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. 2020 Jan 10;6(1):56-63.
doi: 10.1021/acsinfecdis.9b00373. Epub 2019 Nov 28.

Large-Scale Chemical-Genetic Strategy Enables the Design of Antimicrobial Combination Chemotherapy in Mycobacteria

Eachan O Johnson  1   2   3 , Emma Office  1 , Tomohiko Kawate  1   2   3 , Marek Orzechowski  1 , Deborah T Hung  1   2   3
Affiliations

Large-Scale Chemical-Genetic Strategy Enables the Design of Antimicrobial Combination Chemotherapy in Mycobacteria

Eachan O Johnson et al. ACS Infect Dis. .

Abstract

The efficacies of all antibiotics against tuberculosis are eventually eroded by resistance. New strategies to discover drugs or drug combinations with higher barriers to resistance are needed. Previously, we reported the application of a large-scale chemical-genetic interaction screening strategy called PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) for the discovery of new Mycobacterium tuberculosis inhibitors, which resulted in the identification of the small molecule BRD-8000, an inhibitor of a novel target, EfpA [ Johnson et al. ( 2019 ) Nature 517 , 72 ]. Leveraging the chemical genetic interaction profile of BRD-8000, we identified BRD-9327, another structurally distinct small molecule EfpA inhibitor. We show that the two compounds are synergistic and display collateral sensitivity because of their distinct modes of action and resistance mechanisms. High-level resistance to one increases the sensitivity to and reduces the emergence of resistance to the other. Thus, the combination of BRD-9327 and BRD-8000 represents a proof-of-concept for the novel strategy of leveraging chemical genetics in the design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is exploited.

Keywords: antimicrobial resistance; chemical genetics; collateral sensitivity; drug discovery; synergy; tuberculosis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Discovery of a new putative inhibitor of the essential mycobacterial efflux pump, EfpA. (A) Overview of PROSPECT, a sequencing-based, high-throughput chemical-genetic profiling assay. A C-terminal DAS tag, which targets the gene product to degradation by caseinolytic protease (Clp), was integrated at the 3′ end of target genes of interest in the chromosome with concomitant genetic barcoding, which allowed pooling of hypomorph strains. After compound exposure, chromosomal barcodes were PCR amplified, sequenced on the Illumina platform, and analyzed for changes in abundance relative to vehicle controls. For each compound, this generated a vector of strain abundance changes, known as a chemical genetic interaction profile (CGIP). (B) Medicinal chemistry optimization of initial hit BRD-8000, an EfpA inhibitor, yielded BRD-8000.3, a narrow-spectrum antimycobacterial with good wild-type activity. (C) Ranked Pearson correlation of CGIPs with the BRD-8000 CGIP. Each point represents a compound’s CGIP correlation; blue shading indicates the P-value under a permutation test (n = 10 000). Since BRD-8000 had been validated as an EfpA inhibitor, its CGIP could be used as a reference to discover further EfpA inhibitors. The CGIP of BRD-9327 (highlighted in red) had the highest correlation with the CGIP of BRD-8000. (D) Broth microdilution assay of BRD-9327 against wild-type Mtb and its EfpA hypomorph (Mtb efpAKD); open circles show individual replicates (n = 4), filled circles indicate the mean, and error bars show the 95% confidence interval. BRD-9327 showed very little activity against wild-type Mtb, although the EfpA hypomorph was hypersensitive.
Figure 2
Figure 2
Validating EfpA as the target of BRD-9327 using an EtBr efflux assay. (A) Overview of molecular basis of the EtBr assay for determining kinetic inhibition parameters. When intracellular, EtBr (orange) is ∼30-fold more fluorescent than extracellular; thus, EtBr fluorescence is a proxy for intracellular concentration. In living cells, a compound, which is simply a substrate of efflux pumps (green hexagon), will exhibit a competitive mode of EtBr efflux inhibition, since it competes with EtBr for flux through the pumps. However, a compound that has a specific interaction with EfpA (blue hexagon) might also appear to inhibit EtBr efflux competitively but will exhibit an additional non- or uncompetitive modality. In the absence of EfpA, as in a null mutant, this non- or uncompetitive modality will be abolished. (B) EtBr fluorescence decay over time (demonstrating varying efflux rates) at three starting intracellular concentrations and eight BRD-9327 concentrations in Msm. Curves corresponding to global best-fit Michaelis–Menten parameter estimates are shown in red. (C) Global best-fit Michaelis–Menten parameter estimates (±standard deviation) of EtBr efflux inhibition by BRD-9327.
Figure 3
Figure 3
Evolution of Mmar mutants resistant to BRD-8000.3 or BRD-9327. (A) Broth microdilution dose response assay of Mmar and its BRD-8000.3-resistant mutants against BRD-8000.3, demonstrating their high-level resistance to this compound. Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). (B) Amino acid sequence alignment of highly conserved EfpA in Mtb, Mmar, and Msm, with sites conferring resistance to BRD-8000.3 (green) or BRD-9327 (orange) highlighted. (C) Homology model of EfpA with mutations conferring resistance to BRD-8000.3 (green) or BRD-9327 (orange) highlighted. Mesh outlines show possible binding sites of BRD-8000.3 (green) and BRD-9327 (orange), as determined by docking using AutoDock Vina. (D) Broth microdilution dose response assay of Mmar mutants resistant to BRD-8000.3 against BRD-9327, demonstrating the hypersensitivity of Mmar efpA(V319M) and Mmar efpA(A415V). Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). (E) Loewe excess of Mmar growth inhibition at varying combined concentrations of BRD-9327 and BRD-8000.3, demonstrating potentiation of BRD-9327 by BRD-8000.3 between the two EfpA inhibitors.
Figure 4
Figure 4
Resistance to BRD-9327 lowers the resistance frequency to BRD-8000.3. (A) Broth microdilution dose response assay of Mmar and its BRD-9327-resistant mutants against BRD-9327, demonstrating the high-level resistance of efpA mutants and low-level resistance of the mmar_1007 mutant. Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). (B) Broth microdilution dose response assay of Mmar mutants resistant to BRD-9327 against BRD-8000.3, demonstrating the hypersensitivity of Mmar efpA(G328D) and Mmar efpA(A339T). Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). (C) Frequency of wild-type or BRD-9327-resistant mutant colonies growing on agar containing 2×, 4×, or 8× MIC of INH (left) or BRD-8000.3 (right). Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). The dashed line indicates the limit of detection. (D) Growth inhibition from the broth microdilution assay of Mmar (left) and the Loewe excess (right) at varying combined concentrations of BRD-9327 and verapamil, demonstrating modest synergy between the two compounds. (E) Frequency of wild-type or BRD-8000.3 resistant mutant colonies growing on agar containing 2×, 4×, or 8× MIC of INH (left) or BRD-9327 supplemented with verapamil (right). Filled circles show the mean, and error bars indicate the 95% confidence interval (n = 4). The dashed line indicates the limit of detection.
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