RESUMEN
Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of actionâlinezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Oxazolidinonas , Profármacos , Profármacos/farmacología , Profármacos/química , Antituberculosos/farmacología , Antituberculosos/química , Mycobacterium tuberculosis/efectos de los fármacos , Oxazolidinonas/farmacología , Oxazolidinonas/química , Animales , Pruebas de Sensibilidad Microbiana , Ratones , Humanos , Linezolid/farmacología , Linezolid/química , Farmacorresistencia Bacteriana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.
Asunto(s)
Benzofuranos , Mycobacterium tuberculosis , Sintasas Poliquetidas , Antituberculosos/química , Mycobacterium tuberculosis/metabolismo , Benzofuranos/química , Pruebas de Sensibilidad MicrobianaAsunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Antituberculosos/uso terapéuticoRESUMEN
Mycobacterium tuberculosis (Mtb), perhaps more than any other organism, is intrinsically appealing to chemical biologists. Not only does the cell envelope feature one of the most complex heteropolymers found in nature1 but many of the interactions between Mtb and its primary host (we humans) rely on lipid and not protein mediators.2,3 Many of the complex lipids, glycolipids, and carbohydrates biosynthesized by the bacterium still have unknown functions, and the complexity of the pathological processes by which tuberculosis (TB) disease progress offers many opportunities for these molecules to influence the human response. Because of the importance of TB in global public health, chemical biologists have applied a wide-ranging array of techniques to better understand the disease and improve interventions.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Tuberculosis/tratamiento farmacológico , Mycobacterium tuberculosis/metabolismo , Glucolípidos , Membrana Celular/metabolismo , BiologíaRESUMEN
Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.
Asunto(s)
Mycobacterium tuberculosis , Sistema Enzimático del Citocromo P-450/metabolismo , Colesterol/química , Descubrimiento de Drogas , Antituberculosos/farmacología , Antituberculosos/químicaRESUMEN
Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.
Asunto(s)
Mycobacterium tuberculosis , Myxococcales , Antibacterianos/química , Ribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Humanos , Benzoxazoles/farmacología , Antituberculosos/farmacología , Antituberculosos/química , Relación Estructura-Actividad , Pruebas de Sensibilidad MicrobianaRESUMEN
Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
Asunto(s)
Lisina-ARNt Ligasa , Mycobacterium tuberculosis , Tuberculosis , Animales , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/farmacología , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológicoRESUMEN
Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Antibacterianos/farmacología , Microscopía por Crioelectrón , ADN Polimerasa Dirigida por ADN , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Tuberculosis (TB) is one of the world's most deadly infectious diseases resulting in nearly 1.3 million deaths annually and infecting nearly one-quarter of the population. para-Aminosalicylic acid (PAS), an important second-line agent for treating drug-resistant Mycobacterium tuberculosis, has moderate bioavailability and rapid clearance that necessitate high daily doses of up to 12 g per day, which in turn causes severe gastrointestinal disturbances presumably by disruption of gut microbiota and host epithelial cells. We first synthesized a series of alkyl, acyloxy and alkyloxycarbonyloxyalkyl ester prodrugs to increase the oral bioavailability and thereby prevent intestinal accumulation as well as undesirable bioactivation by the gut microbiome to non-natural folate species that exhibit cytotoxicity. The pivoxyl prodrug of PAS was superior to all of the prodrugs examined and showed nearly quantitative absorption. While the conceptually simple prodrug approach improved the oral bioavailability of PAS, it did not address the intrinsic rapid clearance of PAS mediated by N-acetyltransferase-1 (NAT-1). Thus, we next modified the PAS scaffold to reduce NAT-1 catalyzed inactivation by introduction of groups to sterically block N-acetylation and fluorination of the aryl ring of PAS to attenuate N-acetylation by electronically deactivating the para-amino group. Among the mono-fluorinated analogs prepared, 5-fluoro-PAS, exhibited the best activity and an 11-fold decreased rate of inactivation by NAT-1 that translated to a 5-fold improved exposure as measured by area-under-the-curve (AUC) following oral dosing to CD-1 mice. The pivoxyl prodrug and fluorination at the 5-position of PAS address the primary limitations of PAS and have the potential to revitalize this second-line TB drug.
Asunto(s)
Ácido Aminosalicílico , Profármacos , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Ácido Aminosalicílico/efectos adversos , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Disponibilidad Biológica , Ratones , Profármacos/farmacología , Profármacos/uso terapéutico , Tuberculosis/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.
Asunto(s)
Mycobacterium tuberculosis , Benzoxazoles/farmacología , Vías Biosintéticas , Coenzima A , Mycobacterium tuberculosis/genética , FenilalaninaRESUMEN
Phenotypic whole cell high-throughput screening of a â¼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.
Asunto(s)
Antituberculosos/farmacología , Pared Celular/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Sulfonamidas/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Descubrimiento de Drogas , Células Hep G2 , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/metabolismo , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Antimicrobial susceptibility testing is the mainstay of tuberculosis drug development programs. In this chapter, we describe methods for determination of the minimum inhibitory concentration of compounds against Mycobacterium tuberculosis growing in liquid media as a function of carbon source, detergent, and environmental stress imposed by acidic pH as well as reactive nitrogen intermediates. Methods for determining the effect of bovine serum albumin in the growth medium on antimicrobial susceptibility are also described. Finally, we provide a method for antimicrobial susceptibility testing on agar medium.
Asunto(s)
Agar/química , Antituberculosos/farmacología , Medios de Cultivo/farmacología , Mycobacterium tuberculosis/crecimiento & desarrollo , Albúmina Sérica Bovina/metabolismo , Tuberculosis/tratamiento farmacológico , Carbono/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Estrés Fisiológico , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
Isoniazid (INH) remains a cornerstone for treatment of drug susceptible tuberculosis (TB), yet the quantitative structure-activity relationships for INH are not well documented in the literature. In this paper, we have evaluated a systematic series of INH analogs against contemporary Mycobacterium tuberculosis strains from different lineages and a few non-tuberculous mycobacteria (NTM). Deletion of the pyridyl nitrogen atom, isomerization of the pyridine nitrogen to other positions, replacement of the pyridine ring with isosteric heterocycles, and modification of the hydrazide moiety of INH abolishes antitubercular activity. Similarly, substitution of the pyridine ring at the 3-position is not tolerated while substitution at the 2-position is permitted with 2-methyl-INH 9 displaying antimycobacterial activity comparable to INH. To assess the specific activity of this series of INH analogs against mycobacteria, we assayed them against a panel of gram-positive and gram-negative bacteria, as well as a few fungi. As expected INH and its analogs display a narrow spectrum of activity and are inactive against all non-mycobacterial strains evaluated, except for 4, which has modest inhibitory activity against Cryptococcus neoformans. Our findings provide an updated analysis of the structure-activity relationship of INH that we hope will serve as useful resource for the community.
Asunto(s)
Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Isoniazida/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piridinas/química , Relación Estructura-ActividadRESUMEN
Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.
Asunto(s)
Mycobacterium tuberculosis , Péptido Sintasas/antagonistas & inhibidores , Coenzima A , Cisteína/análogos & derivados , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Ácido Pantoténico/análogos & derivados , Péptido Sintasas/genéticaRESUMEN
Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Indoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Triptófano/biosíntesis , Animales , Antituberculosos/química , Antituberculosos/metabolismo , Relación Dosis-Respuesta a Droga , Indoles/química , Indoles/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
Asunto(s)
Antituberculosos/química , Pirimidinonas/química , Animales , Antituberculosos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Semivida , Humanos , Hierro/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Microsomas/metabolismo , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Pirazoles/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Carboxiliasas/antagonistas & inhibidores , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Péptido Sintasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Carboxiliasas/genética , Carboxiliasas/metabolismo , Carboxiliasas/ultraestructura , Coenzima A/biosíntesis , Cristalografía por Rayos X , Pruebas de Enzimas , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Péptido Sintasas/ultraestructura , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.