Enoyl-acyl Carrier Protein Reductase InhA Research Articles

Design, Synthesis and Molecular Docking of Novel Acetophenone-1,2,3-Triazoles Containing Compounds as Potent Enoyl-Acyl Carrier Protein Reductase (InhA) Inhibitors.

New medications are desperately needed to combat rising drug resistance among tuberculosis (TB) patients. New agents should ideally work through unique targets to avoid being hampered by preexisting clinical resistance to existing treatments. The enoyl-acyl carrier protein reductase InhA of M. tuberculosis is one of the most crucial targets since it is a promising target that has undergone extensive research for anti-tuberculosis drug development. A well-known scaffold for a variety of biological activities, including antitubercular activity, is the molecular linkage of a1,2,3-triazole with an acetamide group. As a result, in the current study, which was aided by ligand-based molecular modeling investigations, 1,2,3-triazolesweredesigned and synthesized adopting the CuAAC aided cycloaddition of 1-(4-(prop-2-yn-1-yloxy)phenyl)ethanone with appropriate acetamide azides. Standard spectroscopic methods were used to characterize the newly synthesized compounds. In vitro testing of the proposed compounds against the InhA enzyme was performed. All the synthesized inhibitors completely inhibited the InhA enzyme at a concentration of 10 µM that exceeded Rifampicin in terms of activity. Compounds 9, 10, and 14 were the most promising InhA inhibitors, with IC50 values of 0.005, 0.008, and 0.002 µM, respectively. To promote antitubercular action and investigate the binding manner of the screened compounds with the target InhA enzyme’s binding site, a molecular docking study was conducted.

Novel Hybrid 1,2,4- and 1,2,3-Triazoles Targeting Mycobacterium Tuberculosis Enoyl Acyl Carrier Protein Reductase (InhA): Design, Synthesis, and Molecular Docking.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is still a serious public health concern around the world. More treatment strategies or more specific molecular targets have been sought by researchers. One of the most important targets is M. tuberculosis’ enoyl-acyl carrier protein reductase InhA which is considered a promising, well-studied target for anti-tuberculosis medication development. Our team has made it a goal to find new lead structures that could be useful in the creation of new antitubercular drugs. In this study, a new class of 1,2,3- and 1,2,4-triazole hybrid compounds was prepared. Click synthesis was used to afford 1,2,3-triazoles scaffold linked to 1,2,4-triazole by fixable mercaptomethylene linker. The new prepared compounds have been characterized by different spectroscopic tools. The designed compounds were tested in vitro against the InhA enzyme. At 10 nM, the inhibitors 5b, 5c, 7c, 7d, 7e, and 7f successfully and totally (100%) inhibited the InhA enzyme. The IC50 values were calculated using different concentrations. With IC50 values of 0.074 and 0.13 nM, 7c and 7e were the most promising InhA inhibitors. Furthermore, a molecular docking investigation was carried out to support antitubercular activity as well as to analyze the binding manner of the screened compounds with the target InhA enzyme’s binding site.

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 μg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 μM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

48 - Non-Enzymatic Radical Activation of the Potent Anti-Tubercular Isoniazid by Cu(II)

Isoniazid (INH) is one of the most potent drugs available for tuberculosis treatment. As a pro-drug it requires activation by catalase/peroxidase to produce the reactive isonicotinic acyl radical intermediate responsible for its anti-tuberculosis activity, However, it is not clear whether INH can be activated non-enzymatically by Cu(II). Here we found that INH and Cu(II) together could induce synergistic DNA damage (strand breakage and 8-oxodG formation), while neither of them alone has any effect. DNA damage by INH/Cu(II) could be inhibited by Cu(I)-specific chelator and catalase, but not by SOD and the typical ●OH radical scavengers. Interestingly, ESR spin-trapping method showed that ●OH, ●H and a C-centered radical were produced during Cu(II)-catalyzed oxidation of INH. We proposed that the synergistic DNA damage induced by INH/Cu(II) might be due to the synergistic and site-specific production of ●OH near the binding site of copper and DNA. The C-centered radical was further unequivocally identified by ESR and HPLC-MS as isonicotinic acyl radical, which can react with nicotinamide coenzyme NADH to form the critical isonicotinic acyl-NAD adduct. The adduct can effectively inhibit in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids. This study provided a new insight on non-enzymatic activation of INH by Cu(II) to produce the reactive isonicotinic acyl radical, ●OH and ●H, which may have important biological implications for future research on INH.

Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare.

Multidrug resistant tuberculosis (MDR-TB) has been a topic of growing importance in the last decade. Emergence of MDR-TB has complicated infections caused by human immunodeficiency virus (HIV) and the re-emergence of tuberculosis in the West. MDR-TB is a major therapeutic challenge as the organism is resistant to two key anti-tuberculosis drugs, isoniazid and rifampicin. Programmatic management of drug resistant tuberculosis using second line therapy has been standardized through the development of international guidelines1. In India, the Revised National Tuberculosis Control Programme (RNTCP) advocates second line regimen comprising of six drugs - kanamycin, levofloxacin, ethionamide, pyrazinamide, ethambutol and cycloserine for 6-9 months of intensive phase therapy and levofloxacin, ethionamide, ethambutol and cycloserine for the next 18 months of the continuation phase2. Second line therapy is more complicated than the first line therapy as it has reduced potency, severe side effects and reduced efficacy. Hence, RNTCP advocates that poor response to treatment should prompt an examination of the programmatic, clinical or microbiological causes2. The lack of rapid, accurate and reliable drug susceptibility testing of the second line anti-tuberculosis drugs makes it difficult to examine the microbiological causes of poor response. In vitro results of drug susceptibility testing to the second line drugs also show poor clinical correlation leading the physicians to rely more on their clinical acumen than factual data. Empirical treatment is advocated, not taking into account the risk of medication to which the Mycobacterium tuberculosis isolate may be resistant. Pharmacokinetic variability whereby poor circulating drug concentrations may impair killing of isolates with borderline susceptibility is also not considered3. This may further amplify resistance. For a drug like ethionamide, where drug susceptibility pattern is difficult to determine and the therapy is empirical, development of resistance may be a serious cause of concern. Lakshmi et al4 in this issue have thus taken up a crucial investigation in the wake of emergence of MDR-TB and extensively drug resistant tuberculosis (XDR-TB). Testing for drug susceptibility to ethionamide, a crucial part of second line anti-tuberculosis therapy, is problematic as the difference characterizing drug resistance or susceptibility is small. Moreover, the minimum inhibitory concentration (MIC) is very near to the therapeutic index as commented by Lakshmi et al, in their article in this issue4. In addition, the drug is thermolabile, which makes drug susceptibility testing more difficult5. Due to these issues, there is poor clinical and laboratory correlation5. Inspite of the importance of ethionamide in the treatment of MDR-TB, there are only a few reports on the drug susceptibility testing of this agent. The gold standard of drug susceptibility testing against anti-tuberculosis agents is the proportion method that uses a single critical concentration of drugs to determine resistance or susceptibility. Criteria for measuring resistance such as critical concentrations and critical proportions of drugs affect the treatment predictive value of the drug susceptibility assay. The World Health Organization (WHO) has defined critical concentrations for various second line drugs taking into account the in vitro criteria for resistance using representative clinical samples and recommended a concentration of 40 µg/ml for proportion method of drug susceptibility testing of ethionamide6,7. Lakshmi et al4 also used the critical concentration of 40 µg/ml to perform the drug susceptibility assay by proportion method. However, the thermolabile nature of ethionamide may play a role in reducing the potency of the drug during inspissation of the drug containing medium. Deterioration of the drug during prolonged incubation period required for M. tuberculosis can also be crucial. Technical errors, as mentioned by Lakshmi et al4 like inoculum preparation, incubation and interpretation can add to erroneous results and should be minimized. Various investigators have standardized the critical concentration of ethionamide to be used in assays other than the proportion method, such as MIC and MGIT960. Kim et al8 used a critical concentration of 5 µg/ml for MGIT 960. Mpagama et al3 used 5 µg/ml of ethionamide as the critical concentration to segregate borderline susceptible from resistant through MIC on MycoTB sensititer plates (Trek Diagnostics, Cleveland, Ohio, USA). Lakshmi et al4 recommended a much higher concentration of 80 µg/ml for MIC on Lowenstein-Jensen (L-J) medium. The sensitivity of the MIC improved using 80 µg/ml as the cut-off. Though the MIC method has been observed to be more efficient than the proportion method5, it has not yet been recommended to drive therapeutic decisions. The need of the hour is to generate more data on ethionamide susceptibility profile of a specific geographical region, since it has been reported that geographical variation in strains also leads to a shift in the MIC value9. This would entail re-calibration of MIC values for each geographical region. In future, molecular tests may help in overcoming the problems encountered during testing for ethionamide susceptibility. Structural analogues, ethionamide and isoniazid share the same molecular target, the NADH-dependent enoyl-acyl carrier protein reductase InhA, which is involved in the synthesis of mycolic acids. Hence, cross-resistance to isoniazid and ethionamide may be observed in clinical isolates. Ethionamide acts by inhibiting mycobacterial cell wall mycolic acid synthesis and requires activation by a prodrug activator, NADPH-specific flavin adenosine dinucleotide containing mono-oxygenase encoded by ethA10. The expression of EthA is negatively regulated by EthR, a regulator that interacts directly with the ethA promoter region. Ethionamide resistance has been shown to be associated with mutations at ethA and ethR10,11. It has been reported that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region is associated with high-level resistance to ethionamide among the MDR-TB isolates12. In a study at Mumbai, Vadwai et al13 studied 69 phenotypically determined ethionamide resistant isolates and found that inhA promoter mutation was associated with ethionamide resistance in 21 isolates. They concluded that inhA mutation could be considered as a marker for ethionamide resistance in India. However, since 69.5 per cent of the ethionamide resistant isolates did not have inhA promoter mutations, alternative mechanisms could not be ruled out13. Identifying the molecular basis of ethionamide resistance could be an important focus for future studies. Further studies are needed to address the basis for mechanisms of ethionamide susceptibility. The future holds a lot of promises with newer molecular assays. Considering the problems faced due to phenotypic drug susceptibility profile, future assays for drug susceptibility may include a combination of molecular and conventional assays for second line drug susceptibility testing. What is also needed is better microbiological-clinical correlation for drug susceptibility profiles since there is a vast difference between in vitro and in vivo results. Bigger cohorts correlating the drug susceptibility profile with patient's clinical outcome are also needed to retard the emergence of extensively drug resistant tuberculosis.

Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis.

New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.

N-Substituted 2-Isonicotinoylhydrazinecarboxamides — New Antimycobacterial Active Molecules

This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1–2 μM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC = 4 μM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria.

Mn(III) pyrophosphate as an efficient tool for studying the mode of action of isoniazid on the InhA protein of Mycobacterium tuberculosis.

The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.

The nonenzymatic activation of isoniazid by MnIII-pyrophosphate in the presence of NADH produces the inhibition of the enoyl-ACP reductase InhA from Mycobacterium tuberculosis

Abstract The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III)-pyrophosphate. In the presence of the nicotinamide coenzyme, the INH oxidation produced the formation of INH-NAD(H) adducts and allowed the in vitro inhibition of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway for mycolic acids. Manganese(III)-pyrophosphate is an efficient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations on reactive INH species and resulting InhA inhibitors.