Study of mechanism of interaction of truncated isoniazid-nicotinamide adenine dinucleotide adduct against multiple enzymes of Mycobacterium tuberculosis by a computational approach

Objective/Background: Isoniazid [INH] is one of the effective antituberculosis [TB] drugs used for TB treatment. However, most of the drug-resistant Mycobacterium tuberculosis [MTB] clinical strains are resistant to INH, a first-line antituberculous drug. Certain metabolic enzymes such as adenosylhomocysteinase [Rv3248c], universal stress protein [Rv2623], nicotinamide adenine dinucleotide [reduced]-dependent enoyl-acyl carrier protein reductase [Rv1484], oxidoreductase [Rv2971], dihydrofolate reductase [Rv2763c], pyrroline- 5-carboxylate dehydrogenase [Rv1187] have been identified to bind INH-nicotinamide adenine dinucleotide [INH-NAD] and INH-nicotinamide adenine dinucleotide phosphate adducts coupled to Sepharose resin. These enzymes are reported to be involved in many important biochemical processes of MTB, including cysteine and methionine metabolism, mycobacterial growth regulation, mycolic acid biosynthesis, detoxification of toxic metabolites, folate biosynthesis, etc. The truncated INH-nicotinamide adenine dinucleotide [oxidized] adduct, 4-isonicotinoylnicotinamide, isolated from urine samples of human TB patients treated with INH therapy is proposed to have antimycobacterial activity

Methods: To understand the mechanism of interaction of the truncated INH-NAD adduct, binding energy studies were carried out on the aforementioned six enzymes with known three-dimensional structures using AutoDock4.2

Results: In silico docking analysis of these MTB enzymes with the truncated INH-NAD adduct showed favorable binding interactions with docking energies ranging from -5.29 to -7.07 kcal/mol

Conclusion: Thus, in silico docking study revealed that the INH-NAD adduct, which is generated in vivo after INH activation, may undergo spontaneous hydrolysis to form the truncated INH-NAD adduct and further binds and inhibits multiple enzymes of MTB, in addition to InhA, confirming that INH is an effective anti-TB drug acting at multiple enzymes. Further analysis of amino acid residues in the active site of INH-NAD-binding proteins showed the probable presence of catalytic triad in four enzymes possibly involved in INH binding to the enzyme

Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug

Tuberculosis [TB] is an ancient disease caused by Mycobacterium tuberculosis [MTB], which remains a major cause for morbidity and mortality in several developing countries. Most drug-resistant MTB clinical strains are resistant to isoniazid [INH], a first-line anti-TB drug. Mutation in KatG, a catalase-peroxidase, of MTB is reported to be a major cause of INH resistance. Normally upon activation by KatG, INH is converted to an active intermediate which has antimycobacterial action in MTB. This INH intermediate in the presence of NADH forms INH-NAD adduct which inhibits inhA [2-trans-enoyl-acyl carrier protein reductase] of MTB, thus blocking the synthesis of mycolic acid, a major lipid of the myco-bacterial cell wall. In this docking study, the high binding affinity of INH-NAD adduct towards InhA was observed in comparison with INH alone. In this study, two resistant mutants of KatG [S315Tand S315N] were modeled using Modeller9vlO and docking analysis with INH was performed using AutoDock4.2 and the docking results of these mutants were compared with the wild type KatG. Docking results revealed the formation of a single hydrogen [H] bond between the secondary amine nitrogen [-NH] of INH with Thr or Asn residues in place of Serine at 315 position of KatG mutant strains respectively, whereas in the case of the wild type, there was no H-bond formation observed between INH and Ser315. The H-bond formation may prevent free radical formation by KatG in mutant strains thus the development of resistance to the drug. This in silico evidence may implicate the basis of INH resistance in KatG mutant strains