Potential Use of Nitrate Reductase as a Biomarker for the Identification of Active and Dormant Inhibitors of Mycobacterium tuberculosis in a THP1 Infection Model

Abstract

The development of a macrophage-based, antitubercular high-throughput screening system could expedite discovery programs for identifying novel inhibitors. In this study, the kinetics of nitrate reduction (NR) by Mycobacterium tuberculosis during growth in Thp1 macrophages was found to be almost parallel to viable bacilli count. NR in the culture medium containing 50 mM of nitrate was found to be optimum on the fifth day after infection with M. tuberculosis. The signal-to-noise (S/N) ratio and Z-factor obtained from this macrophage-based assay were 5.4 and 0.965, respectively, which confirms the robustness of the assay protocol. The protocol was further validated by using standard antitubercular inhibitors such as rifampicin, isoniazid, streptomycin, ethambutol, and pyrazinamide, added at their IC(90) value, on the day of infection. These inhibitors were not able to kill the bacilli when added to the culture on the fifth day after infection. Interestingly, pentachlorophenol and rifampicin killed the bacilli immediately after addition on the fifth day of infection. Altogether, this assay protocol using M. tuberculosis-infected Thp-1 macrophages provides a novel, cost-efficient, robust, and easy-to-perform screening platform for the identification of both active and hypoxic stage-specific inhibitors against tuberculosis.

Design and synthesis of 11α-substituted bile acid derivatives as potential anti-tuberculosis agents

“…In a preliminary screening, the antimycobacterial activity of these compounds was assessed at concentrations of 30, 10, 3 μg/mL using XRMA antitubercular screening protocol. 19,20 The antimycobacterial activity results are summarized in Table 1.…”

“…Compounds 2b, 3, 7, and 8 tested in this study exhibited high antitubercular activities against both dormant and active stages of MTB under both in-vitro and ex-vivo conditions. Minimum Inhibitory Concentration (MIC) values of the most active compound (2b) determined against the dormant state were 2.43 and 1.55 μg/mL respectively when measured under invitro 19,20 and ex-vivo 21 conditions. Interestingly, compound 2b was found to be almost equally effective against the actively growing MTB bacilli.…”

Intrinsic therapeutic and biocatalytic roles of ionic liquid mediated self-assembled platinum–phytase nanospheres

“…37 The culture was then incubated for a further 24 h to allow macrophage conversion in the plate. 37 The culture was then incubated for a further 24 h to allow macrophage conversion in the plate.…”

Naturally Occurring Anti-TB Agents: Isolation, Chemical Transformations and In Vitro Antitubercular Activities of Secondary Metabolites of Rhizomes of Alpinia galanga

“…The stock culture was maintained at − 70°C and subcultured once in M. phlei medium before inoculation into the experimental culture. All experiments were performed in triplicate, and IC 50 and MIC values were calculated from their dose-response curves.…”

“…The isolated compounds 1-6 (l " Fig. 1) were assayed in vitro against active and dormant phenotypes of M. tuberculosis H37Ra, respectively, using an established XTT reduction menadione assay (XRMA) antitubercular screening protocol [50][51][52]. The first-line antitubercular drug rifampicin (Sigma) was used as a reference standard and data obtained are presented in l " Table 1.…”