Targeting Phenotypically Tolerant Mycobacterium tuberculosis

Abstract

While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycin-addicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in clinical settings. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of posttreatment relapse. Some promising drugs to treat tuberculosis, such as rifampin and bedaquiline, only kill nonreplicating M. tuberculosisin vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high-throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores.

FIGURE 1

Strategies to evaluate the viability of nonreplicating mycobacteria for high-throughput screening. The arrow color indicates the quality of each readout strategy (considering robustness, ease of use, dynamic range, etc.) as excellent (green arrows), average to poor (black arrows), or infeasible (red line). Compound carryover may result from compound transfer from the nonreplicating assay to replicating assay bacteriologic growth medium or by compound adherence to the bacterial cell wall.

FIGURE 2

Selecting and designing nonreplicating (NR) models. (Left) Nonexhaustive list of models of class I and class II nonreplication. (Right) Variables to consider when designing models. (Center, bottom) Potential activity profiles of nonreplicating actives. The success of compounds targeting nonreplicating mycobacteria is dependent on the interactions among models, variables, and activity profiles. The term “DD Mtb” (differentially detectable M. tuberculosis) is used interchangeably with “viable-but-nonculturable” (VBNC) M. tuberculosis.

FIGURE 3

Compound transformation during screening assays. (a) Predicted, and experimentally validated, points of compound modification that may occur during phenotypic screening. (b) In cell-free, nonreplicating conditions imposed by the multistress model, oxyphenbutazone (left) rapidly transforms in acidic and nitrosative conditions to the intermediate, 4-hydroxy-oxyphenbutazone (center), which further transforms to 4-hydroxy-oxyphenbutazone quinoneimine (right). The electrophilic quinoneimine (red) can react at carbon atoms (green) with intrabacterial nucleophiles such as N-acetyl cysteine (NAC) and/or mycothiol (MSH).

FIGURE 4

Proof-of-concept molecules. Molecules with nonreplicating activity that serve as proof of concept include those that (a) selectively kill nonreplicating mycobacteria; (b) have dual activity, kill mycobacteria in the majority of nonreplicating models, and are effective at treating tuberculosis in animal models; and (c) have selective activity against slowly replicating or nonreplicating mycobacteria and are efficacious in tuberculosis models. n.t., not tested; *, pyrazinamide has activity against slowly replicating mycobacteria; #, experimental data indicate that pyrazinamide is inactive against intracellular mycobacteria in vitro (292, 293). However, pyrazinamide’s dependency on an acidic environment for activity, and potent in vivo activity, suggests that it kills intracellular mycobacteria during animal and human tuberculosis.

FIGURE 5

Canonical and noncanonical targets of dual-active molecules. Dual-active molecules, which have bacteriostatic or bactericidal activity against replicating M. tuberculosis and bactericidal activity against nonreplicating M. tuberculosis, are often presumed to engage the same target under both conditions. Dual-active molecules may exert activity against nonreplicating mycobacteria via novel targets or nonspecific mechanisms. The list of dual-active molecules is not exhaustive.

FIGURE 6a

Replicating and nonreplicating mycobacteria may share common targets. Examples of compounds that engage standard antibiotic target pathways under replicating conditions, and also kill nonreplicating mycobacteria, include inhibitors of the biosynthesis of (a) lipids, (b) DNA, (c) RNA, (d) protein, and (e) peptidoglycan.

FIGURE 7

Quinolines.

FIGURE 8

Quinolones.

FIGURE 9

Compounds targeting the proteostasis and proteolysis pathways.

FIGURE 10

Representative compounds identified by whole-cell high-throughput screening (HTS) against mycobacteria rendered nonreplicating by (a) carbon starvation (54); (b) hypoxia (29); (c) multiple stresses, including low pH, nitric oxide and reactive nitrogen intermediates, hypoxia, and a fatty acid carbon source (28, 53, 145); (d) acidic pH (119); and (e) culture as a biofilm (102).

FIGURE 11

Nitro-containing compounds.

FIGURE 12

Compounds that depolarize the mycobacterial membrane.

FIGURE 13

Salicylanilides are protonophores. (a) The commonly drawn structure of niclosamide (left). Compound S-13, which was used for experimental logP calculations (266), is shown for reference (right). (b) As illustrated by niclosamide, salicylanilides capture protons by forming a stable pseudo-6-membered ring via hydrogen bonding. Once inside the bacterial cell and releasing their proton, they maintain a stable anionic form from electron delocalization. Adapted from Terada (266).

FIGURE 14

Additional compounds that kill nonreplicating mycobacteria.

FIGURE 6b

Replicating and nonreplicating mycobacteria may share common targets. Examples of compounds that engage standard antibiotic target pathways under replicating conditions, and also kill nonreplicating mycobacteria, include inhibitors of the biosynthesis of (a) lipids, (b) DNA, (c) RNA, (d) protein, and (e) peptidoglycan.