RESUMEN
We previously identified a series of triazolopyrimidines with antitubercular activity. We determined that Mycobacterium tuberculosis strains with mutations in QcrB, a subunit of the cytochrome bcc-aa3 supercomplex, were resistant. A cytochrome bd oxidase deletion strain was more sensitive to this series. We isolated resistant mutants with mutations in Rv1339. Compounds led to the depletion of intracellular ATP levels and were active against intracellular bacteria, but they did not inhibit human mitochondrial respiration. These data are consistent with triazolopyrimidines acting via inhibition of QcrB.
Asunto(s)
Mycobacterium tuberculosis , Antituberculosos/farmacología , Citocromos , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RespiraciónRESUMEN
N-furfuryl piperazine ureas disclosed by scientists at GSK Tres Cantos were chosen as antimycobacterial hits from a phenotypic whole-cell screen. Bioisosteric replacement of the furan ring in the GSK Tres Cantos molecules with a phenyl ring led to molecule (I) with an MIC of 1 µM against Mtb H37Rv, low cellular toxicity (HepG2 IC50 ~ 80 µM), good DMPK properties and specificity for Mtb. With the aim of delineating the SAR associated with (I), fifty-five analogs were synthesized and screened against Mtb. The SAR suggests that the piperazine ring, benzyl urea and piperonyl moieties are essential signatures of this series. Active compounds in this series are metabolically stable, have low cellular toxicity and are valuable leads for optimization. Molecular docking suggests these molecules occupy the Q0 site of QcrB like Q203. Bioisosteric replacement of N-furfuryl piperazine-1-carboxamides yielded molecule (I) a novel lead with satisfactory PD, metabolism, and toxicity profiles.
Asunto(s)
Mycobacterium tuberculosis , Antituberculosos/farmacología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Piperazinas/farmacología , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.
Asunto(s)
Antituberculosos/farmacología , Compuestos Aza/farmacología , Compuestos de Boro/farmacología , Hidrocarburos Fluorados/farmacología , Inhibinas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Carga Bacteriana/efectos de los fármacos , Modelos Animales de Enfermedad , Desarrollo de Medicamentos , Femenino , Isoniazida/farmacología , Pulmón/patología , Ratones , Ratones Endogámicos C3H , Pruebas de Sensibilidad Microbiana , Tuberculosis Pulmonar/microbiologíaRESUMEN
There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8-hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure-activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8-hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of <5 µM for some analogs. Small substitutions at C5 resulted in the most potent activity. Substitutions at C2 generally decreased potency, although a sub-family of 2-styryl-substituted analogs retained activity. Representative compounds demonstrated bactericidal activity against replicating M. tuberculosis with >4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC50 of <100 µM). Further development of this series as antitubercular agents should address the cytotoxicity liability. However, the 8-hydroxyquinoline series represents a useful tool for chemical genomics to identify novel targets in M. tuberculosis.
Asunto(s)
Antituberculosos/síntesis química , Hidroxiquinolinas/síntesis química , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxiquinolina/análogos & derivados , Animales , Antituberculosos/química , Antituberculosos/farmacología , Chlorocebus aethiops , Células Hep G2 , Humanos , Hidroxiquinolinas/química , Hidroxiquinolinas/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Relación Estructura-Actividad , Células VeroRESUMEN
Despite increased research efforts to find new treatments for tuberculosis in recent decades, compounds with novel mechanisms of action are still required. We previously identified a series of novel aryl-oxadiazoles with anti-tubercular activity specific for bacteria using butyrate as a carbon source. We explored the structure activity relationship of this series. Structural modifications were performed in all domains to improve potency and physico-chemical properties. A number of compounds displayed sub-micromolar activity against M. tuberculosis utilizing butyrate, but not glucose as the carbon source. Compounds showed no or low cytotoxicity against eukaryotic cells. Three compounds were profiled in mouse pharmacokinetic studies. Plasma clearance was low to moderate but oral exposure suggested solubility-limited drug absorption in addition to first pass metabolism. The presence of a basic nitrogen in the linker slightly increased solubility, and salt formation optimized aqueous solubility. Our findings suggest that the 1,3,4-oxadiazoles are useful tools and warrant further investigation.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Oxadiazoles/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Relación Estructura-ActividadRESUMEN
We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis The compound had an MIC of 2 µM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 µM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 µM) without cytotoxicity against eukaryotic cells (IC50 > 100 µM). We isolated resistant mutants (MIC ≥ 100 µM), which had mutations in Rv1683, Rv3068c, and Rv0047c.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , Antituberculosos/efectos adversos , Línea Celular Tumoral , Farmacorresistencia Bacteriana/genética , Células Hep G2 , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
We identified a di-substituted triazolopyrimidine with anti-tubercular activity against Mycobacterium tuberculosis. Three segments of the scaffold were examined rationally to establish a structure-activity relationship with the goal of improving potency and maintaining good physicochemical properties. A number of compounds displayed sub-micromolar activity against Mycobacterium tuberculosis with no cytotoxicity against eukaryotic cells. Non-substituted aromatic rings at C5 and a two-carbon chain connecting a terminal aromatic at C7 were preferred features; the presence of NH at C7 and a lack of substituent at C2 were essential for potency. We identified compounds with acceptable metabolic stability in rodent and human liver microsomes. Our findings suggest that the easily-synthesized triazolopyrimidines are a promising class of potent anti-tubercular agents and warrant further investigation in our search for new drugs to fight tuberculosis.
Asunto(s)
Antituberculosos/síntesis química , Antituberculosos/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Triazoles/química , Animales , Antituberculosos/química , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
The Mycobacterium tuberculosis cell wall is a complex structure essential for the viability of the organism and its interaction with the host. The glycolipid lipoarabinomannan (LAM) plays an important role in mediating host-bacteria interactions and is involved in modulation of the immune response. The arabinosyltransferase EmbC required for LAM biosynthesis is essential. We constructed recombinant strains of M. tuberculosis expressing a variety of alleles of EmbC. We demonstrated that EmbC has a functional signal peptide in M. tuberculosis. Over- or underexpression of EmbC resulted in reduced or increased sensitivity to ethambutol, respectively. The C-terminal domain of EmbC was essential for activity because truncated alleles were unable to mediate LAM production in Mycobacterium smegmatis and were unable to complement an embC deletion in M. tuberculosis. The C-terminal domain of the closely related arabinosyltransferase EmbB was unable to complement the function of the EmbC C-terminal domain. Two functional motifs were identified. The GT-C motif contains two aspartate residues essential for function in the DDX motif. The proline-rich region contains two highly conserved asparagines (Asn-638 and Asn-652). Mutation of these residues was tolerated, but loss of Asn-638 resulted in the synthesis of truncated LAM, which appeared to lack arabinose branching. All embC alleles that were incapable of complementing LAM production in M. smegmatis were not viable in M. tuberculosis, supporting the hypothesis that LAM itself is essential in M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/genética , Lipopolisacáridos/biosíntesis , Mutación , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Secuencia de Aminoácidos , Antituberculosos/farmacología , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Western Blotting , Etambutol/farmacología , Regulación Bacteriana de la Expresión Génica , Genes Esenciales/genética , Viabilidad Microbiana/genética , Datos de Secuencia Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Señales de Clasificación de Proteína/genética , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is the leading cause of death from a bacterial infection. The 4-aminopiperidine (PIP) series has been reported as having anti-bacterial activity against M. tuberculosis. We explored this series for its potential to inhibit aerobic growth of M. tuberculosis. We examined substitution at the N-1 position and C-4 position of the piperidine and modifications of the piperidine moiety systematically to delineate structure-activity relationships influencing potency. Compounds were tested for growth-inhibitory activity against virulent M. tuberculosis. A selected set of compounds were also tested for its activity against Staphylococcus aureus. RESULTS: The compound with a norbornenylmethyl substituent at the N-1 position and N-benzyl-N-phenethylamine at the C-4 position of the piperidine (1) was the only active compound with a minimum inhibitory concentration (MIC) of 10 µM against M. tuberculosis. Compounds were not active against S. aureus. CONCLUSIONS: We were unable to derive any other analogs with MIC < 20 µM against M. tuberculosis. Therefore we conclude that the lack of activity is a liability in this series precluding it from further development.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/química , Piperidinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Animales , Chlorocebus aethiops , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/fisiología , Staphylococcus aureus/fisiología , Relación Estructura-Actividad , Células VeroRESUMEN
We identified an amino-benzothiazole scaffold from a whole-cell screen against recombinant Mycobacterium tuberculosis under expressing the essential signal peptidase LepB. The seed molecule had 2-fold higher activity against the LepB hypomorph. Through a combination of purchase and chemical synthesis, we explored the structure-activity relationship for this series; 34 analogs were tested for antitubercular activity and for cytotoxicity against eukaryotic cells. We identified molecules with improved potency and reduced cytotoxicity. However, molecules did not appear to target LepB directly and did not inhibit protein secretion. Key compounds showed good permeability, low protein binding, and lack of CYP inhibition, but metabolic stability was poor with short half-lives. The seed molecule showed good bactericidal activity against both replicating and nonreplicating bacteria, as well as potency against intracellular M. tuberculosis in murine macrophages. Overall, the microbiological properties of the series are attractive if metabolic stability can be improved, and identification of the target could assist in the development of this series. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, is a serious global health problem requiring the development of new therapeutics. We previously ran a high-throughput screen and identified a series of compounds with antitubercular activity. In this paper, we test analogs of our hit molecules for activity against M. tuberculosis, as well as for activity against eukaryotic cells. We identified molecules with improved selectivity. Our molecules killed both replicating and nonreplicating bacteria but did not work by targeting protein secretion.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Ratones , Mycobacterium tuberculosis/metabolismo , Antituberculosos/farmacología , Antituberculosos/química , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Relación Estructura-Actividad , Unión Proteica , Pruebas de Sensibilidad MicrobianaRESUMEN
We performed a high-throughput phenotypic whole cell screen of Mycobacterium tuberculosis against a diverse chemical library of approximately 100,000 compounds from the AbbVie corporate collection and identified 24 chemotypes with anti-tubercular activity. We selected two series for further exploration and conducted structure-activity relationship studies with new analogs for the 4-phenyl piperidines (4PP) and phenylcyclobutane carboxamides (PCB). Strains with mutations in MmpL3 demonstrated resistance to both compound series. We isolated resistant mutants for the two series and found mutations in MmpL3. These data suggest that MmpL3 is the target, or mechanism of resistance for both series.
Asunto(s)
Mycobacterium tuberculosis , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/metabolismoRESUMEN
The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 µM). We conducted a structure-activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 µM with no cytotoxicity (IC50 > 100 µM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.
RESUMEN
With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.
RESUMEN
We previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. The overall aim of this study was to determine whether InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis. We tested the ability of two molecules of the diazaborine series to kill non-replicating M. tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Inhibinas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/metabolismoRESUMEN
There is a pressing need to develop novel anti-tubercular drugs. High-throughput phenotypic screening yields chemical series that inhibit bacterial growth. Target identification for such series is challenging, but necessary for optimization of target engagement and the development of series into clinical drugs. We constructed a library of recombinant Mycobacterium tuberculosis strains each expressing a single protein from an inducible promoter as a tool for target identification. The library of 1733 clones was arrayed in 96-well plates for rapid screening and monitoring growth. The library contains the majority of the annotated essential genes as well as genes involved in cell wall and fatty acid biosynthesis, virulence factors, regulatory proteins, efflux, and respiration pathways. We evaluated the growth kinetics and plasmid stability over three passages for each clone in the library. We determined expression levels (mRNA and/or protein) in 396 selected clones. We screened the entire library and identified the Alr-expressing clone as the only recombinant strain, which grew in the presence of d-cycloserine (DCS). We confirmed that the Alr-expressing clone was resistant to DCS (7-fold shift in minimum inhibitory concentration). The library represents a new tool that can be used to screen for compound resistance and other phenotypes.
RESUMEN
OBJECTIVE: Our aim was to identify natural products with anti-tubercular activity. RESULTS: A set of ~ 500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3 to 8.9 µM. Eleven structural analogs had no significant activity (MIC > 20 µM) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Productos Biológicos , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50 > 50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.
Asunto(s)
Compuestos de Boro/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos de Boro/química , Compuestos de Boro/toxicidad , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/toxicidad , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , NADH Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Células THP-1RESUMEN
With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino-thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure-activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.
Asunto(s)
Citocromos c/metabolismo , Morfolinas/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/metabolismo , Tiofenos/química , Tiofenos/farmacología , Animales , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Antituberculosos/toxicidad , Chlorocebus aethiops , Ratones , Relación Estructura-Actividad , Tiofenos/farmacocinética , Tiofenos/toxicidad , Células VeroRESUMEN
New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis. The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents.
RESUMEN
Isoniazid inhibits Mycobacterium tuberculosis InhA and is a key component of drug regimens that treat tuberculosis. However, the high rate of resistance against isoniazid is a contributing factor to the emergence of multi-drug resistance strains of M. tuberculosis. The 4-hydroxy-2-pyridine NITD-916 is a direct inhibitor of M. tuberculosis InhA that has comparable efficacy to isoniazid in mouse models of TB infection but a lower frequency of resistance. To characterize resistance mechanisms against NITD-916 we isolated resistant mutants in H37Rv (Euro-American lineage) and HN878 (East-Asian lineage) strains of M. tuberculosis. The resistance frequency was similar in both strains. Mutations were identified in residues within or near to the active of InhA or in the fabG1inhA promoter region. All mutants were resistant to NITD-916 but were not cross resistant to isoniazid, despite homology to SNPs identified in isoniazid resistant clinical isolates.