Antimicrobial, synergistic and autophagic effects of medicines for Malaria venture pathogen box compounds on resistant strains of Mycobacterium tuberculosis and Neisseria gonorrhoeae

Abstract

Antimicrobial resistance in Mycobacterium tuberculosis and Neisseria gonorrhoeae is emerging globally. Due to the limited treatment options, the World Health Organization has designated M. tuberculosis and N. gonorrhoeae as two critical and high-priority pathogens for research and development of novel antibiotic drugs. Particularly for M. tuberculosis, new agents that can induce autophagy processes directed at clearing intracellular M. tuberculosis from host cells is highly needed. To speed up the discovery and development of novel agents, the Medicines for Malaria Venture (MMV) group developed the Pathogen Box, containing a collection of 400 novel drug compounds. The Pathogen Box was originally assessed primarily for anti-malarial properties but, in the initial screen, has been shown to contain compounds potentially also effective against several other microorganisms, including M. tuberculosis. The aim of this study was to explore the antibiotic potential, including synergistic and autophagic effects, of this diverse compound library of the MMV Pathogen Box. As a first step, the identities and resistance profiles of clinical strains of M. tuberculosis and N. gonorrhoeae selected for use in this study were confirmed, using GeneXpert MTB/RIF and MTBDRplus assays, followed by whole genome sequencing (WGS). Broth microdilution assay was used to determine the pathogen-specific minimum inhibitory and minimum bactericidal concentrations (MICs/MBCs) of the Pathogen Box compounds (PBCs) against reference strains of M. tuberculosis and N. gonorrhoeae. Finally, a checkerboard assay approach was used to determine synergy between the active compounds if used in combination with reference drugs. Time-kill kinetics was performed to determine bactericidal or bacteriostatic activity. Selecting priority compounds for further investigation was based on the following criteria: (1) MIC and MBC for N. gonorrhoeae ≤ 10 µM; and (2) MIC and MBC for M. tuberculosis ≤ 0.625 μM. Five PBCs, MMV676603, MMV687146, MMV687696, MMV687180, and MMV153413, showed potent activity against both susceptible and multidrug-resistant M. tuberculosis strains at MIC and MBC below 0.625 μM. Except for MMV687696, the remaining four PBCs were clearly bactericidal. Combining the PBCs with isoniazid or rifampicin demonstrated either synergistic or additive activity, with fractional inhibitory concentration indexes ranging between 0.18 and 2.60. The five selected anti-TB PBCs recorded low cytotoxicity in murine-derived macrophages and effectively suppressed the growth of intracellular M. tuberculosis. Western blotting analysis was used to assess the potential of the five selected PBCs to induce autophagy against intracellular M. tuberculosis in host cells. All compounds induced some level of LC3 lipidation and LC3II/LC3I, although this was not statistically significant compared to controls. Notably, inhibition of the autophagic flux reversed the anti-mycobacterial activity of MMV676603, MMV687146, and MMV687180. Eight PBCs, MMV676501, MMV002817, MMV688327, MMV688508, MMV024937, MMV687798 (Levofloxacin), MMV021013, and MMV688978 (Auranofin), demonstrated potent activity against resistant strains of N. gonorrhoeae at a MIC and MBC of ≤ 10 µM. All the compounds showed potent bactericidal activity between 4 and 24 hrs, with time-kill kinetics similar to that of ceftriaxone. The N. gonorroheae active PBCs in combination with ceftriaxone showed either synergistic or additive activity with fractional inhibitory concentration indexes ranging between 0.40 to 1.8. Conclusion. This study has identified novel compounds with potent activity against both resistant and susceptible strains of N. gonorrhoeae and M. tuberculosis. The study has also identified compounds that can suppress the growth of intracellular M. tuberculosis with the potential to induce autophagy at high concentrations. Overall, the study results point to promising anti-gonococcal and anti-TB drug leads worthy of further exploration.