Pranita Waghmare

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 mycobacterial 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 (S315T and S315N) were modeled using Modeller9v10 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.

Excretory Secretory Proteins Released during Growth of Mycobacterium tuberculosis (H37Ra), With Diagnostic Potential in Pulmonary and Extra Pulmonary Tuberculosis

TB immunodiagnostics based on antibody and antigen detection for early detection and monitoring tubercular infections at low cost and flexible to adapt to field laboratories are a boon to developing countries. In this context we have explored in-house developed penicillinase based ELISA assays for the detection of antigen, antibody as well as immune-complexed antigen using various excretory secretory antigenic proteins and specific antibodies. Excretory secretory protein antigens such as ES-31, ES-41, ES-43, ES-6, ES-20, ES-100 and EST-6 were studied extensively and found to be useful in various pulmonary and extra pulmonary cases of tuberculosis infection. ES-31 has shown its diagnostic potential in chronic PTB cases, ES-43 in relapse cases and ES-41 in bone and joint TB. Elevated level of ES-20 antigen was observed in patients with weak immune response in TB lymphadenitis. Detection of ES-100 antigen and antibody by penicilinase ELISA was observed to be useful in detection of TB meningitis. ES-6 antigen was shown to be useful in detection of latent infection. These proteins with antigenic properties are utilized for production of specific antibodies against them and observed to be useful in immune diagnostics for detection of specific circulating and immune complexed antigens. Further user friendly peroxidase immunoassay has been standardised and is being routinely used for suspected TB cases attending 1000 bedded Kasturba Hospital attached to medical institute on physician’s request.

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

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.

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 in Understanding Mechanism of Action of Isoniazidand Drug Resistance

Most Multi Drug Resistance and Extremely Drug Resistance clinical strains of Mycobacterium tuberculosis are found to be resistant to the anti-tuberculousis drugs such as Isoniazid and Rifampicin. The mechanism of action and drug resistance due to Isoniazid has been the subject of extensive study. According to Tuberculosis drug resistance mutation database, 22 genes/proteins are associated with Isoniazid resistance such as katG, nat, inhA, ahpc, ndh, kasA etc. Mutation in the gene seems to affect the formation of Isoniazid to its active form or enhancing the catabolism thus making it ineffective. Studies in different laboratories have shown usefulness of computational approach in elucidating the mechanism of action of Isoniazid and development of drug resistance. Computational studies in our laboratory showed that a mutation in KatG (S315T/S315N) prevents free radical formation, thus the development of resistance to the drug. Further, we observed through molecular dynamics simulation approach that mutation (G67R/G207E) in NAT enzyme increases the stability and catalytic ability of the mutant enzyme, thus making the drug ineffective.

Genomics and Proteomics of Virulent, Avirulent and Drug ResistantStrains of Tuberculous Mycobacteria

Mycobacterium tuberculosis (MTB), the causal organism of the oldest infectious disease tuberculosis is the leading cause of morbidity and mortality worldwide. This pathogenic organism has been evolved into variety of strains with diverse genotype, phenotype and pathogenic properties such as MTB H37Rv and CDC1551 strains which are virulent, while MTB H37Ra is an a virulent strain and MTB KZN strain is resistant to different antituberculosis drugs. Due to the advancement in genome sequencing and molecular biology, whole genomes of different MTB strains have been completely sequenced. Genomic as well as proteomic comparison among the sequenced strains will help in understanding the differences between virulent, a virulent and resistant organisms. This article reviews the information available on completely sequenced MTB strains and presents the studies reported by researchers on genomic and proteomic comparison of various MTB strains.

Inhibition of mycobacterial CYP125 enzyme by sesamin and β-sitosterol: An in silico and in vitro study

Background: Cholesterol degradation pathway is one of the important pathways in survival of Mycobacterium tuberculosis (Mtb) bacilli, and steroid C26-monooxygenase (CYP125) enzyme of Mtb associated with this pathway is reported to be novel drug target. This study aims to find out novel, safe, and effective inhibitors against CYP125 from natural phytochemicals with reported anti-tubercular activity. Methods: Bioinformatics approach such as homology modeling, virtual screening, and molecular dynamics (MD) simulation was applied to identify best hits among all the shortlisted 148 compounds. The Mtb H37Ra bacilli growth was measured at optical density at 600 nm in minimal media supplemented with cholesterol and monitored for 10 days. Two promising compounds, namely, sesamin and β-sitosterol, were studied to determine their effective minimum inhibitory concentrations (MICs) in Mtb H37Ra bacilli culture. Results: In virtual screening, 15 compounds showed comparatively better binding affinity than natural substrate (choletst-4-en-3-one). In MD simulation study, the protein structure was observed to be stable in alls the interaction complexes, i.e., with choletst-4-en-3-one, sesamin, and β-sitosterol. The MICs of sesamin and β-sitosterol were observed to be 2 μg/ml, inhibiting the growth of the Mtb bacilli by 51% and 53%, respectively. Conclusions: From the above experimental findings, sesamin and β-sitosterol may be proposed as safe and potential inhibitors of CYP125 resulting in diminished growth of Mtb bacilli.

Mycobacterial Excretory Secretory-31 (ES-31) Protein with Serine Protease and Lipase Activities- A Potential Drug Target against TB Infection

Tuberculosisis the second leading cause of death worldwide. The researchers still finding way to eradicate TB. There is an urgent need to discover new drug which is more effective and less toxic to combat drug resistance. This article reports potential of ES-31 antigen as a new drug target. The characterization of ES-31 antigen showed that ES-31 is a 31 kDa protein antigen and has serine protease as well as lipase activities and shown to be a chymotrypsin-like protein which is having catalytic triad responsible for both activities. Addition of serine protease inhibitors (53-76%), metallo protease inhibitor (46-61%), lipase inhibitor (61%) or anti-ES-31 serine protease antibody (89%) strongly inhibited the MTB H37Ra growth in axenic culture. The importance of excretory secretory ES-31 antigen for the survival of MTB H37Ra and H37Rv bacilli has been shown by 77% and 78% growth inhibition in macrophage culture by protease inhibitor pefabloc. Inhibition of ES-31 leads to growth inhibition of MTB bacilli, suggests that it may be an important drug target for exploring new drugs for tuberculosis.