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

Abstract

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.

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

“…identified as bactericidal to replicating Mtb (13). We identified the mechanism of resistance as N-methylation of compound 14 by a previously uncharacterized methyltransferase, Rv0560c.…”

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

“…In a CFU assay, in addition to serial dilutions that often range up to 10 6 -fold, the agar plate typically dilutes molecules by an additional ~800-fold (10 μl onto 8 ml in a tri-style Petri plate). Our two-stage, multi-stress screening assay for compounds active on non-replicating M. tuberculosis has a carry-over factor of 5-fold, that is, a compound present in the non-replicating stage of the assay is present at one-fifth the original concentration in the outgrowth (replicating) phase of the assay [6][7][8][9] . The CARA mitigates carry-over effects by sequestering small molecules with activated charcoal.…”

“…MIC 90 -style assays typically employ 2-fold dilution series. There are numerous non-replicating models available for mycobacteria [14][15][16]18,24,25 and for illustrative purposes, we are using a multi-stress model of non-replication 6,8,9 . 1.…”

“…We recently developed an in vitro model of Class II non-replicating persistence to mimic conditions confronted by M. tuberculosis in activated macrophages and granulomas. The multi-stress model of Class II persistence includes conditions that slow growth, such as a fatty acid carbon source, and completely halt growth, such as mild acidity (pH 5.0), hypoxia (1% O 2 ), and nitric oxide and other reactive nitrogen intermediates [5][6][7][8][9] . Large-scale screening employing this multi-stress model of non-replication, and other Class II non-replicating models, has yielded diverse molecules whose activity is directed against the non-replicating state [5][6][7][9][10][11][12][13][14][15][16][17][18] .…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…This of course complicates drug development, and warrants the development of new assays to test efficacy of antimicrobials. Screenings for antibiotics active against nonreplicating bacteria are ongoing or have recently been published [400], and it will be exciting to learn about their in vivo activity. Also, better understanding of the phenotype that the bacteria have during transmission could lead to prophylactic treatment to avoid transmission.…”