RESUMO
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.
Assuntos
Antituberculosos , Proteínas de Bactérias , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Antituberculosos/farmacologia , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Relação Estrutura-Atividade , Testes de Sensibilidade Microbiana , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Estrutura Molecular , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/metabolismo , Relação Dose-Resposta a Droga , Simulação de Dinâmica Molecular , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/síntese químicaRESUMO
Antimalarial drugs have been suggested as promising scaffolds with anti-tubercular activities. In this work, we demonstrated, for the first time, the effectiveness of tafenoquine against mycobacteria. Firstly, tafenoquine inhibited the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis with lower MICs values as compared to other antimalarial drugs, such as mefloquine, chloroquine, and primaquine. Importantly, tafenoquine was active against three multi-drug resistant strains of M. tuberculosis with MIC values similar to pan-sensitive strains, suggesting that tafenoquine is capable of evading the major mechanisms of resistance found in drug-resistant clinical isolates of M. tuberculosis. Importantly, tafenoquine displayed a synergistic effect when combined with mefloquine. In addition, tafenoquine displayed an improved activity compared to the groups treated with both isoniazid and rifampicin in the six-week nutrient starved M. tuberculosis cultures. This finding suggests that further investigations of tafenoquine against dormant mycobacteria are worth pursuing. Moreover, different concentrations of tafenoquine ranging from 1.25 to 80 µM displayed different effects against M. tuberculosis, from moderate (reduction of a 1.8 log CFU/mL) to potent bactericidal (reduction of a 4.2 log CFU/mL) activities. Tafenoquine may represent a hit for further drug optimization and for future clinical development as a new anti-mycobacterial agent, especially in cases of resistant and/or dormant forms of tuberculosis.
Assuntos
Aminoquinolinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Reposicionamento de Medicamentos , Sinergismo Farmacológico , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacos , Rifampina/farmacologiaRESUMO
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
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
M. tuberculosis and parasites of the genus Leishmania present the type II fatty acid biosynthesis system (FASII). The pentacyano(isoniazid)ferrate(II) compound, named IQG-607, inhibits the enzyme 2-trans-enoyl-ACP(CoA) reductase from M. tuberculosis, a key component in the FASII system. Here, we aimed to evaluate the inhibitory activity of IQG-607 against promastigote and amastigote forms of Leishmania (Viannia) braziliensis isolated from patients with different clinical forms of L. braziliensis infection, including cutaneous, mucosal and disseminated leishmaniasis. Importantly, IQG-607 inhibited the proliferation of three different isolates of L. braziliensis promastigotes associated with cutaneous, mucosal and disseminated leishmaniasis. The IC50 values for IQG-607 ranged from 32 to 75 µM, for these forms. Additionally, IQG-607 treatment decreased the proliferation of intracellular amastigotes in infected macrophages, after an analysis of the percentage of infected cells and the number of intracellular parasites/100 cells. IQG-607 reduced from 58% to 98% the proliferation of L. braziliensis from cutaneous, mucosal and disseminated strains. Moreover, IQG-607 was also evaluated regarding its potential toxic profile, by using different cell lines. Cell viability of the lineages Vero, HaCat and HepG2 was significantly reduced after incubation with concentrations of IQG-607 higher than 2 mM. Importantly, IQG-607, in a concentration of 1 mM, did not induce DNA damage in HepG2 cells, when compared to the untreated control group. Future studies will confirm the mechanism of action of IQG-607 against L. braziliensis.
Assuntos
Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Animais , Isoniazida/farmacologia , Leishmania braziliensis/crescimento & desenvolvimentoRESUMO
Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.
Assuntos
Citidina Desaminase/genética , Desoxicitidina/genética , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Citidina Desaminase/biossíntese , Desoxicitidina/biossíntese , Técnicas de Inativação de Genes , Humanos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fatores de TempoRESUMO
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
We have previously demonstrated a potent in vitro inhibitory activity for two pentacyano(isoniazid)ferrate(II) compounds, namely IQG-607 and IQG-639, against the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase enzyme. In this study, the activity of these compounds was evaluated using an in vivo murine model of tuberculosis. Swiss mice were infected with M. tuberculosis H37Rv strain and then IQG-607 or IQG-639 (250 mg/kg) was administered for 28 days or 56 days. In addition, a dose-response study was performed with IQG-607 at 5, 10, 25, 50, 100, 200 and 250 mg/kg. The activity of test compounds was compared with that of the positive control drug isoniazid (INH) (25 mg/kg). After 28 days or 56 days of treatment, both IQG-607 and INH significantly reduced M. tuberculosis-induced splenomegaly as well as significantly diminishing the colony-forming units in the spleen and lungs. IQG-607 and INH ameliorated the lung macroscopic aspect, reducing lung lesions to a similar extent. However, IQG-639 did not significantly modify any evaluated parameter. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. IQG-607 might well represent a good candidate for clinical development as a new antimycobacterial agent.