized to analyze the binding affinity between zuclopenthixol and MmpL5 protein. Ethidium bromide (EtBr) accumulation assays were performed to assess the impact of zuclopenthixol on efflux activity in Rv0678-M1 strains. Results: Zuclopenthixol at 1/8×MIC reduced the MIC of CFZ against Rv0678 mutant strain to 1/16 of its original value, reversing CFZ resistance. Synergistic activity between zuclopenthixol and CFZ was observed in different M.tuberculosis strains (FICI=0.25-0.375), with stronger synergistic activity in Rv0678 mutants (FICI=0.25) and MmpL5-MmpS5 overexpression strains (FICI=0.25) compared to H37Rv (FICI=0.3125) and mmpS5 knockout strains (FICI=0.375). Surface plasmon resonance revealed moderate affinity between zuclopenthixol and MmpL5 (Kd=3.075×10^-4 mol), which was weaker than the affinity of CFZ for MmpL5 (Kd=1.218×10^-5 mol). Conclusion: Zuclopenthixol significantly enhances CFZ’s antitubercular activity by inhibiting the MmpL5-MmpS5 efflux system.

Key words: Mycobacterium tuberculosis, Zuclopenthixol, Clofazimine, Molecular mechanisms of pharmacological action