Biology subjects

Coulson, G. B.

Publications and source records attributed to Coulson, G. B..

3 recordsLinked to original sources

bioRxiv2021

AC2P20 selectively kills M. tuberculosis at acidic pH by depleting free thiols

Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a [~]220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.

microbiology↗

bioRxiv2019

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

The Mycobacterium tuberculosis (Mtb) mycolic acid flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole genome sequencing of isolated resistant mutants. Here we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel Mtb growth inhibitors for differential growth inhibition of wild type Mtb as compared to a pool of twenty-four unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular Mtb. Mtb cells treated with these compounds were shown to accumulate trehalose monomycolate and have reduced levels of trehalose dimycolate, supporting MmpL3 as the target. The inhibitors were mycobacteria specific with several also showing activity against the non-tuberculosis mycobacterial species M. abscessus. Cluster analysis of cross resistance profiles generated by dose response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small molecule inhibitors.

microbiology↗

bioRxiv2016

Acidic pH-dependent depletion of Mycobacterium tuberculosis thiol pools potentiates antibiotics and oxidizing agents

Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH and reactive oxygen species (ROS) 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 acidic pH-dependent compound AC2P36 depletes intracellular thiol pools, sensitizes Mtb to killing by acidic pH, and potentiates the bactericidal activity of isoniazid, clofazimine, and oxidizing agents. We show that the pHdependent activity of AC2P36 is associated with metabolic stress at acidic pH and a pHdependent accumulation of intracellular ROS. Mechanism of action studies show that AC2P36 directly depletes Mtb thiol pools. These data support a model where chemical depletion of Mtb thiol pools at acidic pH enhances sensitivity to oxidative damage, resulting in bacterial killing and potentiation of antibiotics.

microbiology↗