Recent studies suggest that host defense mechanisms like autophagy, inflammation, oxidative stress and apoptosis in macrophages plays a significant role in host defense against intracellular pathogens like viruses, fungi, protozoan, and bacteria, including Mycobacterium tuberculosis (M. tb). It is still unclear if micromolecules inducing host defense mechanisms could be an attractive approach to combat the intracellular burden of M. tb. Hence, the present study has investigated the anti-mycobacterial effect of apoptosis mediated through phenotypic screening of micromolecules Shows Ac-93253 a potent candidate against the mycobacteria in THP-1 cells. Through MTT and trypan blue exclusion assay, 0.5 &muM of Ac-93253 was found to be noncytotoxic even after 72 h of treatment in phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. We have found that Ac-93253 treatment does not affect autophagy regulation, ROI, or RNI generation in uninfected and mycobacteria-infected dTHP-1 cells. At the same time point and same concentration, inflammation was also not affected upon Ac-93253 treatment. Significant regulation in the expression of various pro-apoptotic genes like Bcl-2, Bax, and Bad and the cleaved caspase 3 was observed upon treatment with a non-cytotoxic dose of Ac-93253. Ac-93253 treatment also leads to DNA fragmentation and increased phosphatidylserine accumulation in the plasma membrane's outer leaflet. Further, Ac-93253 also effectively reduced the growth of mycobacteria in infected macrophages, Z-VAD-FMK a broad range apoptosis inhibitor significantly brought back the mycobacterial growth in Ac-93253 treated macrophages. Ac-93253 treatment manipulates the mitochondrial membrane potential, CsA a mitochondrial membrane potential stabilizer, substantially inhibits the apoptosis and abrogates the anti-mycobacterial effect of Ac-93253. These findings suggest apoptosis may be the probable effector response through which Ac-93253 manifests its anti-mycobacterial property.

Keywords: Tuberculosis Mycobacteria Ac-93253 Host directed therapy Mitochondrial membrane potential Apoptosis