visualization-charcoal-agar-resazurin-assay-for-semi-quantitative

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Weill Cornell Medical College

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, synthesis and structure-activity relationships of a series of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a replicating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and 72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI). As illustrated by compound 70 (MIC90 < 0.049 μg/mL, SI > 512), the 5-nitrofuranyl group was compatible with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay, making this class of compounds good candidates for further evaluation and target identification.

Benzimidazole-based compounds kill Mycobacterium tuberculosis.

It is urgent to introduce new drugs for tuberculosis to shorten the prolonged course of treatment and control drug-resistant Mycobacterium tuberculosis (Mtb). One strategy toward this goal is to develop antibiotics that eradicate both replicating (R) and nonreplicating (NR) Mtb. Naturally occurring (+)-calanolide A was active against R-Mtb. The present report details the design, synthesis, antimycobacterial activities, and structure-activity relationships of synthetic calanolides. We identified potent dual-active nitro-containing calanolides with minimal in vitro toxicity that were cidal to axenic Mtb and Mtb in human macrophages, while sparing Gram-positive and -negative bacteria and yeast. Two of the nitrobenzofuran-containing lead compounds were found to be genotoxic to mammalian cells. Although genotoxicity precluded clinical progression, the profound, selective mycobactericidal activity of these calanolides will be useful in identifying pathways for killing both R- and NR-Mtb, as well as in further structure-based design of more effective and drug-like antimycobacterial agents.

Synthetic calanolides with bactericidal activity against replicating and nonreplicating Mycobacterium tuberculosis.

The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days-about 2 weeks sooner than required to count CFU-fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.

Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.

Identification of compounds that target metabolically diverse subpopulations of Mycobacterium tuberculosis (Mtb) may contribute to shortening the course of treatment for tuberculosis. This study screened 270,000 compounds from GlaxoSmithKline's collection against Mtb in a nonreplicating (NR) state imposed in vitro by a combination of four host-relevant stresses. Evaluation of 166 confirmed hits led to detailed characterization of 19 compounds for potency, specificity, cytotoxicity, and stability. Compounds representing five scaffolds depended on reactive nitrogen species for selective activity against NR Mtb, and two were stable in the assay conditions. Four novel scaffolds with activity against replicating (R) Mtb were also identified. However, none of the 19 compounds was active against Mtb in both NR and R states. There was minimal overlap between compounds found active against NR Mtb and those previously identified as active against R Mtb, supporting the hypothesis that NR Mtb depends on distinct metabolic pathways for survival.

Identification of Novel Anti-mycobacterial Compounds by Screening a Pharmaceutical Small-Molecule Library against Nonreplicating Mycobacterium tuberculosis.

Article	Year
Benzimidazole-based compounds kill Mycobacterium tuberculosis. European journal of medicinal chemistry\| PubMed ID: 24556148	2014
Synthetic calanolides with bactericidal activity against replicating and nonreplicating Mycobacterium tuberculosis. Journal of medicinal chemistry\| PubMed ID: 24694175	2014
Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy\| PubMed ID: 26239979	2015
N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27432954	2016
Identification of Novel Anti-mycobacterial Compounds by Screening a Pharmaceutical Small-Molecule Library against Nonreplicating Mycobacterium tuberculosis. ACS infectious diseases\| PubMed ID: 27623055	2015

Selin Somersan-Karakaya

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