A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

Abstract

Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

A Drug Repositioning Approach Reveals that Streptococcus mutans Is Susceptible to a Diverse Range of Established Antimicrobials and Nonantibiotics

“…bacteria in high-throughput screens of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, as well as M. tuberculosis (18,23). Screening for compounds using the AK assay offers numerous advantages: specific detection of bactericidal drugs; increased sensitivity over standard growth-based or alamarBlue assays; detection of bioactive molecules below the MIC (22); and, importantly, the ability to detect antibiofilm activity of drugs (18).…”

Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model

“…There is a clear and desperate need for new medicines to treat TB. As working with M. tuberculosis limits research and preclinical drug development to those laboratories around the world with the resources and facilities to safely handle the bacterium, non-tuberculous mycobacteria such as M. marinum are widely used as surrogates 5, 7, 41, 42 . A major drawback of using M. marinum for antimycobacterial compound screening is that the in vitro resistance profile of the bacterium is very different to M. tuberculosis .…”

“…Due to the difficulties and dangers involved in culturing M. tuberculosis , an airborne Biosafety Level 3 pathogen, faster-growing and less pathogenic mycobacterial species, such as M. smegmatis and M. marinum , are routinely exploited for TB research and anti-mycobacterial drug discovery 5–8 . M. marinum is a pathogen of ectotherms (fish, amphibians and reptiles) that produces a tuberculosislike disease 9 .…”

New Urea Derivatives as Potential Antimicrobial Agents: Synthesis, Biological Evaluation, and Molecular Docking Studies

“…Samples were prepared in DMSO and water to a final testing concentration of 32 µg/mL or 20 µM (unless otherwise indicated in the datasheet), in 384-well, non-binding surface plate (NBS) for each bacterial/fungal strain, in duplicates (n = 2); the final DMSO concentration was kept to a maximum of 1% DMSO [29][30][31][32][33]. All sample preparations for the antimicrobial studies were done using liquid handling robots.…”