Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics

Abstract

Summary Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular ROS at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.

Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance

“…Previously, two HTS were conducted, targeting the two component regulatory systems, DosRST and PhoPR 28, 29, 30 . In addition to inhibitors targeting these pathways, a series of compounds was identified that inhibited Mtb growth independent of the targeted pathways 9, 28, 30 .…”

“…Previously, two HTS were conducted, targeting the two component regulatory systems, DosRST and PhoPR 28, 29, 30 . In addition to inhibitors targeting these pathways, a series of compounds was identified that inhibited Mtb growth independent of the targeted pathways 9, 28, 30 . A series of high throughput assays were then conducted to prioritize these compounds (Supplemental Figure 1) including confirming hits, testing for eukaryotic cytotoxicity in primary murine bone marrow-derived macrophages (BMMΦ, ≤10% cytotoxicity), and testing for the ability of the compounds to inhibit Mtb growth inside BMMΦ (≥25% growth inhibition).…”

“…Unless otherwise specified, streptomycin resistant strains of Mtb Erdman or CDC1551 were cultured in 7H9 media supplemented with 10% OADC (v/v) with 0.05% Tween-80 (v/v) in standing T25, T75 or T150 flasks at 37 °C with 5% CO 2 . Spectrum of activity studies in different bacterial species (Table 2) were conducted as described by Coulson et al, 30 , with the exception of the M. abscessus studies which are described in the supplemental methods.…”

Genetic and metabolic regulation ofMycobacterium tuberculosisacid growth arrest

“…Moreover, small molecules inhibiting pH-homeostasis have also been identified [52]. Additionally, it was recently shown that Mtb has enhanced sensitivity to thiol and oxidative stress at acidic pH, indicating that pH-dependent changes in redox homeostasis are vulnerable pathways for new drug development [53]. The discovery that acid growth arrest drives drug tolerance and can be genetically disrupted supports the potential to develop small molecules targeting acid growth arrest.…”

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development

“…Class 2 hits were defined as compounds exhibiting >50% inhibition of both fluorescence and growth. These compounds are killing Mtb or slowing its growth, one of which AC2P36 has been shown to be a pH‐selective inhibitor of Mtb growth . GFP quenchers were experimentally identified and removed from the list of hits.…”