RESUMO
New effective compounds to treat tuberculosis are urgently needed. IQG-607 is an orally active anti-tuberculosis drug candidate, with promising preliminary safety profile and anti-mycobacterial activity in both in vitro and in vivo models of tuberculosis infection. Here, we evaluated the mutagenic and genotoxic effects of IQG-607, and its interactions with CYP450 isoforms. Moreover, we describe for the first time a combination study of IQG-607 in Mycobacterium tuberculosis-infected mice. Importantly, IQG-607 had additive effects when combined with the first-line anti-tuberculosis drugs rifampin and pyrazinamide in mice. IQG-607 presented weak to moderate inhibitory potential against CYP450 isoforms 3A4, 1A2, 2C9, 2C19, 2D6, and 2E1. The Salmonella mutagenicity test revealed that IQG-607 induced base pair substitution mutations in the strains TA100 and TA1535. However, in the presence of human metabolic S9 fraction, no mutagenic effect was detected in any strain. Additionally, IQG-607 did not increase micronucleus frequencies in mice, at any dose tested, 25, 100, or 250 mg/kg. The favorable activity in combination with first-line drugs and mild to moderate toxic events described in this study suggest that IQG-607 represents a candidate for clinical development.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Ferrosos/efeitos adversos , Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Aberrações Cromossômicas , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Compostos Ferrosos/administração & dosagem , Isoniazida/administração & dosagem , Isoniazida/efeitos adversos , Isoniazida/farmacologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Testes de Mutagenicidade , Mycobacterium tuberculosis/genética , Salmonella typhimurium/genética , Tuberculose/microbiologiaRESUMO
IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG(S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Humanos , Isoniazida/farmacologia , Mutação/genética , Mycobacterium tuberculosis/genética , Sequenciamento Completo do Genoma/métodosRESUMO
Tuberculosis (TB) is one of the most fatal diseases and is responsible for the infection of millions of people around the world. Most recently, scientific frontiers have been engaged to develop new drugs that can overcome drug-resistant TB. Following this direction, using a designed scaffold based on the combination of two separate pharmacophoric groups, a series of menadione-derived selenoesters was developed with good yields. All products were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv and attractive results were observed, especially for the compounds 8a, 8c and 8f (MICs 2.1, 8.0 and 8.1 µM, respectively). In addition, 8a, 8c and 8f demonstrated potent in vitro activity against multidrug-resistant clinical isolates (CDCT-16 and CDCT-27) with promising MIC values ranging from 0.8 to 3.1 µM. Importantly, compounds 8a and 8c were found to be non-toxic against the Vero cell line. The SI value of 8a (>23.8) was found to be comparable to that of isoniazid (>22.7), which suggests the possibility of carrying out advanced studies on this derivative. Therefore, these menadione-derived selenoesters obtained as hybrid compounds represent promising new anti-tubercular agents to overcome TB multidrug resistance.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Selênio/farmacologia , Vitamina K 3/farmacologia , Animais , Antituberculosos/síntese química , Antituberculosos/química , Chlorocebus aethiops , Humanos , Modelos Moleculares , Selênio/química , Tuberculose/tratamento farmacológico , Células Vero , Vitamina K 3/análogos & derivados , Vitamina K 3/síntese químicaRESUMO
The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis.
RESUMO
The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B1R-/-, B2R-/- and double B1R/B2R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B1R and B2R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B2R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B1R/B2R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B1R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg9-BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B1R (des-Arg9-BK and SSR240612) and B2R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis.
Assuntos
Antituberculosos/administração & dosagem , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Dioxóis/administração & dosagem , Pulmão/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Receptor B1 da Bradicinina/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Tuberculose Pulmonar/tratamento farmacológico , Administração Oral , Animais , Carga Bacteriana , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Antagonistas de Receptor B2 da Bradicinina/administração & dosagem , Modelos Animais de Doenças , Feminino , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células RAW 264.7 , Receptor B1 da Bradicinina/deficiência , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/microbiologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
New effective compounds for tuberculosis treatment are needed. This study evaluated the effects of a series of quinoxaline-derived chalcones against laboratorial strains and clinical isolates of M. tuberculosis. Six molecules, namely N5, N9, N10, N15, N16, and N23 inhibited the growth of the M. tuberculosis H37Rv laboratorial strain. The three compounds (N9, N15 and N23) with the lowest MIC values were further tested against clinical isolates and laboratory strains with mutations in katG or inhA genes. From these data, N9 was selected as the lead compound for further investigation. Importantly, this chalcone displayed a synergistic effect when combined with moxifloxacin. Noteworthy, the anti-tubercular effects of N9 did not rely on inhibition of mycolic acids synthesis, circumventing important mechanisms of resistance. Interactions with cytochrome P450 isoforms and toxic effects were assessed in silico and in vitro. The chalcone N9 was not predicted to elicit any mutagenic, genotoxic, irritant, or reproductive effects, according to in silico analysis. Additionally, N9 did not cause mutagenicity or genotoxicity, as revealed by Salmonella/microsome and alkaline comet assays, respectively. Moreover, N9 did not inhibit the cytochrome P450 isoforms CYP3A4/5, CYP2C9, and CYP2C19. N9 can be considered a potential lead molecule for development of a new anti-tubercular therapeutic agent.
Assuntos
Antituberculosos/farmacologia , Chalconas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Proteínas de Bactérias/genética , Catalase/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/patogenicidade , Ácidos Micólicos/antagonistas & inibidores , Oxirredutases/genética , Quinoxalinas/farmacologia , Tuberculose/genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Simulação por Computador , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Termodinâmica , Animais , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chlorocebus aethiops , Humanos , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/fisiologia , Oxirredutases/metabolismo , Células RAW 264.7 , Tuberculose/microbiologia , Células VeroRESUMO
Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5-8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.
Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Mefloquina/farmacologia , Mefloquina/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazóis/farmacologia , Oxazóis/uso terapêutico , Animais , Carga Bacteriana , Modelos Animais de Doenças , Interações Medicamentosas , Reposicionamento de Medicamentos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
In this work, the antitubercular activity of a pentacyano(isoniazid)ferrate(II) compound (IQG-607) was investigated using a macrophage model of Mycobacterium tuberculosis infection. Importantly, treatment of M.-tuberculosis-infected macrophages with IQG-607 significantly diminished the number of CFU compared with the untreated control group. The antitubercular activity of IQG-607 was similar to that observed for the positive control drugs isoniazid and rifampicin. Nevertheless, higher concentrations of IQG-607 produced a significantly greater reduction in bacterial load compared with the same concentrations of isoniazid. Analysis of the mechanism of action of IQG-607 revealed that the biosynthesis of mycolic acids was blocked. The promising activity of IQG-607 in infected macrophages and the experimental determination of its mechanism of action may help in further studies aimed at the development of a new antimycobacterial agent.