RESUMEN
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
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
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.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacología , Ensayos Analíticos de Alto Rendimiento , Mycobacterium tuberculosis/genética , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.
Asunto(s)
Amidas/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Glucosa/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Permeabilidad de la Membrana Celular , Farmacorresistencia Bacteriana/genética , Eliminación de Gen , Lípidos/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Magnesio/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Homeostasis/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
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
Mycobacterium tuberculosis ( MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure-activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.
Asunto(s)
Mycobacterium tuberculosis/enzimología , NADH Deshidrogenasa/química , Quinazolinonas/química , Estructura Molecular , NADH Deshidrogenasa/antagonistas & inhibidores , Quinazolinonas/síntesis química , Quinazolinonas/farmacología , Relación Estructura-ActividadRESUMEN
Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization.
Asunto(s)
Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Antituberculosos/síntesis química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Ligandos , Modelos Moleculares , Estructura Molecular , Oxidorreductasas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Resonancia por Plasmón de SuperficieRESUMEN
The availability of suitable diverse fragment- and lead-oriented screening compounds is key for the identification of suitable chemical starting points for drug discovery programs. The physicochemical properties of molecules are crucial in determining the success of small molecules in clinical development, yet reports suggest that pharmaceutical and academic sectors often produce molecules with poor drug-like properties. We present a platform to design novel, high quality and diverse fragment- and lead-oriented libraries with appropriate physicochemical properties in a cost-efficient manner. This approach has the potential to assist the way libraries are constructed by significantly addressing the historical uneven exploration of chemical space for drug discovery. Additionally, this platform can teach undergraduates and graduates about compound library design.
Asunto(s)
Diseño de Fármacos , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Estabilidad de Medicamentos , Modelos Químicos , Estructura Molecular , SolubilidadRESUMEN
We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Sitios de Unión , Transporte de Electrón/efectos de los fármacos , Complejo IV de Transporte de Electrones/genética , Metabolismo Energético/efectos de los fármacos , Técnicas de Inactivación de Genes , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Oxazinas/química , Estructura Terciaria de Proteína , Piridinas/farmacología , Xantenos/químicaRESUMEN
A versatile one-pot nitro-Mannich/lactamization cascade for the direct synthesis of 1,3,5-trisubstituted 4-nitropyrrolidin-2-ones has been developed. The reaction is easy to perform and broad in scope, and high levels of diastereoselectivity can be achieved.
Asunto(s)
Nitrocompuestos/química , Pirrolidinonas/síntesis química , Catálisis , Estructura Molecular , Pirrolidinonas/química , EstereoisomerismoRESUMEN
The profile of a series of triazine and pyrimidine based ROCK inhibitors is described. An initial binding mode was established based on a homology model and the proposed interactions are consistent with the observed SAR. Compounds from the series are potent in a cell migration assay and possess a favorable kinase selectivity. In vivo activity was demonstrated for compound 1A in a spontaneous hypertensive rat model.
Asunto(s)
Antihipertensivos/química , Hipertensión/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Triazinas/química , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Sitios de Unión , Simulación por Computador , Modelos Animales de Enfermedad , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Ratas , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
An efficient three-component nitro-Mannich/lactamization cascade of methyl 3-nitropropanoate with in situ formed acyclic imines for the direct preparation of pyrrolidinone derivatives has been developed. The reaction is easy to perform, broad in scope, and highly diastereoselective and may be extended to cyclic imines allowing the direct formation of polycyclic pyrrolidinone derivatives.