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.

INTRODUCTION

The high prevalence of tuberculosis (TB), coupled with growing antibiotic resistance, highlights the need to develop new TB drugs (1). With the recent approval of pretomanid and delamanid for TB treatment (2, 3), nitro-containing compounds have emerged as important agents to control TB. Pretomanid and delamanid are classified as nitroimidazoles. Other antitubercular nitro-containing chemical scaffolds include benzothiazinones, dinitrobenzamides, nitrobenzamides, and nitrofurans, among others (2, 4, 5). Some compounds from these series such as PBTZ-169 and BTZ-043 have been shown to be efficacious in clinical trials for TB treatment (3).

Pretomanid and delamanid kill Mycobacterium tuberculosis (Mtb) by targeting essential cellular processes such as respiration or cell wall biogenesis and are effective against non-replicating Mtb (6 - 11). They are prodrugs and require reductive activation by the mycobacterial-specific deazaflavin-dependent nitroreductase (Ddn) (7, 12, 13). Their prodrug status enables them to specifically inhibit the growth of the infecting Mtb while limiting dysbiotic effect on the host microbiome. Despite their promising use for TB treatment, pretomanid and delamanid have some limitations. There are reports of Mtb isolates that are naturally resistant to either drug due to genetic polymorphism in Ddn or other genes in the F₄₂₀ biosynthesis pathway, and the F₄₂₀-dependent glucose-6-phosphate dehydrogenase-1 (fdg1) (14, 15). Fdg1 mediates one of the earliest steps in the pentose phosphate pathway of mycobacteria. It uses F₄₂₀, instead of the canonical NAD(P), in catalyzing its reaction. In this process, F₄₂₀ is reduced and can be used by Ddn in the activation of pretomanid or delamanid (7, 10, 12, 13, 16, 17). Clinical strains that have developed resistance to either pretomanid or delamanid have been isolated in different parts of the world (9, 14). The pharmacokinetic profile and side effects of the compounds can make them less ideal for certain patients. Delamanid has a relatively poor oral bioavailability and can have a modest effect on QT prolongation (2, 9, 18). Due to these challenges, there are ongoing efforts to develop new antitubercular nitro-containing compounds with improved properties.

Our lab previously conducted a whole-cell high-throughput screen of the ~340,000 compound Molecular Libraries Small Molecular Repository (MLSMR) for inhibitors of the DosRST two-component regulatory system (19). From this primary high throughput screen, we identified compounds that inhibited Mtb growth independent of the targeted pathway. We noted many of these growth-inhibiting compounds contained a nitro group as a presumptive pharmacophore. The goal of this study is to decipher the possible mechanisms of action of 10 nitro-containing compounds that inhibit mycobacterial growth and prioritize analogs for continued development. Here, we provide a genetic basis for the antimycobacterial activities of the compounds. We show that, like pretomanid and delamanid, several of the nitrofurans depend on cofactor F₄₂₀-dependent enzymes for activity. Unlike the nitroimidazoles that depend only on Ddn, these nitrofurans partially depend on Ddn and possibly a second, unknown F₄₂₀-dependent enzyme for activation. Additionally, we show that the nitrofurans are active against Mycobacterium abscessus (Mab), whereas pretomanid had limited inhibition of Mab growth. Other nitro-containing compounds, including dinitrobenzamides and a nitrofuran in this study, are proposed to target decaprenyl-phosphoryl-ribose 2′-epimerase 1 (DprE1), an essential protein involved in cell wall biogenesis. These putative DprE1 inhibitors were active against both Mtb and Mycobacterium smegmatis (Msm). Lastly, we demonstrate that a novel nitrofuran compound, HC2210, is effective, when delivered orally, in a chronic murine Mtb infection model.

RESULTS

New nitro-containing compounds have potent antitubercular activities

As part of our efforts toward developing a mechanistic understanding of the antimycobacterial activity of the small molecules discovered from the previous high-throughput screen of the MLSMR, we selected 10 nitro-containing compounds (Fig. 1) and characterized their mechanisms of action. Six of these compounds are nitrofurans, and they include HC2209 (1-(4-fluorophenyl)-4-[(5-nitro-2-furyl)methyl]piperazine), HC2210 (1-[(5-nitro-2-furyl)methyl]-4-(4-nitrophenyl)piperazine oxalate), HC2211 (1-[(5-nitro-2-furyl)methyl]-4-phenylpiperazine), HC2233 (N-{4-[4-(2-methylpropanoyl)piperazin-1-yl]phenyl}-5-nitrofuran-2-carboxamide), HC2234 (N-{4-[4-(2,2-dimethylpropanoyl)piperazin-1-yl]phenyl}-5-nitrofuran-2-carboxamide), and HC2250 (N′-[(E)-(5-nitrofuran-2-yl)methylidene]-2-phenoxyacetohydrazide). Previously, nitrofuran piperazine, nitrofuran triazine and other nitrofuran compounds have been reported as Mtb growth inhibitors (2, 20 - 23) . The other four compounds, with their nitro groups attached to a parent benzene ring, are dinitrobenzamides, and they include HC2217 (N-(2-morpholin-4-yl-2-thiophen-2-ylethyl)-3,5-dinitrobenzamide), HC2226 (N-(cyclopropylmethyl)-3,5-dinitrobenzamide), HC2238 (N-[(4-fluorophenyl)methyl]-4-methyl-3,5-dinitrobenzamide), and HC2239 (N-[2-(3-methoxyphenoxy)ethyl]-3,5-dinitrobenzamide). Notably, related dinitrobenzamide compounds have previously been described as DprE1 inhibitors (3, 4, 24).

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.

An in vitro dose-response study against Mtb shows that all the compounds are relatively potent with half-maximal effective concentrations (EC₅₀) ranging from 0.05 to 6.86 µM (Fig. 2; Table 1). Of particular interest is HC2210, a nitrofura compound that has an EC₅₀ of 50 nM. By comparison, in this assay, HC2210 is >2× more potent than isoniazid (EC₅₀ = 140 nM) and >12× more potent than pretomanid (EC₅₀ = 620 nM). During infection, Mtb can replicate inside macrophages; therefore, we tested compound activity against intracellular Mtb and for cytotoxicity against murine bone marrow-derived macrophages. In a dose-response study, all the nitro-containing compounds exhibited high potency against intracellular Mtb and had limited eukaryotic cytotoxicity (Table 1). These data demonstrate that the compounds can selectively inhibit intracellular Mtb with no or limited cytotoxicity on macrophages.

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.

TABLE 1.

Potency of nitro-containing compounds against mycobacterial and non-mycobacterial species^a

Compounds	In vitro EC50 (µM) of compounds
									Ex vivo EC50 (µM)	CC50 (µM)
	Mtb	Msm	Mab	Escherichia coli	Pseudomonas aeruginosa	Staphylococcus aureus	Enterococcus faecalis	Proteus vulgaris
HC2209	0.94	>200	<5.12	>200	>200	>200	>200	>200	<0.13	>80
HC2210	0.05	>200	<0.82	56.83	>200	>200	>200	>200	<0.13	>32
HC2211	1.89	>200	2.81	>200	>200	>200	>200	>200	<0.33	>80
HC2217	<2.05	<2.05	>200	>200	>200	>200	>200	>200	<0.13	>80
HC2226	6.86	<32	>80	>200	>200	>200	>200	>200	<0.83	>80
HC2233	3.78	>200	<80	>200	>200	>200	>200	>200	<0.33	>80
HC2234	3.20	>200	<32	>200	>200	>200	>200	>200	<32	>80
HC2238	0.54	1.25	>80	>200	>200	>200	>200	>200	<0.13	>80
HC2239	1.15	0.63	>80	>200	>200	>200	>200	>200	<0.13	>80
HC2250	4.52	<5.12	>80	>200	>200	>200	>200	25.48	<0.33	>80
Pretomanid	0.62	>80	<80	>200	>200	>200	>200	>200	ND	ND
Isoniazid	0.14	39.88	ND	ND	ND	ND	ND	ND	ND	ND
Ethambutol	10.41	<0.819	ND	ND	ND	ND	ND	ND	ND	ND

^a ND, not determined; Mtb, Mycobacterium tuberculosis; Msm, Mycobacterium smegmatis; Mab, Mycobacterium abscessus; Ex vivo EC₅₀ (µM), EC₅₀ of compounds against intracellular Mtb in bone marrow-derived macrophages; and CC₅₀ (µM), macrophage cytotoxicity.

Next, we sought to determine whether the compounds were bactericidal or bacteriostatic against Mtb. For most antibiotics, it is important to note that this classification system depends on the dose and time allowed for treatment (25, 26). Treatment of Mtb with HC2210, pretomanid, and isoniazid showed the compounds are bactericidal at the tested concentrations (Fig. 3A). Three other nitrofurans in this study (HC2233, HC2234, and HC2250) were also bactericidal at the tested concentrations and time points, with 50 µM of HC2233 or HC2234 completely sterilizing the culture after 4 or 10 days of treatment, respectively (Fig. 3B). For the dinitrobenzamides, we tested HC2217, HC2226, and HC2238. At 4 days of incubation, all the compounds exhibited bactericidal activity even at the lowest test concentrations (Fig. 3C). We noticed interesting differences at 10 days of incubation. While HC2238 continued to kill the pathogen at 10 days of incubation, HC2217 and HC2226 start to lose their bactericidal activity at this time point. In fact, the two test concentrations of HC2217 completely lose their bactericidal profile at this time point. While we do not know the exact cause of this lost activity, we presume that it may be due to the instability of the compounds.

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.

HC2210, HC2233, HC2234, and HC2250 are active against non-replicating Mtb

In response to different environmental signals such as hypoxia during infection, Mtb can transition into a non-replicating persistent (NRP) state that is non-responsive to many antibiotics (27, 28). One of the goals of modern TB chemotherapy is to develop drugs that can kill Mtb in this dormant state (19, 29). Using a hypoxic shift-down assay (30), we investigated the effect of the nitro-containing compounds on the survival of NRP Mtb. All the tested dinitrobenzamides (HC2217, HC2226, HC2238) had no impact on the viability of the pathogen relative to the DMSO-vehicle control (Fig. 3D). Isoniazid, a cell wall inhibitor, was used as a control in this assay and was inactive against NRP bacteria. In contrast, all the tested nitrofuran compounds (HC2210, HC2233, HC2234, HC2250) significantly reduced the viability of the NRP bacteria relative to the control, with HC2233 and HC2234 again showing sterilizing activity, suggesting that these compounds may be inhibiting essential cellular activities during Mtb dormancy.

HC2209, HC2210, and HC2211 are cofactor F₄₂₀-dependent nitrofurans

Due to the presence of one or more nitro groups in these compounds, we reasoned that, like other nitro-containing compounds, they might be prodrugs that need mycobacterial proteins for activation. Isolation of resistant mutants has previously been used to identify activating enzymes (2, 12, 24, 31). Spontaneous mutants resistant to HC2210 were isolated on media supplemented with either 0.1 or 0.3 µM HC2210 with a frequency of ~1.6 × 10⁻⁶ similar to what we observed for pretomanid (1.8 × 10⁻⁶).

Ten resistant colonies from each plate were isolated and confirmed for resistance against HC2210 (Fig. S1; Fig. 4). Notably, two resistance patterns were observed from the dose-response curves: (i) partial resistance with an EC₅₀ of 5 µM and (ii) total resistance at all concentrations tested. The partially resistant mutants were isolated from both the 0.1 and 0.3 µM HC2210 selection plates, while the fully resistant clones were only observed in the 0.3 µM selection plate. The absence of fully resistant clones from the 0.1 µM HC2210 plate may be due to a lower selective pressure to evolve full resistance to the compound.

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.

To ascertain mutations that cause these resistance patterns, we sequenced the genomes of the isolated resistant mutants. For the fully resistant clones, we identified nonsense, insertion, and deletion mutations of fdg1, while the partially resistant clones harbored missense mutations or deletion in ddn (Table 2). Since we selected mutants in these genes, we hypothesized that HC2210 shares a related activation mechanism with pretomanid and delamanid. Notably, partial resistance of the ddn mutants for HC2210 suggests that a second nitroreductase may be required for its activation, as was previously observed for nitro-containing triazines (2). As expected, cross-resistance screening of two fdg1 spontaneous mutants against pretomanid showed a full loss of activity of the drug (Fig. 4). The ddn spontaneous mutants also showed full resistance to high concentrations of pretomanid, further highlighting the role of the nitroreductase in the activation of the compound. Cross-resistance profiling of the spontaneous mutants also showed HC2209 and HC2211 to be dependent on Fdg1 and Ddn for activation (Fig. S2), with fdg1 mutants providing full resistance and the ddn mutants providing partial resistance.

TABLE 2.

Mutations of ddn and fdg1 in resistant clones

Resistance	Mutant strain #	SNP location (nt)	Gene	Nucleotide change	Amino acid substitution
Partial	100.1	3,967,990	ddn	GGG→GAG	G34E
	100.2	3,968,082	ddn	TAC→GAC	Y65D
	100.3	3,966,767	[fadA5], ddn [ERDMAN_3893]	Δ2,315 bp
	100.4	3,967,990	ddn	GGG→GAG	G34E
Full	300.1	492,852	fgd1	TAC→TAA	Y118*
	300.2	490,918	[pks6], fgd1, [pta]	Δ2,760 bp
	300.3	492,727	fgd1	(C)5→6	Coding (231/1,011 nt)
	300.5	493,331	fgd1	CAG→TAG	Q279*

The three other nitrofurans in this study (HC2233, HC2234, and HC2250) retained their full potency against the fdg1 and ddn spontaneous mutants (Fig. 4; Fig. S2). This suggests that they do not depend on the F₄₂₀ machinery for their activity. The same can be said for all the dinitrobenzamides since they did not show any change in their potency against the spontaneous mutants (Fig. S3), consistent with their presumed target of DprE. Overall, HC2209, HC2210, and HC2211 are the only compounds in this study that depended on the F₄₂₀ bioreductive activation system.

Mutations in dprE1 confer resistance to the nitrofuran HC2250 and dinitrobenzamides

Dinitrobenzamides are known DprE1 inhibitors (3, 4, 24, 32). To determine if the compounds are potential DprE1 inhibitors, we isolated resistant mutants to HC2238 and confirmed their resistance in a dose-response study (Fig. S4; Fig. 5). Whole-genome sequencing identified the mutants harbored single-nucleotide variants leading to a C384S substitution in DprE1. DprE1 is a conserved protein that catalyzes an essential epimerization step during the synthesis of mycobacterial arabinogalactan (24, 33 - 35). Cross-resistance profiling of the mutants against other dinitrobenzamides in this study further confirmed that they share the same likely target (Fig. 5; Fig. S4). As expected, the mutants did not show any cross-resistance against a common cell wall inhibitor such as ethambutol (Fig. S4), indicating that they target different proteins in the cell wall biogenesis pathway.

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.

Since HC2233, HC2234, and HC2250 remained the only compounds in this study whose mechanism of action remained unknown, we attempted to select resistant mutants on agar plates amended with the respective compounds at various concentrations. However, these efforts were unsuccessful. We also examined their inhibitory activity against the dprE1 mutants. HC2233 and HC2234 retained their full potency against the mutants, indicating that they likely do not target DprE1 or that other mutations are required for resistance (Fig. 5; Fig. S4). HC2250 had reduced potency in these mutants, indicating that it might be a DprE1 inhibitor (Fig. 5). Recently, Batt et al. showed that nitrofurans can also target DprE1 (36). Together, these findings support potential for developing nitrofuran scaffolds as DprE1 inhibitors.

All the nitro-containing compounds have a narrow spectrum of activity

Several of the nitro compounds need a mycobacterial-specific target or system for activation; therefore, we hypothesized that they would have a narrow spectrum of activity. To test this hypothesis, we carried out a dose-response study of the compounds against Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Enterobacter faecalis, and Staphylococcus aureus. We also used pretomanid as a control. As expected, pretomanid did not affect any of the test organisms, indicating a narrow spectrum of activity (Table 1). Similarly, the nitro-containing compounds had a narrow spectrum of activity displaying little or no effect on the tested pathogens not belonging to the genus Mycobacterium (Table 1).

HC2209, HC2210, and HC2211 are active against M. abscessus

The inhibitory activity of the prioritized compounds was next tested against the mycobacterial species, Msm and Mab. These organisms retain some degree of genome homology with Mtb, suggesting that the compounds may also inhibit these mycobacterial species. Interestingly, we observed different inhibitory profiles for the nitro-containing scaffolds with respect to the test species.

Pretomanid and the F₄₂₀-dependent nitrofurans had no inhibitory effects on Msm (Table 1). This agrees with previous reports on the loss of activity of pretomanid against Msm (14, 37, 38). However, when tested against Mab, the F₄₂₀-dependent nitrofurans diverged from pretomanid (Table 1; Fig. S5). While pretomanid did not inhibit the pathogen even at high concentrations, the F₄₂₀-dependent nitrofurans showed potency against the pathogen (EC₅₀ = 0.81–5 µM) that is better or comparable to amikacin (EC₅₀ = 5.4 µM). Studies are currently underway to decipher the mechanisms of action of these nitrofurans against Mab.

While the putative DprE1 inhibitors (HC2217, HC2226, HC2238, HC2239, and HC2250) retain their activity against Msm, they lose their activity against Mab (Table 1). Indeed, the isolation and whole-genome sequencing of Msm-resistant mutants further confirmed that the compounds are likely DprE1 inhibitors in Msm (Fig. S6 and S7; Supplemental Data 1). From the genome sequence analysis, we also observed that mutations in the regulator, MSMEG_6503, caused resistance against the tested putative DprE inhibitors. This corroborates previous studies that show mutations in MSMEG_6503 lead to the overexpression of a nearby nitroreductase, NfnB, in Msm (24, 39). NfnB can subsequently inactivate the exposed nitro groups of the compounds, reducing their potency. The Msm-resistant mutants retained their susceptibility to other cell wall inhibitors such as isoniazid and ethambutol (Fig. S7), further confirming the different cellular targets of the compounds.

HC2210 is orally bioavailable and efficacious in a chronic murine Mtb infection model

Based on the promising drug-like potency of HC2210, we examined its efficacy in a murine model of chronic tuberculosis. C57Bl/6 mice were aerosol infected with Mtb Erdman, and the infection was allowed to progress for 39 days before initiating treatment. For treatment, one group was treated by oral gavage with HC2210 at 75 mg/kg, dosed once daily, 5 days a week. The other groups were either treated twice daily with rifampicin (10 mg/kg) as a positive control or sham control (corn oil/DMSO). After 4 weeks of treatment, compared to the vehicle control group, HC2210 reduced the bacterial burden by 1.1-log and 1.2-log CFU in the lungs and spleens of infected mice, respectively (Fig. 6). Overall, these data show HC2210 is orally bioavailable and efficacious in a mouse model of Mtb infection and support its further development.

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.

DISCUSSION

The nitro-containing compounds in this study have potent antimycobacterial activities against both Mtb and Mab. Other nitrofurans or dinitrobenzamides have been previously described (2, 4, 5, 24, 21 - 23); however, several of the tested compounds are chemically distinct and based on their potency warranted further characterization. Using a genetic selection method, we found that nitrofurans such as HC2209, HC2210, and HC2211 depend on the fdg1 activation system for their antimicrobial activities. This system of activation is also used by pretomanid and delamanid, two clinically approved TB drugs. Fdg1 provides the reduced form of cofactor F₄₂₀ that Ddn uses to activate the nitro-containing compounds into active metabolites. To date, Ddn is the only nitroreductase that has been described in the activation of pretomanid and delamanid (7, 9 - 14 - 16 - 17). We further confirmed this with the full loss of activity of pretomanid when tested against the ddn mutants in this study. Interestingly, the Fdg1-dependent nitrofurans did not fully lose their potency against the ddn mutants. They retained some levels of antimycobacterial activities at high concentrations (Fig. 4; Fig. S2). This suggests the presence of other Fdg1-dependent reductases (FDORs) that may be playing a role in the activation of the compounds. A similar observation was made by Wang et al. (2) for JSF-2019, another nitrofuran. Deletion of fdg1 led to a large loss in the activity of JSF-2019, while perturbation of ddn only led to a slight potency loss. This led the authors to suggest that Ddn is not the primary reductase for JSF-2019. In this case, we observed a large potency loss when we tested the ddn spontaneous mutants against the nitrofurans. We propose Ddn as the primary nitroreductase for these Fdg1-dependent nitrofurans and suggest a possible role for other secondary FDORs in the activation of the compounds. Many computationally and functionally annotated FDORs exist in the literature (37, 40, 41), but only Ddn is involved in the activation of different antimycobacterial nitro compounds. Interestingly, CGI-17341, a parent nitroimidazole molecule for pretomanid and delamanid (11, 42, 43), depends on Fdg1 but not Ddn for activation (12, 16). This same conclusion was made in another study that associated a full loss of antitubercular activity of some nitrofurans with spontaneous mutations in fdg1 or the F₄₂₀ biosynthesis pathway (44). These compounds, however, retained their efficacy against a ddn mutant. Taken together, these studies suggest the possibility of uncovering other clinically relevant FDORs.

The Fdg1-dependent nitrofurans were also different from pretomanid in their activity against the growth of Mab. While pretomanid did not have any inhibitory effect on Mab, HC2209, HC2210, and HC2211 retained their activity against the pathogen. Mab is a challenging to treat pathogen that is non-responsive to many antibiotics. The intrinsic resistance of Mab limits the chemotherapeutic strategies for treating the infection (45). Among other factors, the intrinsic resistance of Mab may be attributed to its highly efficient efflux system. Genetic polymorphic differences may also explain the lack of activity of pretomanid against the pathogen. Indeed, phylogenetic analysis and multiple sequence alignment showed a low homology or relatedness between the Ddn in Mtb and Mab (38). However, these reasons do not fully explain why we see differences in the susceptibility of Mab to pretomanid and the Fdg1-dependent nitrofurans described here. We suggest two hypotheses to further explain the susceptibility of Mab to these nitrofurans. Ddn of Mtb and its Mab homolog may share residues that interact with these nitrofurans but not pretomanid. This can be tested through detailed biochemical studies and co-crystallization of the compounds with the Ddn of both species. Unfortunately, researchers have been unable to isolate co-crystals of pretomanid with Ddn (7). Only the crystal structure of Mtb Ddn has been solved, and molecular docking has been used to identify residues that interact with pretomanid (7, 14, 16, 37). Our second hypothesis, reinforced by the partial resistance of the Mtb ddn mutants to the tested nitrofurans, is that Mab may be using an unknown FDOR to activate these compounds. This FDOR may be found in both species but can only activate the nitrofurans described in this study. This hypothesis can be tested by the selection of spontaneous mutants or targeted disruption of candidate FDORs in Mab and testing for resistance. This study also identified two nitrofurans—HC2233 and HC2234—which did not depend on either Fdg1 or Ddn for activation and do not elicit resistance in DprE1 mutants. It is possible that these compounds may not be prodrugs and do not require a nitroreductase for activation or they are prodrugs that require unknown activation systems.

The other five nitro-containing compounds in this study (HC2217, HC2226, HC2238, HC2239, and HC2250) are proposed to be DprE1 inhibitors. These inhibitors were potent against Mtb and Msm, probably owing to the high homology of DprE1 between both species (46, 47). Generally, DprE1 inhibitors can be classified as covalent or non-covalent inhibitors (34, 47). Resistance to covalent DprE1 inhibitors is usually characterized by the substitution of C387/384 to different residues (33, 34, 47). Due to the generation of C384S spontaneous mutants resistant to these compounds, we can speculate that they may be covalent DprE1 inhibitors. However, this can only be conclusively determined with detailed biochemical and structural analyses. Additionally, HC2250 seems to be different from other DprE1 inhibitors in terms of its bactericidal activity against NRP bacilli. During dormancy, cell wall biosynthesis, replication, or translation is minimized. Hence, drugs that target these physiologic activities in actively replicating cells are less effective against Mtb in the NRP state. As expected, NRP Mtb tolerated all the tested dinitrobenzamides (HC2217, HC2226, and HC2238) since these compounds likely target DprE1, an enzyme in the cell wall biosynthesis pathway. However, HC2250, a putative DprE1 inhibitor, continued to kill the bacilli even in the NRP phase. This suggests that HC2250 may also be targeting a cellular process that is needed by Mtb during dormancy. In the isolated dprE1 Msm mutants (Data S1), we observed partial resistance of F346C spontaneous mutants against the tested DprE1 inhibitors. The putative DprE1 inhibitors did not have any inhibitory activity against Mab, agreeing with a study done with PBTZ169, a covalent DprE1 inhibitor that has undergone clinical trials for TB treatment (35).

Apart from identifying potential chemical probes that can be used to further understand Mtb physiology, a critical end goal of most drug discovery efforts is to move potent compounds from the lab into the clinic. The in vitro inhibitory activities of most antitubercular compounds can be difficult to translate into in vivo potency due to the complex nature of Mtb infection and the pharmacokinetic considerations (2, 48). These factors lead to a high attrition rate for most antitubercular agents. In this pilot study, without optimizations, HC2210 significantly reduced the burden of Mtb in both the lung and spleen of the infected mice when delivered orally. This finding shows the promise of HC2210 as a potential TB drug. HC2210 is a nitrofuran with a piperazine backbone. HC2210 has two nitro groups, and an important question is whether one or both nitro groups are necessary for the full antimycobacterial activity of the compound. Second, are there functional groups in the compound that might pose a significant metabolic liability in a human host? These questions seem to have been partly answered in an earlier structure-activity relationship (SAR) study of antimycobacterial nitrofuranyl methyl piperazine series (20). The furan ring was preferred to other heterocycles in order to maintain the antitubercular activities of the compounds, while the piperazine ring was preferred to substituents such as morpholine or piperidine. These nitrofuranyl series only had one nitro group, and as will be expected, the removal of the nitro group abolished the antitubercular activities of the compounds. This SAR study established the nitro group as the pharmacophore, but it does not show if the presence of two nitro groups may contribute to the overall potency of the compounds. The review of the related HC2209, HC2210, and HC2211 structures showed that the three compounds are similar except that HC2210 has two nitro groups. The potency of HC2210 is in the nanomolar range, while those of HC2209 and HC2211 are in the low micromolar range, indicating that the additional nitro group of HC2210 may play a role in its high potency. Structure-activity relationship studies involving the synthesis of new analogs will be needed to address this question.

Overall, this report characterizes 10 antitubercular nitro-containing compounds from the MLSMR collection and showed their potential development as TB drugs. Genetic analyses provided evidence supporting distinct mechanisms of action. Some compounds are putative DprE1 inhibitors, while others depend on Fdg1 for activation and likely inhibit Mtb following mechanisms similar to pretomanid. We also highlighted the possibility of unknown FDORs involved in further activating HC2209, HC2210, and HC2211. Notably, for HC2233 and HC2234, we could only rule out resistance in mutants we have isolated in fdg, ddn, and dprE1. A drawback of this chemical genetics approach is the need for more biochemical experiments to define the specific mechanisms of action. However, this problem is remedied by the established body of biochemical and structural knowledge already available on the subject. Additionally, the resistance of the ddn and fdg1 spontaneous mutants to pretomanid serves as a probe-based confirmation that these genes are driving the resistance of some of the compounds reported in this study. A significant takeaway from this study is the possibility of developing HC2210 as an orally bioavailable TB drug. The compound is also active against Mab, a pathogen that is recalcitrant to most drugs, highlighting the possible use of this compound to treat the infection.

MATERIALS AND METHODS

Culture conditions, strains, and compounds

Unless otherwise specified, streptomycin-resistant or wild-type Erdman and CDC1551 Mtb strains were used. The strains were maintained in 7H9 Middlebrook medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.05% Tween 80, and with or without 0.2% cycloheximide and were incubated at 37°C and 5% CO₂ in standing vented flasks. M. smegmatis mc²155 and M. abscessus ATTCC 19977 were grown shaking in 7H9/OADC media at 35-37°C. Other cultures used in this study include Staphylococcus aureus Wichita (29213) or Seattle (25923), Escherichia coli (Migula), Pseudomonas aeruginosa (Schroeter), Proteus vulgaris (Hauser emend. Judicial Commission), and Enterococcus faecalis (Andrewes and Horder). Except for E. faecalis which was grown in either brain heart infusion medium or Luria-Bertani (LB), all the non-mycobacterial cultures were grown exclusively in LB broth at 35-37°C.

Antimycobacterial compounds were purchased from commercial vendors that supply compounds with >90% purity. HC2209,HC2210, and HC2211 were supplied by Chembridge; HC2217 by Enamine; HC2226 from Chemdiv; HC2233 and HC2234 from Specs; and HC2238, HC2239, and HC2250 from Vitas-M. To authenticate the supplied compounds, the mass of the compounds was examined by electrospray ionization (ESI) or atmospheric pressure chemical ionization mass spectrometry in the positive mode. All of the tested compounds had observed masses matching the predicted masses (Table S1). For HC2210, the oxalic acid cannot be detected using the ESI method.

In vitro dose-response study in M. tuberculosis and spectrum of activity in other mycobacteria and non-mycobacterial species

Mtb cultures were aliquoted (0.2 mL) into 96-well assay plates to an initial optical density (OD) of 0.1. Starting at 80 µM, the cultures were treated with an 8-point (2.5-fold) dilution series of the test compounds (HC2209, HC2210, HC2211, HC2217, HC2226, HC2233, HC2234, HC2238, HC2239, HC2250, pretomanid, isoniazid, and ethambutol). For the comparative study of the most potent compounds (HC2210, pretomanid, and isoniazid), a 12-point (twofold) dilution series starting from 40 µM were used. The treated cultures were incubated for 6 days at 37°C and in 5% CO₂. After incubation, the OD of the cultures was measured in a plate reader (PerkinElmer Enspire) at 595 nm, and the growth of the cultures was normalized based on the OD relative to a rifampicin-positive control (100% growth inhibition) and a DMSO-negative control (0% growth inhibition). The half-maximal effective concentrations (EC_50s) of each compound were determined by fitting the normalized data to a four-parameter logistic equation using the GraphPad Prism software package.

For Msm and Mab, the cultures were diluted to an initial OD of 0.1 and aliquoted into 96-well plates (0.2 mL) or 384-well plates (0.05 mL). This is followed by the treatment of the cultures with 2.5-fold serial dilutions of the compounds starting from either 200 or 80 µM. The cultures were incubated for 3 days before measuring the OD. Growth was normalized based on a positive control (kanamycin for Msm or amikacin for Mab) and a DMSO negative control. The EC_50s of the compounds were determined by fitting the normalized data to a four-parameter logistic equation using the GraphPad Prism software package.

For the non-mycobacterial cultures, an initial OD of 0.05 was prepared and aliquoted into 96-well plates (0.2 mL) or 384-well plates (0.05 mL). The cultures were treated with 2.5-fold serial dilutions of the compounds starting from either 200 or 80 µM and were incubated for 5–8 h before measuring the OD. Except for P. aeruginosa which was normalized with tobramycin-positive control (100% inhibition), other cultures were normalized with kanamycin (100% inhibition). DMSO was used as the negative control (0% inhibition). The normalized data were fitted to a four-parameter logistic equation to calculate the EC_50s of the compounds using the GraphPad Prism software package.

Kinetic killing assays

For Mtb, an initial OD of 0.1 OD was prepared and dispensed in 0.2 mL aliquots into 96-well assay plates. The cultures were treated with two different doses of the compounds, with an equivalent volume of DMSO used as a negative control. After 4- and 10-day incubation at 37°C and in 5% CO₂, the cultures were diluted serially in phosphate-buffered saline-Tween-80 solution and plated for colony forming units (CFU) in 7H10/OADC agar quadrant plates. The bactericidal activity was determined by comparing the CFU of the initial inoculum to the bacterial CFU after treatment.

Hypoxic shift-down assay to test activity against NRP Mtb

The hypoxic shift-down assay (30) was used to generate NRP bacilli and was performed as previously described with slight modifications (19). Briefly, 0.2 mL aliquots of CDC1551 (hspX′::GFP) culture in 7H9/OADC medium was dispensed into 96-well assay plates to an initial OD of 0.25. The cultures were incubated at 37°C in an anaerobic chamber (BD GasPak). At 4 days of incubation, cultures have become completely anaerobic as indicated by the methylene blue indicator turning to colorless. This was considered to be the first day of anaerobiosis. Aliquots of cultures from day 1 were collected and plated onto 7H10/OADC to quantify the initial CFU. Subsequently, 20 µM of the test compounds was added to the cultures and incubated for 10 days in the anaerobic chamber. DMSO was used as the negative control. The surviving bacterial CFU at different treatments was enumerated at day 10 by plating onto 7H10/OADC agar.

Isolation of resistant mutants

The isolation and confirmation of resistant mutants were done as previously described (49). Briefly, 1 × 10⁹ CFU streptomycin-resistant Erdman culture was plated onto 7H10/OADC agar plates containing 0.3 or 0.1 µM HC2210. The plates were incubated at 37°C until colonies appeared. Colonies were randomly picked from each plate and grown in 7H9/OADC broths. The broth cultures were subjected to a dose-response study using HC2210 as previously described above. Resistance was confirmed by an increase in the EC_50s of the mutants when compared to that of the Erdman streptomycin-resistant culture.

To generate mutants resistant to HC2238, mutant #300.2 (Δfdg1) from the above setup was used. Briefly, 1 × 10⁹ of mutant #300.2 was plated onto 7H10/OADC agar plates supplemented with 5 or 20 µM of HC2238 and incubated at 37°C until colonies appeared. Colonies were grown in broth cultures and subjected to a dose-response study with HC2238 as the test compound. Resistance was confirmed by an increase in the EC₅₀ values of the spontaneous mutants with respect to that of mutant #300.2. The same protocol was used in generating HC2238- and HC2217-resistant mutants in M. smegmatis background except that the agar plates were amended with 10 or 20 µM of the compounds.

Whole-genome sequencing and analysis

The genomic DNAs of the confirmed resistant mutants and an Erdman streptomycin-resistant control (or Msm wild-type control) were extracted and submitted for Illumina-based whole-genome sequencing. The breseq computational pipeline was used to analyze the sequence reads and identify single-nucleotide variations (50). Erdman reference genome (for Mtb) or mc²155 (for Msm) was used in the analysis. After subtracting the mutations shared by the resistant mutants with the Erdman streptomycin-resistant control (for HC2210-resistant mutants), mutant #300.2 control (for HC2238-resistant mutants), or mc²155 WT (for Msm), all the unique mutations in the resistant mutant strains are presented in Supplemental Data 1 (for Msm) or Supplemental Data 2 (for Mtb).

Inhibitory activity against intracellular M. tuberculosis

A previously described protocol was adapted in testing the efficacy of the compounds against intracellular M. tuberculosis (49). Briefly, primary bone marrow-derived macrophages were harvested from C57BL/6 mice and distributed into 96-well assay plates in preparation for mycobacterial infection. The macrophages were infected for 1 h with CDC1551 luciferase reporter strain, followed by treatment with different concentrations of the nitro-containing compounds (80–0.136 µM). Rifampicin and DMSO were used as negative and positive controls, respectively. After incubating the samples for 6 days at 37°C and 5% CO₂, bacterial survival was measured in a luciferase readout assay. The EC_50s of the compounds against intracellular M. tuberculosis were determined by fitting the normalized data to a four-parameter logistic equation using the GraphPad Prism software package.

Eukaryotic cytotoxicity assay

Murine primary bone marrow-derived macrophages were distributed into 96-well assay plates as described above. Different concentrations of the indicated inhibitors, ranging from 80–0.136 µM, were used in treating the macrophages. Cells were treated with DMSO as a positive control, while 4% Triton X-100 served as the negative control. Following a 3-day incubation of the macrophages at 37°C and in 5% CO₂, the viability of the cells was assessed with the CellTiter-Glo (Promega) luciferase assay kit. The half-maximal cell cytotoxicity concentration (CC₅₀) values were calculated by fitting the normalized data into a non-linear four-parameter least squares regression model in the GraphPad prism package.

Evaluation of the efficacy of HC2210 is a chronic murine TB infection model

All animal studies were approved by the Michigan State University Institutional Animal Care and Use Committee. Female ~8-week-old C57BL/6 mice purchased from Jackson Laboratories were used in this study. Low-dose infection was initiated by aerosol exposure to 100 CFU of M. tuberculosis Erdman strain using a Glas-Col aerosol inhalation exposure device. One day after infection, five mice were euthanized, and the lungs were aseptically collected to assess the initial infection dose. The remaining mice were randomly distributed into three groups of eight mice and allowed for 38 days to develop a chronic infection. Treatment was then initiated by administering the mice with oral doses of the vehicle (corn oil/5% DMSO), 75 mg/kg of HC2210, or 10 mg/kg rifampicin through oral gavage. HC2210 was administered once daily, while rifampicin and vehicle doses were given twice daily. The mice were treated 5 days a week, with a 2-day resting period. The treatment lasted 4 weeks after which the mice were euthanized. The lungs and spleens were aseptically removed and homogenized, and the mycobacterial burdens were assessed by enumerating CFUs. The mean differences between the groups were compared using an unpaired Student’s t-test.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00474-23.

Supplemental Data 1. aac.00474-23-s0001.xlsx.

Single nucleotide variants in M. smegmatis resistant mutants

Supplemental Data 2. aac.00474-23-s0002.xlsx.

Single nucleotide variants in M. tuberculosis resistant mutants

Supplemental figures. aac.00474-23-s0003.pdf.

Supplemental figures

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