Isoniazid with Multiple Mode of Action on Various Mycobacterial Enzymes Resulting in Drug Resistance

Bioinformatics Centre, Biochemistry & JBTDRC, MGIMS, Sevagram, Maharashtra, India

*Corresponding Author:

Harinath BC
JB Tropical Disease Research Centre, Mahatma Gandhi Institute of Medical Sciences
Sevagram, 442 102 (Wardha) Maharashtra, India
Tel: 91-7152–284341-284355
Fax: (07152)-284038
E-mail: bc_harinath@yahoo.com

Received date: September 11, 2016; Accepted date: October 17, 2016; Published date: October 20, 2016

Citation: Jena L, Nayak T, Deshmukh S, Wankhade G, Waghmare P, et al. (2016) Isoniazid with Multiple Mode of Action on Various Mycobacterial Enzymes Resulting in Drug Resistance. J Infect Dis Ther 4:297. doi:10.4172/2332-0877.1000297

Copyright: © 2016 Jena L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Isoniazid (INH), is one of the drugs shown to be effective and has been extensively used in TB control. Interestingly tuberculosis showed predominant drug resistance to isoniazid and thus lead to multi drug therapy in TB treatment. However, isoniazid is still advocated in latent TB and use as prophylactic in HIV infection and in children for prevention of TB. It is of interest that different studies revealing interaction of isoniazid with around 117 enzymes of mycobacteria influencing metabolic pathways by number of ways in addition to inhibiting mycolic acid synthesis and thus affecting growth of mycobacteria. The purpose of this review is to present the various mechanisms of action of isoniazid at different enzymes of MTB causing drug resistance.

Keywords

Tuberculosis; Isoniazid; Drug resistance; Mycobacterium; Metabolic pathway

Introduction

Tuberculosis (TB), being an oldest infectious disease, has been a major health problem worldwide. It is caused by Mycobacterium tuberculosis (MTB) which infects around one third of the world’s population. According to WHO Global Tuberculosis Report 2015, there were around 9.6 million people with active TB infection and amongst them 12% were HIV-positive. Further, in 2014 there were only 1,23,000 reported cases of multidrug-resistant TB (MDR-TB) amongst 4,80,000 cases [1]. The occurrences of extensively drug-resistant (XDR) tuberculosis have also been a rising risk in different regions around the globe [2]. The isoniazid (INH), also known as isonicotinyl hydrazine, one of the effective anti-TB drugs used for tuberculosis treatment is found to be resistant in different clinical strains of MTB [3]. Further, according to various studies, 82 different enzymes of mycobacteria associated with the interaction of INH, resulting in mutation and isoniazid drug resistance (Table 1) [4,5].

Table 1: Mutations in MTB Genes / Proteins reported to be associated with Isoniazid resistance [4,5].

As INH has been used as a first-line drug in the prevention and treatment of TB [6], its mechanism of action has been studied for more than five decades. It is reported to produce various highly reactive compounds [7] which then target multiple enzymes of MTB [8]. Thus, the complex mode of action of isoniazid with number of enzymes needs study. Further, it is useful to understand how mutation in different MTB enzymes affects drug-enzyme interaction. This communication reviews the various mechanism of action of a single drug isoniazid at different MTB enzymes leading to drug resistance.

Mechanisms of Action of Isoniazid

Activation of INH by KatG and formation of INH-NAD(P) adduct

KatG of MTB encoded by Rv1908c has 740 amino acids in its protein sequence, is a multifunctional enzyme, showing both a catalase and a peroxynitritase activities [9,10]. Besides playing an important role in the intracellular survival of the pathogen within macrophages, it protects against reactive nitrogen and oxygen species produced by phagocytic cells [10]. Being a pro-drug, INH is activated by the catalase– peroxidase KatG and MnCl₂ and forms isonicotinoyl radical or anion which then reacts with NAD+ and NADP+ [11], and subsequently generates INH-NAD(P) adducts [12]. Amongst these adducts, the INH-NAD reported to inhibit the enoyl-ACP reductase enzyme (InhA) whereas INH-NADP inhibit dfrA - encoded dihydrofolate reductase [13] and MabA (3-oxoacyl-ACP reductase) [14].

Inhibition of InhA by INH-NAD adduct

INH-NAD adduct was reported to inhibit InhA of MTB encoded by Rv1484 which is reported to block the synthesis of mycolic acid, a major lipid of the mycobacterial cell wall.

Our in silico docking study between InhA and truncated INH-NAD adduct, demonstrated that the adduct binds with InhA by forming a hydrogen bond with its substrate binding residue Tyr158 [15] which correlates the in vitro study by Nguyen et al. reporting that the INHNAD adduct as a potential inhibitor of InhA [16].

Truncated INH-NAD adduct

After profiling the MTB proteome using both the INH-NAD and INH-NADP adducts coupled to Sepharose solid supports, Argyrou et al. identified seventeen proteins (Table 2) that bind to these adducts with high affinity [13]. Further, the truncated form of INH–NAD adduct (4-isonicotinoylnicotinamide, 4-INN,) reported to have potential antimycobacterial activity [17]. The in silico docking study of truncated INH–NAD adducts with six MTB enzymes with known three-dimensional (3D) structure out of 17 proteins (Table 2), showed considerable binding affinity and thus revealing the truncated INH– NAD adducts as effective inhibitors for these proteins [15].

Table 2: High Affinity INH-NAD (P) - binding proteins from Mycobacterium tuberculosis [13,14].

Acetylation of INH by NAT

The arylamine N-acetyltransferase (NAT) of MTB also reported to have direct interaction with INH like KatG. As a drug-metabolizing enzyme, NAT acetylates INH and forms INH to a therapeutically inactive form i.e. N-acetylate INH [18]. Payton et al. observed that the over expression of NAT leads to increased INH resistance in Mycobacterium smegmatis [19]. Further, when the gene was knockedout, the bacteria showed increased sensitivity to INH [19].

Isoniazid Drug Resistance

Mutation in different mycobacterial enzymes associated with INH resistance

The mechanism of INH resistance has been the focus of extensive study. It is broadly reported that INH resistance in MTB has been associated with mutations in different genes [7] such as katG, inhA, kasA, ahpC etc. Mutation in NADH dehydrogenase, encoded by ndh also reported to be linked with INH resistance [20]. Further, mutation in promoter region of inhA strongly linked with extensively drugresistant tuberculosis [21].

Besides 22 genes of MTB reported in Tuberculosis Drug Resistance Mutation Database, which were associated with INH resistance [4], Shekar et al. identified 60 novel genes associated with INH resistance in INH-resistant clinical isolates of MTB by their whole genome sequencing [5]. Most of the genes are associated with important metabolic pathways of MTB such as biosynthesis of secondary metabolites, antibiotics, amino acid, fatty acid; carbon metabolism; cell wall biosynthesis; glycolysis/gluconeogenesis, glutathione metabolism, glyoxylate and dicarboxylate metabolism, lipid biosynthesis, lipid metabolism, metabolic pathway, microbial metabolism in diverse environments, mycolic acid, nitrotoluene degradation, oxidative phosphorylation etc [22,23].

As KatG is associated with activation of INH, mutation in its gene plays an important role in INH resistance. Among various mutations identified in KatG, mutation at S315T and S315N has been widely reported in INH resistance strains. Our computational studies also showed that KatG mutations (S315T/S315N) prevent free radical formation that leads to drug resistance [24].

Two mutations in NAT enzymes (G207E and G67R), reported in MTB clinical isolates associated with INH resistance. From the molecular dynamics (MD) simulation analysis of NAT wild type and mutants (G67R and G207E) models, it was observed that these mutations increases the stability of the binding interfaces of enzyme by providing extra electrostatic interaction with neighboring amino acids. This stability might facilitate in rapid acetylation of INH and detoxification and leads to isoniazid resistance [25].

The mycobacterial NADH pyrophosphatase (NudC) reported to have an important role in the degradation of INH-NAD adduct (the active forms of isoniazid) and ETH-NAD adduct (active form of ethionamide (ETH)) that leads to INH and ETH inactivation. The polymorphism P237Q leads to loss of enzymatic activity and thus leads to INH and ETH resistance [26,27]. Further, a silent mutation in mabA (Rv1483), at nucleotide position 609 (g609a), leads to INH resistance in MTB [28].

Mycothiol (MSH), a major low molecular mass thiol in mycobacteria has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Four genes such as mshA (Rv0486), mshB (Rv1170), mshC (Rv2130c) and mshD (Rv0819) involves in Mycothiol biosynthesis. Buchmeier et al. observed that the MTB mutant (613 bp deletion within the mshB gene) showing increase resistance to Isoniazid [29]. Further, the mshA (Rv0486) gene of MTB encoding glycosyltransferase involved in the first step of mycothiol biosynthesis [10]. Jagielski et al. observed that a defective mshA gene (frame shift mutation - insC1283) might contribute to the increase in isoniazid resistance [30].

Other Enzymes Associated with INH Resistance

The MTB glf (Rv3809c) gene encoding UDP-galactopyranose mutase, catalyzes the conversion of UDP-galactopyranose into UDPgalactofuranose through a 2-keto intermediate. It was reported that the over expression of Glf enzyme bound with the modified form of INH or by sequestering a factor such as NAD+ required for INH activity and thus might contribute to INH resistance [31]. Further, Pasca et al. observed that over expression of transmembrane transport protein MmpL7 encoded by mmpL7 (Rv2942) gene, in Mycobacterium smegmatis leads to high level INH resistance [32]. Yang et al. observed that InbR, a transcriptional regulatory protein which is encoded by Rv0275c, directly bind with INH and involved in isoniazid resistance [33]. Pandey et al. observed the over expression of Rv1475c (acn) gene, encoding Aconitate hydratase-A enzyme in clinical isolate of MTB resistant to rifampicin, isoniazid, ethambutol and kanamcyin [34] which suggested that Rv1475c might have some important role in INH resistance [35].

Though INH resistance is mostly due to the chromosomal mutations in the target genes, around 20-30% of INH resistant MTB isolates do not have mutations in any of the genes linked with resistance to INH [36] which suggests that other mechanism(s), namely efflux pump systems of MTB may be involved in INH resistance. The over expression of efflux pump genes such as efpA, mmpL7, mmr, p55 and the Tap-like gene Rv1258c etc are shown to contribute for INH resistance [36]. Further, efpA, jefA (Rv2459), drrA, drrB, mmr, Rv1250, Rv1634 and Rv0849 were reported to be over expressed under isoniazid or rifampicin stress [37]. Besides some other genes of MTB (Table 3) of an INH-sensitive strain, are also observed to be induced by isoniazid or ethionamide treatment [38].

Table 3: Genes induces by INH or ethionamide treatment of an INH-sensitive strain [38].

Though, the in depth molecular mechanism of INH resistance in number of mycobacterial proteins from drug resistance strains is yet to be thoroughly understood, many studies proposed the association of INH with number of MTB proteins in different ways such as direct activation by KatG, acetylation by NAT, inhibiting different enzymes through adduct formation, inducing different enzymes etc. Further, mutations in many enzymes of MTB are reported to be associated with INH resistances which are supposed to associate with different important metabolic pathways of MTB. The consequence of mutation of those enzymes on respective pathways needs further study so as to reveal other mechanisms of INH resistance.

References

1. World Health Organization (2015) WHO Global Tuberculosis Report 2015.
2. Shah NS, Wright A, Bai GH, Barrera L, Boulahbal F, et al. (2007) Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis 13: 380-387.
3. Marrakchi H, Lanéelle G, Quémard A (2000) InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FASII. Microbiology 146: 289-296.
4. Sandgren A, Strong M, Muthukrishnan P, Weiner BK, Church GM, et al. (2009) Tuberculosis drug resistance mutation database. PLoS Med 10: e2.
5. Shekar S, Yeo ZX, Wong JC, Chan MK, Ong DC, et al. (2014) Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis. PLoS One 15: e102383.
6. Middlebrook G, Cohn ML (1953) Some observations on the pathogenicity of isoniazid-resistant variants of tubercle bacilli. Science 118: 297-299.
7. Jena L, Wankhade G, Waghmare P, Harinath BC (2016) Computational Approach in Understanding Mechanism of Action of Isoniazid and Drug Resistance. Mycobact Dis 6: 202.
8. Santos LC (2012) Review: the molecular basis of resistance in Mycobacterium tuberculosis. OJMM 2: 24-36.
9. Heym B, Zhang Y, Poulet S, Young D, Cole ST (1993) Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis. J Bacteriol 175: 4255-4259.
10. Lew JM, Kapopoulou A, Jones LM, Cole ST (2011) TubercuList--10 years after. Tuberculosis 91:1-7.
11. Zhao X, Yu H, Yu S, Wang F, Sacchettini JC, et al. (2006) Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant. Biochemistry 45: 4131-4140.
12. Nguyen M, Claparols C, Bernadou J, Meunier B (2001) A fast and efficient metal-mediated oxidation of isoniazid and identification of isoniazid-NAD(H) adducts. ChemBioChem 2: 877-883.
13. Argyrou A, Jin L, Siconilfi-Baez L, Angeletti RH, Blanchard JS (2006) Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis. Biochemistry 45: 13947-13953.
14. Timmins GS, Deretic V (2006) Mechanisms of action of isoniazid. Mol Microbiol 62: 1220-1227.
15. Jena L, Deshmukh S, Waghmare P, Kumar S, Harinath BC (2015) Study of mechanism of interaction of truncated isoniazid-nicotinamide adenine dinucleotide adduct against multiple enzymes of Mycobacterium tuberculosis by a computational approach. Int J Mycobacteriol 4: 276-283.
16. Nguyen M, Quémard A, Broussy S, Bernadou J, Meunier B (2002) Mn(III) pyrophosphate as an efficient tool for studying the mode of action of isoniazid on the InhA protein of Mycobacterium tuberculosis. Antimicrob Agents Chemother 46: 2137-2144.
17. Mahapatra S, Woolhiser LK, Lenaerts AJ, Johnson JL, Eisenach KD (2012) A novel metabolite of antituberculosis therapy demonstrates host activation of isoniazid and formation of the isoniazid-NAD+ adduct. Antimicrob Agents Chemother 56: 28-35.
18. Sandy J, Holton S, Fullam E, Sim E, Noble M (2005) Binding of the anti-tubercular drug isoniazid to the arylamine N-acetyltransferase protein from Mycobacterium smegmatis. Protein Sci 14: 775-782.
19. Payton M, Auty R, Delgoda R, Everett M, Sim E (1999) Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance. J Bacteriol 181: 1343-1347.
20. Lee AS, Teo AS, Wong SY (2001) Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 45: 2157-2159.
21. Müller B, Streicher EM, Hoek KG, Tait M, Trollip A, et al. (2011) inhA promoter mutations: a gateway to extensively drug-resistant tuberculosis in South Africa? Int J Tuberc Lung Dis 15:344-351.
22. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28: 27-30.
23. Caspi R, Billington R, Ferrer L, Foerster H, Fulcher CA, et al. (2016) The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res 44: D471-D480.
24. Jena L, Waghmare P, Kashikar S, Kumar S, Harinath BC (2014) Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug. Int J Mycobacteriol 3: 276-282.
25. Jena L, Deshmukh S, Nayak T, Wankhade G, Harinath BC (2016) Effect of G67R and G207E mutations on stability of Arylamine N-acetyltransferase in isoniazid resistance strains of Mycobacterium tuberculosis revealed by molecular dynamics simulation study. Eur J Biomed Pharmaceut Sci 3: 487-494.
26. Wang XD, Gu J, Wang T, Bi LJ, Zhang ZP, et al. (2011) Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation. Mol Microbiol 82:1375-1391.
27. Vilchèze C, Jacobs WR Jr (2014) Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis: Genes, Mutations, and Causalities. Microbiol Spectr 2: MGM2-0014-2013.
28. Ando H, Miyoshi-Akiyama T, Watanabe S, Kirikae T (2014) A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis. Mol Microbiol 91: 538-547.
29. Buchmeier NA, Newton GL, Koledin T, Fahey RC (2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 47:1723-1732.
30. Jagielski T, BakuÅ?a Z, Roeske K, KamiÅ?ski M, Napiórkowska A, et al. (2014) Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates. J Antimicrob Chemother 69: 2369-2375.
31. Chen P, Bishai WR (1998) Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance. Infect Immun 66: 5099-5106.
32. Pasca MR, Guglierame P, De Rossi E, Zara F, Riccardi G (2005) mmpL7 gene of Mycobacterium tuberculosis is responsible for isoniazid efflux in Mycobacterium smegmatis. Antimicrob Agents Chemother 49: 4775-4777.
33. Yang M, Gao CH, Hu J, Zhao L, Huang Q, et al. (2015) InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG. Sci Rep 5: 13969.
34. Pandey R, Rodriguez GM (2012) A ferritin mutant of Mycobacterium tuberculosis is highly susceptible to killing by antibiotics and is unable to establish a chronic infection in mice. Infect Immun 80: 3650-3659.
35. Parsa K, Hasnain SE (2015) Proteomics of multidrug resistant Mycobacterium tuberculosis clinical isolates: a peep show on mechanism of drug resistance & perhaps more. Indian J Med Res 141: 8-9.
36. Rodrigues L, Machado D, Couto I, Amaral L, Viveiros M (2012) Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex. Infect Genet Evol 12: 695-700.
37. Li G, Zhang J, Guo Q, Jiang Y, Wei J, et al. (2015) Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates. PLoS One 10: e0119013.
38. Wilson M, DeRisi J, Kristensen H-H (1999) Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proc Natl Acad Sci USA 96: 12833-12838.