Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis

Abstract

The REMA and HyRRA assays will be useful for anti-tubercular anti-dormancy compound screening and drug susceptibility testing in a safe, reliable, easy and cost-effective manner particularly in low resource countries. The application of the assays in M. smegmatis or M. bovis BCG offers the distinct advantage of rapidly and safely screening anti-tubercular compounds in a high-throughput format.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

“…These species are known to exhibit different degrees of susceptibility to rifampin and other anti-TB drugs (50). We have considered the following possibilities to account for the differential susceptibility to imipramine and norclomipramine.…”

Boronic-aurone Derivatives as Anti-Tubercular Agents: Design, Synthesis and Biological Evaluation

“…The utility of Alamar Blue as a phenotypic high-throughput cell viability screen for identification of potentially new TB leads for screening of clinical isolates of MTB has been demonstrated [23,24], among others. In addition, several studies have also shown the reliability of Alamar blue cell viability assay for detection of drug resistant MTB [25,30].…”

“…The resazurin-based assay is a simple, inexpensive, and effective method with very high diagnostic accuracy, and lends itself to easy use in countries or even laboratories with limited resources [23,24]. Whole-cell screening is getting renewed attention in the drug discovery process compared to target-based method [31,32].…”

“…The resazurin microtiter assay (REMA) was performed in 7H9 medium containing Middlebrook broth and 0.5% glycerol and supplemented with oleic acid, albumin, dextrose, and catalase (Difco TM ) as previously reported [23][24][25]. Clear 96-well plates were inoculated with 100 μL of drug diluted with 90 µl of 7H9 medium and 10 µL DMSO (giving essentially 50 µL of drug).…”

Antitubercular In Vitro Drug Discovery: Tools for Begin the Search

“…An inexpensive alternative is the combination of in vitro model of mycobacterial dormancy with colorimetric methods, recently, Khan & Sarkar (2008) have developed a dormant stage specific antitubercular screening protocol in microplate format using Wayne's hypoxic model and nitrate reductase activity in M. bovis BCG (Bacillus Calmette-Guérin) culture. In addition, Taneja & Tyagi (2007) used resazurin reduction assay to develop screening for searching anti-dormancy and anti-tubercular compounds (Khan & Sarkar, 2008;Taneja & Tyagi, 2007).…”