Research ArticleTuberculosis

Direct inhibitors of InhA are active against Mycobacterium tuberculosis

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Science Translational Medicine  07 Jan 2015:
Vol. 7, Issue 269, pp. 269ra3
DOI: 10.1126/scitranslmed.3010597


New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)–dependent manner and blocked the enoyl substrate–binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB.


Tuberculosis (TB) infections caused by Mycobacterium tuberculosis (Mtb) continue to be a major public health threat, particularly in the developing world. Resistance to multiple drugs, together with the HIV, has created new challenges in the management of TB. In 2012, about 8.6 million people developed TB, including ~400,000 who had multidrug-resistant TB (MDR-TB), with 1.3 million deaths (1). Globally, ~4% of newly diagnosed TB cases and 20% of those previously treated for TB have MDR-TB (1). Hence, there is an immediate need to address the growing problem of clinical drug resistance with new therapeutic entities active against Mtb. Despite some recent successes with several new chemical entities (2), the high attrition rate in drug development and clinical testing requires continued efforts to find better drugs.

Inhibition of the mycobacterial enoyl reductase InhA is one of the most effective means of killing Mtb, as clinically demonstrated by isoniazid, the most potent TB drug. Unfortunately, both MDR and extensively drug-resistant (XDR) Mtb isolates are resistant to isoniazid, predominantly due to mutations in KatG, the catalase-peroxidase involved in the activation of isoniazid (3). This has led to extensive efforts to identify direct InhA inhibitors (47). Over the last two decades, these efforts have yielded many potent structurally diverse direct InhA inhibitors, but so far with limited success in achieving an orally active candidate with in vivo efficacy. Here, we report the identification of a new class of small-molecule mycobactericidal agents, the 4-hydroxy-2-pyridones, using phenotypic screening. These compounds blocked the target InhA without requiring bioactivation. The lead candidate, NITD-916, showed in vivo efficacy and was active against common MDR-TB clinical isolates. Our results suggest that the 4-hydroxy-2-pyridones are an attractive candidate for lead optimization in the quest for new drugs to treat TB.


Identification of 4-hydroxy-2-pyridones and microbiological profiling

A whole-cell high-throughput screen of the ~2.3 million Novartis compound collection against Mtb H37Ra resulted in 20,000 hits with activity >50% inhibition at 12.5 μM concentration. Promiscuous pan-active compounds (8), scaffolds of known anti-TB compounds, cytotoxic compounds against mammalian cells (Huh7 or HepG2), compounds containing undesirable functional groups, and compounds with molecular weight >500, clogP <1 or >4 were deprioritized, resulting in one of the hits NITD-529, a new anti-TB compound (Fig. 1A). NITD-529, 4-hydroxy-6-isobutyl-3-phenylpyridin-2(1H)-one, is a small and polar molecule with moderate activity against Mtb H37Rv (MIC50, 1.5 μM) and good solubility (table S1). Structure-activity relationship studies with several 4-hydroxy-2-pyridone analogs (9, 10) revealed the importance of the pyridone core 4-hydroxy group and R6 lipophilic group (Fig. 1A) for Mtb activity, which led to the identification of NITD-564 and NITD-916 (Fig. 1A). NITD-916, a dimethylcyclohexyl derivative at the R6 position, is 30 times more potent than the initial screening hit NITD-529. The anti-TB activity of NITD-916 is five to eight times more potent than isoniazid (MIC50, 0.33 μM) and PA-824 (MIC50, 0.4 μM) (11), and is comparable to bedaquiline (MIC50, 50 nM) (12). 4-Hydroxy-2-pyridone analogs showed both concentration- and time-dependent bactericidal activity against in vitro replicating Mtb and were also active against Mtb within macrophages (Fig. 1, B and C). The in vitro cidal activity profile of NITD-916 showed rapid killing at concentrations greater than 0.2 μM, similar to isoniazid at 0.5 μM. Viable bacterial counts with isoniazid treatment increased from day 3 to 5, potentially due to the emergence of resistance. However, no such increase in bacterial counts was observed with 4-hydroxy-2-pyridone analogs, possibly suggesting lower mutation frequency. 4-Hydroxy-2-pyridones were also shown to be active against both slow-growing [Mtb, Mycobacterium bovis BCG (Bacille Calmette-Guérin)] and fast-growing (Mycobacterium smegmatis) mycobacterial species (table S2). They showed a narrow spectrum of antibacterial activity, with no activity against tested Gram-positive and Gram-negative bacterial species (table S2). The antimycobacterial activity of 4-hydroxy-2-pyridones was restricted to actively replicating Mtb; they were not active in the Wayne model for nonreplicating persistent hypoxic Mtb (table S2), implying that these molecules block an essential step in active metabolism of Mtb.

Fig. 1. Chemical structures of 4-hydroxy-2-pyridones and in vitro and in vivo antimycobacterial activity of these compounds.

(A) Chemical structures of 4-hydroxy-2-pyridone analogs used in this study. MIC50 values against Mtb are given in parentheses (μM). R1-6 numbering is shown in the NITD-529 structure. (B) Concentration-dependent bactericidal kill kinetics activity of NITD-529 and NITD-916 against in vitro replicating Mtb, compared to that of isoniazid. (C) Concentration-dependent activity of NITD-916, NITD-529, and isoniazid (INH) against Mtb in intracellular activated THP-1 macrophages during a 5-day drug exposure. IC90 and IC99 values are indicated by stippled lines. Both kill kinetics and intramacrophage analysis were performed in biological replicates (n = 2), and results are shown as mean values with SEs.

4-Hydroxy-2-pyridones are also active against six different clinical MDR-TB isolates that are distributed into five prominent clusters representing global populations of Mtb strains (13). The minimum concentration (MIC) required to inhibit 99% growth of the diverse drug-resistant clinical isolates (MDR 1 to 6) by NITD-529, NITD-564, and NITD-916 was in a similar range to that needed to inhibit 99% growth of wild-type Mtb H37Rv (Table 1). The MIC activity of NITD-916 against the MDR-Mtb strains ranged from 0.04 to 0.16 μM (Table 1), demonstrating the potential of 4-hydroxy-2-pyridones for use against MDR-TB strains.

Table 1. Activity of 4-hydroxy-2-pyridone analogs against a panel of drug-resistant TB clinical isolates.

S, streptomycin; I, isoniazid; R, rifampicin; PZA, pyrazinamide; M, moxifloxacin.

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Identification and validation of the molecular target

In an attempt to identify the molecular target of 4-hydroxy-2-pyridones, we isolated and characterized mutants that were spontaneously resistant to NITD-529. The observed frequency of resistance in Mtb H37Rv against a 10× MIC of NITD-529 and NITD-916 was 1 × 10−8, which was nearly two log orders lower than that for isoniazid (Table 2). Five independent Mtb-resistant mutants selected against NITD-529 were at least 25-fold more resistant to NITD-529 than the parental H37Rv strain and also were cross-resistant to both NITD-564 and NITD-916 (Table 2). All of these mutants were equally sensitive to streptomycin. Similarly, the frequency of resistance of M. bovis BCG to NITD-916 was 1 × 10−8, and NITD-916–resistant mutants retained full sensitivity to streptomycin (table S3). Surprisingly, one of the Mtb pyridone-resistant mutants (529-B2) and two from M. bovis BCG (529-2 and 916-B1) showed significant (MIC50, >3-fold) resistance to both isoniazid and ethionamide.

Table 2. Cross-resistance and whole-genome sequencing analysis of 4-hydroxy-2-pyridone–resistant mutants of Mtb.

In genetically complemented strains, MIC50 fold shift with the Mtb H37Rv strain is given in parentheses. +, positive for catalase activity; nd, not determined. I, isoniazid; E, ethionamide; S, streptomycin; WT, wild type.

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To further elucidate the genetic basis of the action and resistance of 4-hydroxy-2-pyridones, we carried out whole-genome sequencing of three Mtb-mutant strains. Whole-genome sequencing revealed two independent single-nucleotide polymorphisms in the inhA gene encoding the NADH [reduced form of nicotinamide adenine dinucleotide (NAD+)]–dependent enoyl–ACP (acyl carrier protein) reductase compared to the parental strain Mtb H37Rv (Table 2). In the 529-B2 mutant, a missense mutation encoding S94A in InhA was observed, whereas 529-S1 and 529-S3 mutants shared a common D148G mutation in InhA. In addition, sequencing of inhA in the remaining Mtb and BCG mutants revealed more missense mutations, that is, G96A, D148E, M161I, M161A, and T17A (Table 2 and table S3). To genetically validate InhA as the molecular target of NITD-916, we overexpressed wild-type or mutated copies of inhA under the control of the hsp60 promoter on a nonintegrative plasmid pMV262. Overexpression of mutated copies of InhA (D148G, D148E, or S94A) in wild-type Mtb H37Rv resulted in >10-fold shift in MIC, whereas overexpression of wild-type InhA resulted in a marginal 1.7- to 2.2-fold shift in MIC for both pyridones and isoniazid (Table 2). Thus, resistance-conferring mutations in InhA seem to exert a dominant effect over the wild-type protein. None of the mutations affected the MIC for streptomycin. Together, these data suggested that a mutation in InhA may be responsible for resistance to the 4-hydroxy-2-pyridones.

InhA is an essential component of the FAS-II (fatty acid synthase-II) complex and is necessary for mycolic acid biosynthesis. Mycolic acids are long-chain fatty acids (C60–80) that are major constituents of a mycobacterial cell wall (14). The mycobacterial FAS-II pathway differs significantly from the mammalian FAS-I pathway, which uses a multienzyme complex, in contrast to the distinct enzymes that separately accomplish each step of the bacterial acyl chain elongation cycle. In the last step, the double bond of the enoyl-ACP is reduced to acyl-ACP by InhA. InhA is a well-known clinically validated target of the TB drugs isoniazid and ethionamide (6). Isoniazid is a prodrug activated by the mycobacterial catalase-peroxidase enzyme (KatG) to its acyl radical form, which reacts with NAD [nicotinamide adenine dinucleotide (oxidized form)] to form an adduct that inhibits InhA (15, 16). All of the tested 4-hydroxy-2-pyridone–resistant mutants showed a catalase-positive phenotype, and all the MDR-TB clinical isolates tested that contained a mutation in katG were fully sensitive to 4-hydroxy-2-pyridones (Table 1). All five InhA mutations that conferred pyridone resistance were highly conserved across mycobacterial species (fig. S1), among them only S94 and M161 showed low-level cross-resistance to isoniazid and ethionamide; the other mutations (T17, G96, and D148) remained fully sensitive to both drugs (Table 2 and table S3). An MDR-TB clinical isolate with a mutation in InhA::I194T was resistant not only to isoniazid but also to 4-hydroxy-2-pyridones (Table 2).

It is well established that inhibition of Mtb InhA by isoniazid results in the depletion of mycolic acids from the cell wall with a concomitant accumulation of fatty acids (17). [14C]Acetate metabolic labeling experiments revealed that the treatment of mycobacterial cells with NITD-529 and NITD-916 resulted in a dose-dependent inhibition of mycolates, with a concomitant accumulation of fatty acids (Fig. 2, A and B, and fig. S2). However, no significant changes in the lipid profiles were observed with 4-hydroxy-2-pyridone–resistant mutants of Mtb (Fig. 2 and fig. S2). Together, our data suggests that NITD-916 potentially targets InhA and that its mechanism of action is independent of KatG activation.

Fig. 2. Mechanism of action of pyridones.

(A and B) Fatty acid (A) and mycolic acid (B) lipid profiles of Mtb after exposure to 4-hydroxy-2-pyridones. Fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were prepared after [14C]acetate metabolic labeling and analyzed by thin-layer chromatography and phosphorimaging. (C and D) Binding of NITD-529 (C) and NITD-564 (D) to the apo-InhA (triangles), the InhA-NAD+ complex (circles), and the InhA-NADH complex (squares) was measured by ITC.

In vitro inhibition and biophysical interaction of 4-hydroxy-2-pyridones with InhA

To confirm that InhA is the molecular target of 4-hydroxy-2-pyridones, enzyme inhibition studies in biochemical assays were carried out. In these experiments, NITD-529 (MIC, 1.54 μM) and NITD-564 (MIC, 0.16 μM) inhibited the InhA enzyme activity with an IC50 (50% inhibition concentration) of 9.60 and 0.59 μM, respectively (Table 3). Although NITD-916 showed about three times more cellular potency than NITD-564, no shift in the enzyme IC50 was observed, potentially due to the difference in cell permeability because NITD-916 has more than a log unit higher logP than NITD-564. The 4-methoxy cell-inactive analog (NITD-560; MIC, >20 μM) was also inactive against the InhA enzyme (Fig. 1A and Table 3). Similarly, a para-chloro substitution on the R3 position (NITD-520; MIC, >20 μM) was also inactive against the InhA enzyme, unlike a meta-chloro substitution (NITD-716) (Table 3). Overall, with a limited set of compounds, the InhA enzyme inhibition by 4-hydroxy-2-pyridones correlated with their Mtb MIC. In vitro, the InhA enzyme IC50s of 4-hydroxy-2-pyridone analogs were 4- to 10-fold higher than Mtb cellular activity. The difference in InhA and cellular IC50 may be due to intracellular accumulation, differential sensitivity of InhA between in vitro and in vivo conditions, and potential direct or indirect effects including secondary molecular targets of 4-hydroxy-2-pyridones inside the cells.

Table 3. Interaction of 4-hydroxy-2-pyridones with InhA.

— indicates not determined.

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Isothermal titration calorimetry (ITC) revealed that NITD-529 bound to InhA only in the presence of NADH, but not in the presence of NAD or enzyme alone (Fig. 2, C and D). NITD-529 and NITD-564 bound to the InhA-NADH complex with a Kd (dissociation constant) of 25 and 0.56 μM, respectively (Fig. 2 and Table 3). Compound binding to NADH fits well with one binding site per monomer. Differential scanning fluorimetry also confirmed that 4-hydroxy-2-pyridones preferentially bound to the InhA-NADH complex similar to PT166 (18) and in contrast to PT70 (19), a diphenyl ether derivative that forms a stable ternary complex with NAD+ (Table 3). NITD-529 binding to the InhA-NADH complex led to a modest increase (ΔTm +4.4°C) in thermal stability compared to binding to the InhA-NAD+ complex; in contrast, the thermal stability of the diphenyl ether was higher with NAD+Tm −6.6°C) (Table 3). Similar NADH-dependent binding has recently been shown for methylthiazoles (7). Collectively, the enzymology and biophysical binding data demonstrate that 4-hydroxy-2-pyridones are direct InhA inhibitors and that they preferentially bind to the InhA-NADH complex.

Crystal structure of cofactor-bound InhA with NITD-564 and NITD-916

To further understand the molecular interaction of 4-hydroxy-2-pyridones with InhA, co-crystal structures of ternary complexes with NADH–NITD-564 and NADH–NITD-916 were solved at resolutions of 2.9 and 3.2 Å, respectively (table S4 and Fig. 3). The refined structures are similar, with a root mean square deviation for all Cα atoms of 0.199 Å, and the ligands NITD-564 and NITD-916 were bound in the same position and orientation (Fig. 3A). Consistent with the ITC and thermal shift data, both NITD-916 and NITD-564 bound to the enzyme-NADH (E-NADH) complex (Fig. 3B). The co-crystal structure revealed five key interactions of 4-hydroxy-2-pyridones with InhA and the NADH cofactor: (i) the aromatic pyridone ring of 4-hydroxy-2-pyridones π-stacks against the pyridine ring of the cofactor NADH; (ii) the oxygen at the 4-hydroxy group of NITD-916 hydrogen bonds with the 2′-hydroxyl moiety of the nicotinamide ribose sugar and the hydroxyl of Y158 of InhA, a conserved residue of the enoyl reductase active-site triad; (iii) the N of the pyridone core interacts with the S of M199 (Fig. 3, B and C); (iv) the R3 phenyl ring is exposed to a narrow pocket of the enzyme; and (v) the cyclohexyl or dimethylcyclohexyl moiety of NITD-564 and NITD-916, respectively, occupies the large hydrophobic pocket constituting the side chains of F149, M155, Y158, M199, G192, P193, I215, L218, and W222 (Fig. 3B). Consequently, at the R6 position, when the isopropyl group in NITD-529 was replaced by cyclohexyl (NITD-564), it provided a better interaction between the ligand and these hydrophobic side chains, leading to a 10-fold increase in anti-Mtb potency, as well as in the potency against the InhA enzyme (Table 3). Replacing the cyclohexyl with the more hydrophobic dimethylcyclohexyl (NITD-916) further enhanced Mtb activity by 10-fold. NITD-916 binding to InhA led to ordering of the substrate-binding loop encompassing residues 196 to 211 (Fig. 3A). All five pyridone-resistant mutations mapped in InhA (T17, S94, G96, D148, and M161) were within 6 Å of the NADH-binding site (Fig. 3C).

Fig. 3. Structural analysis of the 2-pyridone–binding site in the InhA-NADH complex.

(A) Superimposed crystal structures of InhA–NADH–NITD-564 (yellow) and InhA–NADH–NITD-916 (orange); respective 2-pyridone ligands are shown in green and cyan. Substrate-binding loop encompassing residues 196 to 211 is shown in red. (B) Close-up of NITD-916 (cyan)–binding pocket in InhA-NADH complex, with protein polar (cyan) and hydrophobic (gray) surfaces shown. The side chains of Y158 and M199 residues are shown. The distance (in Å) between the ligand and side chains of Y158 and M199 and the 2′-OH on the ribose sugar of NADH are highlighted by dotted lines. (C) Hydrogen bonding interactions of NITD-916 with critical residues in the active site of InhA. Side chains of amino acid residues responsible for NITD-916 resistance (T17, S94, G96, D148, and M161) are shown. (D) The InhA–NITD-916 (green) structure overlaid with the fatty acyl substrate (cyan, 1BVR), along with other direct InhA inhibitors, namely, triclosan derivative (orange, 3FNG), alkyl diphenyl ether (gray, 2X23), pyrrolidine carboxamides (pink, 2H7I), and methylthiazoles (blue, 4BQP). (E) InhA–NITD-916 structure (red) overlaid with co-crystal structures of fatty acyl substrate (blue, 1BVR) and alkyl diphenyl ether (yellow, 2X23). NITD-916, fatty acyl substrate, and alkyl diphenyl ether ligands are colored in green, cyan, and gray, respectively. The shift in the conformation of the substrate-binding loop is shown by an arrow.

Superimposition of the InhA–NITD-916 structure with the enoyl substrate–bound form (20) revealed that NITD-916 partly occupies the fatty acyl substrate–binding pocket, and the dimethylcyclohexyl group forms hydrophobic interactions with the substrate-binding loop (Fig. 3D). Thus, it is likely that the binding of the NITD-916 to InhA-NADH complex blocks enoyl substrate access to its binding site on the enzyme. Earlier efforts to identify direct InhA inhibitors to overcome KatG-mediated resistance yielded many inhibitors that blocked the lipid-binding site (47, 19) (fig. S3). Overlaying a few of these inhibitors (7, 2123) along with the fatty acyl substrate in the NITD-916 structure revealed that these inhibitors all occupied the enoyl substrate–binding site (Fig. 3D). Recently, the natural product pyridomycin has also been shown to be a direct InhA inhibitor occupying both NADH and enoyl substrate–binding sites (5, 24). The InhA–NITD-916 structure attained an open substrate-binding loop conformation similar to the enoyl substrate–bound structure (20), unlike PT70 (23), which has a closed conformation (Fig. 3E). Thus, the co-crystal structures confirmed the NADH-dependent binding of 4-hydroxy-2-pyridones and showed that these ligands partly occupy the enoyl substrate–binding pocket.

Pharmacology of 4-hydroxy-2-pyridones

Considering the lengthy treatment period for MDR-TB (more than 18 months), cytotoxicity, cardiotoxicity, or mutagenicity of compounds would impede the progression to clinical testing (25). Hence, it is important to identify the potential safety liabilities of 4-hydroxy-2-pyridones. In a battery of in vitro assays, these compounds were found to have an adequate cellular selectivity, displayed no mutagenic or cardiotoxicity potential, and showed no in vitro safety pharmacological liabilities (table S1). Any new TB drug has to be clinically administered in combination with other antitubercular or antiretroviral medications, so the absence of drug-drug interactions is critical for clinical development (25). NITD-916 and other pyridone analogs did not inhibit the major CYP450 isoenzyme 3A4 in either reversible or time-dependent inhibition assays and also did not induce hPXR (human pregnane X receptor) activation (table S1).

NITD-916 has low aqueous solubility and high permeability in vitro with a low to moderate metabolic clearance in mouse and human hepatic microsomes (table S1). Considering the highly lipophilic nature of the compound, a special lipid-based microemulsion preconcentrate (MEPC) formulation was required for oral pharmacokinetic (PK) studies (26). Upon intravenous administration, the compound showed a low total systemic clearance and low volume of distribution (Vd, 0.54 liter/kg) but good oral bioavailability (66% at 25 mg/kg) in rodents (table S5). The plasma concentration of NITD-916 in vivo (table S5) was greater than the in vitro MIC against Mtb, justifying mouse efficacy studies. Nonetheless, it is worth noting that the compound distribution in the lungs was lower than that in plasma [at a dose of 25 mg/kg, the ratio of Cmax in lungs to Cmax in plasma was 0.2, and the area under the curve (AUC) ratio was 0.4]. In the acute and established murine efficacy models, all animals tolerated 1 month of daily dosing of NITD-916 in MEPC formulation. In the acute model, NITD-916 showed a dose-dependent in vivo activity in both lungs and spleen (Fig. 4A). Treatment of mice with NITD-916 for 1 month at a dose of 100 mg/kg resulted in 1.92 and 2.82 log CFU (colony-forming unit) reduction in lung and spleen tissues, respectively, compared to untreated control animals. The efficacy of NITD-916 (100 mg/kg) observed in the acute infection model is comparable to rifampicin (10 mg/kg) and ethambutol (100 mg/kg), but inferior to isoniazid (25 mg/kg). In an established infection mouse model, 4 weeks of treatment with NITD-916 (100 mg/kg) reduced the bacterial burden by 0.95 log in the lungs (Fig. 4B). The in vivo efficacy of NITD-916 in the established model was comparable to the first-line TB drug ethambutol (100 mg/kg) and a potential drug candidate, PA-824 (25 mg/kg). However, under similar conditions, rifampicin and isoniazid were significantly more potent than NITD-916. Despite its very high plasma protein binding, low volume of distribution, and low lung-to-plasma ratio, the lead candidate NITD-916 showed dose-dependent in vivo efficacy in the lungs of infected mice when given orally.

Fig. 4. In vivo efficacy of NITD-916 in Mtb-infected mouse models.

(A and B) BALB/c mice were infected intranasally with 103 H37Rv strain of Mtb, and animals were orally treated at the indicated doses (mg/kg) for 4 weeks after 1 or 4 weeks of infection in acute (A) or established (B) efficacy models, respectively. Efficacy of test compounds was measured by Δlog CFUs compared to untreated controls independently for lung and spleen. Statistical evaluation was done using one-way analysis of variance (ANOVA), and the data were analyzed using Tukey’s multiple comparison test. Statistical significance was accepted with P values <0.05. All bars labeled with the same letter of the alphabet do not differ significantly. INH, isoniazid; RIF, rifampicin; EMB, ethambutol.


Isoniazid, which primarily targets InhA, is one of the key pillars of TB chemotherapy. Considering the frequent emergence of KatG-mediated isoniazid resistance in patients, several groups have identified structurally diverse direct InhA inhibitors (6, 27). However, translating the potent enzyme inhibitors into compounds with anti-Mtb activity and the physicochemical properties required for achieving optimal bioavailability and in vivo efficacy remains a significant challenge. Here, using unbiased phenotypic screening, we report the identification of a new class of anti-mycobactericidal agents, the 4-hydroxy-2-pyridones, that act as potent and direct InhA inhibitors. These compounds do not require KatG-mediated activation; isoniazid-resistant MDR/XDR-TB clinical isolates with katG mutations were fully susceptible to the lead compound NITD-916. Moreover, the 4-hydroxy-2-pyridones have appropriate physicochemical properties and a mode of binding that translated into potent anti-TB activity and in vivo efficacy in both acute and established mouse infection models.

InhA is highly conserved in eubacteria with 27 to 33% amino acid sequence identity (fig. S1). Sequence alignment of enoyl reductase homologs across multiple bacterial species revealed that the five NITD-916–resistant InhA mutations are highly conserved in the genus mycobacterium, but not in other Gram-positive and Gram-negative bacteria, conceivably explaining the narrow spectrum of activity (table S2). Moreover, other pathogenic mycobacteria such as Mycobacterium leprae and Mycobacterium ulcerans, which cause leprosy and buruli ulcer, respectively, also share a nearly identical InhA sequence and thus are expected to be susceptible to 4-hydroxy-2-pyridones. It has been shown previously that InhA S94A and D148G mutants have 7- to 14-fold less affinity for NADH than the wild-type enzyme (24, 28). This suggests that the resistance to NITD-916 arises because of remodeling of the cofactor-binding site in InhA. Recently, resistance to other direct InhA inhibitors, the methylthiazoles, has been mapped to G96 and M103, other conserved residues in the vicinity of the NITD-916–binding pocket (7). Likewise, resistance to pyridomycin, a natural product known to block both the NADH and enoyl substrate–binding pockets of InhA, has been mapped to residue D148 (5, 24). Thus, the mutation data offer strong genetic evidence for molecular target engagement by 4-hydroxy-2-pyridones. The in vitro frequency of spontaneous 4-hydroxy-2-pyridone–resistant mutants of Mtb is 100 times lower than for isoniazid (Table 2), suggesting a lower risk for developing drug resistance.

Unlike most other ligands that are known to bind to the InhA-NAD product complex (for example, triclosan, alkyl diphenyl ethers, and pyrrolidine carboxamides), NITD-916 preferentially binds to the InhA-NADH complex [like methylthiazole (7) and pyridone inhibitors of other FabIs (18)] and occupies part of the lipid substrate–binding site. The affinity (Km) of InhA enzyme for NADH is about two log orders higher than that for NAD+ (7); therefore, ligands that bind to the E-NADH complex are likely to be more efficient than those that bind to the enzyme-NAD complex. Structural data indicate that NITD-916 binds to the InhA-NADH complex with multiple hydrogen bonds, π-stacking, and hydrophobic interactions. Thus, we propose that binding of 4-hydroxy-2-pyridones to the InhA-NADH complex inhibits the fatty acid elongation step, resulting in blocking of the biosynthesis of mycolic acids, weakening of the cell wall mycolyl-arabinogalactan-peptidoglycan complex, and ultimately lysis of Mtb (fig. S4).

NITD-916 is a promising small-molecule lead candidate that is not mutagenic, lacks cardiotoxicity, and does not show CYP (cytochrome P450)–mediated drug-drug interactions. Addressing the solubility and lipophilicity concerns of 4-hydroxy-2-pyridones will be critical to move away from lipid-based formulation and also to de-risk the drug during clinical development. Phosphate ester prodrugs are typically designed to enhance their aqueous solubility to allow a more favorable oral administration and are generally rapidly hydrolyzed by intestinal alkaline phosphatases (29). As a proof of concept, we have synthesized NITD-113, a 4-hydroxy methyl phosphate ester prodrug of NITD-916, which improved the aqueous solubility by two log orders (fig. S5A). The oral mouse PK of NITD-113 in simple 0.5% carboxymethylcellulose (CMC) aqueous formulation resulted in good bioconversion to NITD-916, and the drug exposure achieved with the CMC formulation was similar to NITD-916 in the MEPC formulation (fig. S5B). In addition, preliminary analysis of the binding mechanism of NITD-916 that suggested replacing R3 phenyl with suitable ring systems could potentially address solubility and increase basicity, leading to a higher volume of distribution and lower binding to plasma proteins. Thus, the co-crystal structure of NITD-916 with the InhA-NADH complex has opened up avenues for structure-based rational lead optimization. The real value of identifying the molecular target of a phenotypic hit has yet to be exploited.

The molecular target of 4-hydroxy-2-pyridones, InhA, is a well-recognized clinically validated target. High-throughput phenotypic screening approaches and target deconvolution of NITD-916 by whole-genome sequencing of spontaneous resistance mutants are well-established strategies to elucidate the mechanisms of action of compounds in infectious diseases. Other groups have previously discovered direct inhibitors of InhA and have helped to characterize the ligand-binding pocket of InhA.

Nonetheless, the “rediscovery” of InhA as the molecular target of a phenotypic screening hit with an NADH-dependent mode of binding and in vitro and in vivo potency emphasizes the power of combining cell-based screening with target deconvolution approaches. Given that the lead compound NITD-916 has a low volume of distribution and a reduced lung-to-plasma ratio, further medicinal chemistry optimization will be required to achieve the necessary drug exposure in the lung cavities of TB patients. The biochemical and structural characterization of NITD-916 bound to InhA reported here should facilitate further structure-guided rational approaches to identify better preclinical candidates.


Study design

The objective of this study was to identify a new chemical entity that is active against MDR and XDR-TB. First, a phenotypic high-throughput screening of a library of 2.3 million compounds against Mtb was performed. Further, microbiological profiling of promising hits led to the identification of NITD-529, a 4-hydroxy-2-pyridone with moderate activity against Mtb including MDR-TB clinical isolates. Next, several analogs of 4-hydroxy-2-pyridones were evaluated to improve their activity and optimize PK properties, resulting in NITD-916. Subsequently, the mechanism of action and structural characterization of the ligand-target molecular interactions confirmed that 4-hydroxy-2-pyridones were direct InhA inhibitors. NITD-916 was then evaluated in mouse for in vivo efficacy. Mice were infected and randomized in different groups before drug treatment either 1 or 4 weeks after infection. Animal allocations were not blinded to study scientists because of personnel considerations. Both in vitro and in vivo experiments with Mtb H37Rv were carried out in a biosafety level 3 high-containment laboratory, and all protocols were approved by the Novartis Institute for Tropical Diseases (NITD) institutional biosafety committee. All in vitro experiments were repeated at least two times unless otherwise stated in the figure legends. All in vivo studies were performed in accordance with the state and institutional guidelines and were approved by the NITD Institutional Animal Care and Use Committee. Guidelines for humane endpoints were strictly followed for all in vivo experiments.

Strains and growth conditions

Mtb H37Rv [American Type Culture Collection (ATCC) 27294], Mtb H37Ra, M. bovis BCG Pasteur (ATCC 35734), M. smegmatis MC2 155, and derivative strains were maintained in Middlebrook 7H9 broth medium supplemented with 0.05% Tween 80 and 10% ADS supplement.

High-throughput screening, MIC determination, kill kinetics analysis, and catalase assay

High-throughput screening of Novartis compound collection was carried out using Mtb H37Ra strain, MIC50 against Mtb H37Rv, and kill kinetics analysis was determined as previously (26). For determining growth inhibition against diverse MDR Mtb clinical isolates (13), pellet formation method was used (26). MIC is defined as the minimum concentration of the drug required to inhibit 50% of H37Rv growth or 99% of growth in MDR clinical isolates after 5 or 10 days of incubation, respectively. Catalase activity was assayed using a mixture of H2O2 (15%) and Tween 80 (10%) in water as described earlier (30).

Cytotoxicity determination and intramacrophage activity

Cytotoxicity against HepG2 and THP-1 cell lines was determined as previously described (26). For intramacrophage activity, 5 × 105 THP-1 cells were differentiated using 50 nM phorbol 12-myristate 13-acetate for 48 hours. The differentiated cells were activated with 10 μM interferon-γ for 4 hours and were infected with Mtb at a multiplicity of infection of 1:1. At 1 hour after infection, extracellular mycobacteria were removed by washing twice with warm phosphate-buffered saline (PBS) and replaced with fresh medium with or without compounds. The number of viable intracellular mycobacteria was determined by plating after lysing the macrophages with 500 μl of 0.1% Triton X-100 at 1 hour, 5 days after infection. Inhibitory concentration 90 (IC90) and inhibitory concentration 99 (IC99) were defined as the lowest concentration of drug resulting in 90 and 99% reduction in CFU, respectively (26).

Efficacy studies in mouse models of acute and established infection

Acute and established in vivo efficacy studies were carried out as described previously (26). Briefly, BALB/c mice were infected intranasally with 103 Mtb H37Rv, and animals were orally treated for 4 weeks after 1 or 4 weeks of infection for acute and established efficacy model, respectively. Ethambutol (100 mg/kg), rifampicin (10 mg/kg), isoniazid (25 mg/kg), and PA-824 (25 mg/kg) were used as control. Bacterial loads in lung and spleen (mean ± SD from six mice per group and per time point) were analyzed at 4 weeks after treatment by enumerating CFUs. Statistical evaluation was done using one-way ANOVA, and the data were analyzed using Tukey’s multiple comparison test. Statistical significance was accepted with P values <0.05.

Generation of pyridone-resistant mutants and whole-genome sequencing

Mtb H37Rv strain was plated on 7H11 agar plates containing 10× and 20× MIC50 of the test compounds. After 3 weeks of incubation at 37°C, single isolated colonies were propagated in drug-free 7H9 broth. The resistance phenotype to the pyridones was confirmed by testing MIC50 values against NITD-529, NITD-564, and NITD-916. Whole-genome sequencing and single-nucleotide polymorphism analysis of the spontaneous resistant mutants were carried out as described earlier (26, 31).

Lipid labeling, extraction, and analysis

Radiolabeling of Mtb lipids with [14C]acetate was carried out by adding [1,2-14C]acetic acid sodium salt (1 μCi/ml) to a 5-ml mid-log phase [A600 nm optical density (OD) = 0.3] H37Rv cells treated with the indicated concentrations of drugs/inhibitors for 2 hours. After 1 hour, [14C]acetate labeling cultures were centrifuged, and the cell pellet was used directly to analyze total cellular lipids using the methyl esterification protocol for preparing MAMEs and FAMEs as described earlier (32).

Recombinant expression and genetic complementation of Mtb InhA

Mtb inhA gene (Rv1484) was amplified by polymerase chain reaction (PCR) (Pfx polymerase; Invitrogen) from H37Rv genomic DNA using forward (5′-CTTTAAGAAGGAGATATCATATGACAGGACTGCTGGACGGC-3′) and reverse (5′-GACGCCGGATCCTAGAGCAATTGGGTGTGCGC-3′) primers. PCR-amplified fragments were cloned into Nde I and Bam HI sites of pET15(b) vector to obtain pET15b-inhA (33). Mtb InhA in pET15b vector encoding C-terminal His tag was transformed into BL21 (DE3) cells, and protein expression was induced at 0.6 OD with 0.1 mM isopropyl-β-d-thiogalactopyranoside for 16 hours. Soluble recombinant His6-InhA protein was purified on a nickel affinity binding column in buffer containing 20 mM tris-HCl (pH 8.0) and 500 mM NaCl. Subsequently, the protein was subjected to gel filtration on Superdex 200 in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 1 mM TCEP [tris(2-carboxyethyl)phosphine] buffer. The purified protein was concentrated to ~10 mg/ml in 20 mM Hepes (pH 7.0), 150 mM NaCl, 1 mM TCEP and was used for ITC binding and x-ray crystallographic studies. For genetic complementation, Mtb inhA gene was amplified from the genomic DNA isolated from wild types or mutants using forward (5′-TAGGATCCATGACAGGACTGCTGGACGG-3′) and reverse (5′-CGGAATTCCTAGAGCAATTGGGTGTGCGCG-3′) primers. PCR-amplified fragments were cloned into Bam HI and Eco RI sites of pMV262 vector, transformed into Mtb H37Rv, and selected for kanamycin-resistant recombinant clones.

Binding assay

ITC experiments were performed with an iTC200 (GE Healthcare) at 25°C, with 10 to 20 μM InhA in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 2 mM TCEP in the sample cell. The compound at 125 to 500 μM was diluted, from a 10 mM stock in dimethyl sulfoxide (DMSO), into the same buffer and titrated into the InhA sample supplemented with the same concentration of DMSO. NADH from a 5 mM stock dissolved in water was added to both the cell and syringe at a final concentration of 50 μM. Typically, 15 injections of 2.6 μl of compound were injected into the sample cell at 2.5-min intervals. The data were fitted to a single-site binding equation using Origin. The Thermofluor stability assay was performed on a Bio-Rad CFX96 Real-Time System, using 30 mM Pipes buffer (pH 6.8) containing 150 mM NaCl and 1.0 mM EDTA. Briefly, a 20-μl solution of 7.4 μM InhA, 2.5 mM cofactor (NADH or NAD+), and 25 μM inhibitors was incubated on a 96-well plate at room temperature for 2 hours before adding 5× SYPRO Orange protein gel stain. A thermocycle was run from 25° to 90°C. At the end of each 0.20°C increment for 10 min, the fluorescence intensity of the plate was read after 10 min. Diphenyl ether was used as a positive control for compounds binding to NAD+, and 4-pyridone analog was used as a positive control for compounds binding to NADH.

InhA enzyme assay

Trans-2-dodecenoyl-CoA (DD-CoA) was synthesized from trans-2-dodecenoic acid. IC50 was performed with 25 μM trans-2-dodecenoyl-CoA, 100 nM InhA, 0 to 40 μM inhibitor in 30 mM Pipes (pH 6.8) buffer containing 150 mM NaCl and 1 mM EDTA as described earlier (34). Initial velocity (vi) was measured for the first 10% of the reaction. Percentage activity was plotted as a function of inhibitor concentration into the following equation:Embedded Image

Crystallization and structure determination

InhA in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 2 mM TCEP at 10 mg/ml was mixed with 1 mM NADH (from a 20 mM stock dissolved in water) and 500 μM compound (from a 10 mM stock in DMSO) before setting up hanging-drop crystallization trials. This is about a 1:3:1.5 ratio of protein to NADH to compound. Crystals grew in 10% PEG (polyethylene glycol) 4000, 0.1 M Hepes (pH 7.0), and 0.2 M ammonium acetate. Crystals appeared within a week and were transferred to a cryoprotectant containing the same solution supplemented with 25% DMSO before rapid cooling in liquid nitrogen. X-ray diffraction data were collected at beamline X10SA of the Swiss Light Source, integrated using iMOSFLM (35), and scaled using SCALA (36), part of the CCP4 suite (Winn 2011). The structures were solved by molecular replacement using the structure of InhA [Protein Data Bank (PDB) ID 3OEW] (37) as a search model. Model building was done with COOT (38), and the structures were refined using REFMAC5 (39), using noncrystallographic symmetry restraints.



Fig. S1. Multiple sequence alignment of InhA protein from mycobacterial spp., along with enoyl-ACP reductase homologs from other representative eubacteria.

Fig. S2. Effect of 4-hydroxy-2-pyridones on mycolic acid profiles in wild-type and pyridone-resistant M. bovis BCG mutants.

Fig. S3. Summary of reported direct InhA inhibitors including NAD-isoniazid adduct and isoniazid.

Fig. S4. A proposed mechanism of action for 4-hydroxy-2-pyridones.

Fig. S5. In vivo PK analysis of NITD-113, a phosphate ester prodrug of NITD-916.

Table S1. In vitro PK and safety profiling data for NITD-529, NITD-564, and NITD-916.

Table S2. Minimum inhibitory concentration (MIC50) of pyridones against various mycobacterial species and broad-spectrum Gram-positive and Gram-negative bacteria.

Table S3. Cross-resistance and InhA sequencing analysis of M. bovis BCG 4-hydroxy-2-pyridone–resistant mutants.

Table S4. Summary of data collection and refinement statistics.

Table S5. In vivo lung and plasma PK data for NITD-916 in mice.

References (40, 41)


  1. Acknowledgments: We would like to thank B. Kreisworth for TB clinical isolates; G. De Pascale, M. Sachdeva, and J. Leeds for broad-spectrum antibacterial testing; and S. Nilar and D. McNeeley for feedback on the manuscript. We would like to thank V. Lim, Z. Chen, B. H. Lee, P. Thayalan, N. Mahesh, S. Ravindran, and other colleagues from NITD and Genomics Institute of the Novartis Research Foundation (GNF) for their support. Funding: This work was supported by NITD and GNF. P.J.T. and W.Y. are supported in part by NIH grant GM102864. We thank the beamline scientists at the Swiss Light Source for assistance with data collection. Author contributions: U.H.M., S.P.S.R., L.R.C., B.H.T., S.H.N., K.K., and D.B. performed/analyzed mycobacterial cell–based assays; U.H.M., S.P.S.R., B.H.T., S.W.B., and J.R.W. performed/analyzed whole-genome sequencing; U.H.M., B.H.T., and S.H.N. designed/performed genetic complementation studies; U.H.M. and S.P.S.R. performed/analyzed lipid profiling; P.J.T. and W.Y. performed/analyzed InhA Thermofluor binding and enzyme inhibition studies; U.H.M., C.G.N., B.H.T. and S.H.N. performed protein expression, crystallography, and ITC binding studies; S.B.L. and F.B. performed/analyzed animal PK studies; U.H.M., S.P.S.R., and M.H. performed/analyzed mouse efficacy studies; R.R.K., P.S.N., N.L.M., and P.W.S. performed the cheminformatic analysis, and designed and synthesized analogs; U.H.M., S.P.S.R., R.R.K., L.R.C., F.B., D.B., R.G., P.W.S., and T.T.D. supervised and directed the work; U.H.M., S.P.S.R., C.G.N., and T.T.D. wrote the paper; all authors discussed the results and commented on the manuscript. Competing interests: U.H.M., S.P.S.R., R.R.K., and N.L.M. are named as inventors on the patent application WO2014/093606A1 titled “Pyridone derivatives and uses thereof in the treatment of tuberculosis.” The other authors declare no competing interests. Data and materials availability: Atomic coordinates and structure factors for InhA–NADH–NITD-564 and InhA–NADH–NITD-916 complex structures have been deposited in PDB under accession codes 4R9R and 4R9S, respectively. All requests for compounds are subject to a material transfer agreement.
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