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. 2021 Jan 8;7(1):141-152.
doi: 10.1021/acsinfecdis.0c00675. Epub 2020 Dec 15.

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis

Shipra Grover  1 , Curtis A Engelhart  1 , Esther Pérez-Herrán  2 , Wei Li  3 , Katherine A Abrahams  4 , Kadamba Papavinasasundaram  5 , James M Bean  6 , Christopher M Sassetti  5 , Alfonso Mendoza-Losana  2 , Gurdyal S Besra  4 , Mary Jackson  3 , Dirk Schnappinger  1
Affiliations

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis

Shipra Grover et al. ACS Infect Dis. .

Abstract

MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis (Mtb), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of Mtb by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome bc1-aa3 oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.

Keywords: antibiotics; drug discovery; molecular genetics; mycolic acids; respiration; targeted whole-cell screen.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Consequences of two-way regulation of mmpL3 on growth, protein expression, and susceptibility to on-target MmpL3 inhibitors. (A) Growth and (B) protein levels of MmpL3 in mmpL3-TetON in the absence and presence of inducer ATc. (C–F) Dose–response profiles of MmpL3 inhibitors against mmpL3-TetON (orange) in the absence (open, dashed) and presence (closed, solid) of ATc (500 ng/mL) vs WT (black). Data are representative of three experiments; values are averages of two technical replicates and error bars represent the standard error (SE) of the mean. See also Table S2.
Figure 2
Figure 2
Silencing of MmpL3 expression alters cell envelope permeability to specific off-target inhibitors. (A–E) Dose–response profiles of mmpL3-TetON (orange) in the absence (open, dashed) and presence (closed, solid) of ATc (500 ng/mL) against high molecular weight antibiotics (A) rifampicin and (B) clarithromycin; and to different classes of (C–E) β-lactams. Data are representative of three experiments; values are averages of three technical replicates and error bars represent the SE of the mean. (F) Impact of MmpL3 depletion on accumulation of EtBr. The strains were incubated with 8 μg/mL of EtBr in PBS supplemented with 0.05% Tyloxapol and 0.4% Glucose. Reads were collected every minute until 60 min at 37 °C. See also Figure S2.
Figure 3
Figure 3
Susceptibility profile of mmpL3-TetON against the on-target hits. (A–C) Impact of selected screening hits on mmpL3-TetON (orange) and WT (black). For mmpL3-TetON, measurements were performed without (open squares, dotted lines) and with (closed squares, solid lines) ATc (500 ng/mL). Data are representative of three biological and two technical replicates. See also Figure S3 and Table S1.
Figure 4
Figure 4
Impact of MmpL3 expression on membrane potential and the activity of ETC inhibitors. (A) Effect on membrane potential (ΔΨ). DiOC2(3) accumulation was assessed in mmpL3-TetON in the presence and absence of ATc (500 ng/mL) before (solid) and after (hollow) exposure to 500 μM CCCP for 30 min at 37 °C. Student’s unpaired t test (p < 0.01) was performed to determine statistical significance of differences between the mutant groups denoted with **. (B,C) Dose–response profile of mmpL3-TetON (orange) in absence (open, dashed) and presence (closed, solid) of ATc (500 ng/mL) against (B) the ATP synthase inhibitor bedaquiline and (C) the cytochrome bc1-aa3 inhibitor Q203. Data are representative of three experiments; values are averages of three technical replicates and error bars represent the SE of the mean.
Figure 5
Figure 5
Partial expression of MmpL3 identifies a sulfanylacetamide as an inhibitor of cytochrome bc1-aa3 oxidase. Dose–responses profiles of the cytochrome bd oxidase knockout (orange, open, dashed) and complemented strain (orange, closed, solid) vs WT (black) against (A) bedaquiline, (B) Q203, and (C) GSK1859936A. Data are representative of three experiments; values are averages of three technical replicates and error bars represent the SE of the mean.
Figure 6
Figure 6
CCI7967 is an MmpL3 inhibitor. (A) Structure of CCI7967. (B) Dose–response profile of mmpL3-TetON (orange) in the absence (open, dashed) and presence (closed, solid) of ATc (500 ng/mL). (C) Cross-resistance depicted as the ratio of IC50 between the mutant and WT for different mutations in MmpL3 for CCI7967, GSK2200150A (Spiros), and GSK1180781A (THPP). (D–F) Isobolograms of CCI7967 tested in combination with (D) β-lactam, cefadroxil; (E) RNA polymerase inhibitor, rifampicin; and (F) MmpL3 inhibitor, SQ109.
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