Discovery and characterization of antimycobacterial nitro-containing compounds with distinct mechanisms of action and in vivo efficacy

Abstract

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F₄₂₀ machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F₄₂₀-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.

Keywords: DprE1; Mycobacterium abscessus; Mycobacterium tuberculosis; deazaflavin-dependent nitroreductase; nitrofuran.

Fig 1

Nitro-containing compounds that inhibit Mtb growth. (A) Fdg1-dependent nitrofurans. (B) Fdg1-independent nitrofurans. (C) Dinitrobenzamides that are putative DrpE inhibitors. (D) Pretomanid, a nitro-containing FDA-approved TB drug.

Fig 2

Nitro-containing compounds inhibit Mtb growth in a dose-dependent manner. (A) Dose-response curves for HC2210 inhibition of Mtb growth relative to pretomanid and isoniazid. (B) Dose-response curves for other nitrofurans. (C) Dose-response curves for the dinitrobenzamides. The dotted line represents the growth inhibition of the negative control (DMSO). The error bars represent the standard deviations of two to three biological replicates. All experiments were independently conducted at least twice with similar results.

Fig 3

In vitro time and concentration-dependent killing of Mtb. (A) Comparing the bactericidal activity of HC2210 with those of pretomanid and isoniazid shows that it is weakly bactericidal. (B) The other tested nitrofurans killed Mtb in a dose- and time-dependent manner. For both time points, HC2233 completely sterilized the culture at 50 µM. Hence, the line is not shown in the graph. After 10 days of treatment, 50 µM of HC2234 completely sterilized the culture below the limit of detection. Hence, the graph line ended at 4 days. (C) The time- and dose-dependent killing of Mtb by the tested dinitrobenzamides. (D) The bactericidal activity of the compounds against non-replicating Mtb in a hypoxic shift-down assay. The upper black dotted lines in A–C represent the starting cell concentration of 1.7 × 10⁸ CFU/mL. The limit of detection in this assay is ~20 CFU. The error bars represent the standard deviations of two technical replicates for (A–C) or two biological replicates for (D). Asterisks denote statistically significant differences between the compared groups in an unpaired Student’s t-test (**P value ≤0.01). ns, statistically non-significant with P value >0.05.

Fig 4

Resistance of the ddn and fdg1 spontaneous mutants against the tested nitrofurans and pretomanid. fdg mutants provide full resistance, and ddn mutants provide partial resistance against HC2210. Pretomanid entirely loses its activity in the tested fdg and ddn mutants. fdg and ddn mutants did not provide resistance to HC2234 or HC2250. The dotted lines represent the growth inhibition of the negative control (DMSO). The error bars represent the standard deviations of three biological replicates. All experiments were independently repeated three times with similar results.

Fig 5

Resistance to dinitrobenzamides and HC2250 in dprE1 mutants. HC2238 and HC2226 lose activity against the spontaneous dprE1 mutants, while partial resistance is observed toward HC2250. HC2234 is active against the tested dprE mutant. The dotted lines represent the growth inhibition of the negative control (DMSO). The Δfdg mutant is included as a control showing the compounds are independent of the F₄₂₀-dependent activation. The error bars represent the standard deviations of three biological replicates. All experiments were independently conducted two times with similar results.

Fig 6

HC2210 delivered orally reduces Mtb survival in a chronic model of Mtb infection. Mycobacterial burden is reduced in the lung and spleen of infected C57Bl/6 mice following 4 weeks of treatment with HC2210. HC2210 treatment was performed by oral gavage once daily, 5 days a week at 75 mg/kg. Rifampin treatment was twice daily, 5 days a week at 10 mg/kg. p.i is acronym for post-infection. 1 day p.i is the mycobacterial burden of the mice a day after infection. 39 days p.i is the mycobacterial burden of the untreated mice 39 days post-infection, prior to treatment. The vehicle control is 95% corn oil/5% DMSO. Asterisks denote statistically significant differences between the compared groups in an unpaired Student’s t-test (**P value ≤0.01; ***P value ≤0.001). ns, statistically non-significant with P value >0.05.