Chapter 15 : Targeting Phenotypically Tolerant Mycobacterium tuberculosis

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.

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.

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).

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.

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.

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.

Quinolines.

Quinolones.

Compounds targeting the proteostasis and proteolysis pathways.

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 ).

Nitro-containing compounds.

Compounds that depolarize the mycobacterial membrane.

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 ).

Additional compounds that kill nonreplicating mycobacteria.