Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

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.

Handling the Hurdles on the Way to Anti-tuberculosis Drug Development

“…The thermodynamic signatures of non-covalent interactions were determined by protein fluorescence spectroscopy and van't Hoff analyses of ligands binding to MtInhA:NADH binary complex. Five new chemotypes inhibited in vitro M. tuberculosis H37Rv growth (Martinelli et al, 2017). Two of them, named Labio_16 and Labio_17 (Figure 7), also inhibited the in vitro growth of PE-003 multidrug-resistant strain.…”

“…Nineteen molecules from an initial data set of ∼1 million that could bind to MtInhA:NADH binary complex were identified (Pauli et al, 2013). The inhibition constants and mode of action were determined by steadystate kinetics for seven of these compounds that inhibited MtInhA activity (Martinelli et al, 2017). The thermodynamic signatures of non-covalent interactions were determined by protein fluorescence spectroscopy and van't Hoff analyses of ligands binding to MtInhA:NADH binary complex.…”

“…In addition, in Zebrafish model Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. A model was built for the MtInhA:NADH:Labio_16 ternary complex (Martinelli et al, 2017). Crystal structure determination of this ternary complex are currently underway to provide experimental data on which to base medicinal chemistry efforts on this likely hit for the development of chemotherapeutic agents to treat TB.…”

Zebrafish as a Tool in the Study of Sleep and Memory-related Disorders

“…The use of zebrafish as an experimental model for several areas of study is already well documented, including biochemical [ 114 - 116 ], behavioral [ 117 , 118 ], and toxicological [ 119 - 121 ], being considered an important model animal for drug screening [ 122 , 123 ]. Findings in this species have contributed to the development of new technologies and sophisticated methodological strategies, such as the generation of CRISPR-Cas 9 mutants, morpholinos, and microarray technology [ 124 - 126 ], since after genome sequencing of this animal, the presence of 82% of genes related to human diseases was revealed [ 17 ].…”