RESUMO
A library of thirty N-substituted tosyl N'-acryl-hydrazones was prepared with p-toluenesulfonyl hydrazide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes. The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the collection showed Minimum Inhibitory Concentration ≤10 µM, of which the most active members (MIC of 1.25 µM) were exclusively E isomers. In order to validate the mechanism of action of the most active acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that consistently doubled those obtained on the wild type strain. Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible cytotoxicity risk and ADME-Tox profile. Based on their simple preparation and interesting antimycobacterial activity profile, the newly prepared aza-acrylates are promising candidates for antitubercular drug development.
Assuntos
Antituberculosos/farmacologia , Hidrazonas/farmacologia , Compostos de Tosil/farmacologia , Animais , Antituberculosos/síntese química , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Chlorocebus aethiops , Hidrazonas/síntese química , Hidrazonas/metabolismo , Isoniazida/química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Compostos de Tosil/síntese química , Compostos de Tosil/metabolismo , Células VeroRESUMO
Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, ß-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and ß-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the ß-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.
Assuntos
Acetilcoenzima A/metabolismo , Malonil Coenzima A/metabolismo , Policetídeo Sintases/metabolismo , Acilação , Animais , Domínio Catalítico , Patos , Cinética , Filogenia , Policetídeo Sintases/química , Policetídeo Sintases/genética , Domínios Proteicos , Especificidade por SubstratoRESUMO
Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.