Abstract
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.
Highlights
Tuberculosis (TB), caused mainly by Mycobacterium tuberculosis, still is one of the major threats in public health worldwide
It should not display any antagonism with other TB drugs to maintain a treatment with at least three active drugs, and be able to eradicate in different physiological stages, such as non-replicating and asymptomatic latent M. tuberculosis
The half-maximum inhibitory concentration (IC50) of MtInhA enzyme activity and determination of minimum inhibitory concentration (MIC) to arrest in vitro growth of M. tuberculosis H37Rv and PE-003 strains were carried out to select promising chemical compounds The in vitro mode of inhibition of MtInhA activity by steady-state kinetics was determined for these selected chemical compounds
Summary
Tuberculosis (TB), caused mainly by Mycobacterium tuberculosis, still is one of the major threats in public health worldwide. A new anti-TB candidate should be more active than the existing drugs to reduce time of treatment, be effective against MDR-TB and XDR-TB, and be compatible with current anti-retroviral therapy[4] It should not display any antagonism with other TB drugs to maintain a treatment with at least three active drugs, and be able to eradicate in different physiological stages, such as non-replicating and asymptomatic latent M. tuberculosis. We propose that the Labio_16 compound may be a lead compound for further efforts to develop anti-TB agents owing to its inhibitory activity of MtInhA enzyme, spontaneous and favorable binding process, efficacy against H37Rv and PE-003 (a drug-resistant strain), intracellular activity in a macrophage model, and lack of detectable cytotoxic and cardiotoxic effects. Labio_16 is a drug candidate not a pro-drug as isoniazid needs to be activated by the mycobacterial catalase-peroxidase KatG to form an isonicotinyl-NAD adduct that inhibits MtInhA enzyme activity[13]. It should be pointed out that further efforts will have to be pursued to show whether or not MtInhA is the molecular target of Labio_16 chemical compound
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