RESUMO
Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F420-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.
Assuntos
Nitrofuranos , Nitroimidazóis , Animais , Camundongos , Nitrocompostos , Nitrofuranos/farmacologia , Carga BacterianaRESUMO
The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosisM. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Benzamidas/farmacologia , Benzotiazóis/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/farmacologia , Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzamidas/síntese química , Benzotiazóis/síntese química , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Fatores Corda/antagonistas & inibidores , Fatores Corda/biossíntese , Fatores Corda/metabolismo , Farmacorresistência Bacteriana/genética , Galactanos/metabolismo , Expressão Gênica , Ensaios de Triagem em Larga Escala , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Mycobacterium abscessus/crescimento & desenvolvimento , Mycobacterium abscessus/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Piridinas/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequenciamento Completo do GenomaRESUMO
Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills M. tuberculosis in a time- and dose-dependent manner in vitro and inhibits M. tuberculosis growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mmpL3 mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized Mycobacterium membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating M. tuberculosis, thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of M. tuberculosis treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
IMPORTANCE: MmpL3 is a protein that is required for the survival of bacteria that cause tuberculosis (TB) and nontuberculous mycobacterial (NTM) infections. This report describes the discovery and characterization of a new small molecule, MSU-43085, that targets MmpL3 and is a potent inhibitor of Mycobacterium tuberculosis (Mtb) and M. abscessus survival. MSU-43085 is shown to be orally bioavailable and efficacious in an acute model of Mtb infection. However, the analog is inactive against Mtb in chronically infected mice. Pharmacokinetic and metabolite identification studies identified in vivo metabolism of MSU-43085, leading to a short half-life in treated mice. These proof-of-concept studies will guide further development of the MSU-43085 series for the treatment of TB or NTM infections.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não TuberculosasRESUMO
Dissectol A is a rearranged terpene glycoside isolated from several flowering plants. Starting from glucose, the densely functionalized bicyclic structure has been prepared via site-selective oxidation and an intramolecular allylic alkylation reaction with an enediolate as the nucleophile. Despite earlier reports, dissectol A is not effective at inhibiting DevRS signaling in whole-cell Mycobacterium tuberculosis and does not inhibit growth of the bacterium.
RESUMO
Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here, we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular reactive oxygen species at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.