Mycobacterium Tuberculosis Enzyme Research Articles

Exploring the interaction mechanism between potential inhibitor and multi-target Mur enzymes of mycobacterium tuberculosis using molecular docking, molecular dynamics simulation, principal component analysis, free energy landscape, dynamic cross-correlation matrices, vector movements, and binding free energy calculation

Multi-targeting enzyme approaches are considered to be the most significant in suppressing pathogen growth and disease control for MDR and XDR-resistant Mycobacterium tuberculosis. The multiple Mur enzymes involved in peptidoglycan biosynthesis play a key role in a cell’s growth. Firstly, homology modeling was employed to construct the 3 D structure of the Mur enzymes. The computational approaches, including molecular docking and molecular dynamics simulations and MM-PBSA methods, were performed to explore the detailed interaction mechanism to evaluate the inhibitory activity against targeted proteins. The computational calculations revealed that the best-docked phytochemical compound (gallomyricitrin) inhibits the selected targets: Mur enzymes by forming stable hydrogen bonds. The analysis of RMSD, RMSF, Rg, PCA, DCCM, cross-correlation network, FEL, H-bond, and vector movement reveal that the docked complex of MurA, MurI, MurG, MurC, and MurE is more stable compared to MurB, MurF, MurD, and MurX docked complexes during MD simulations. Moreover, FEL exposed that gallomyricitrin stabilized to the minimum global energy of Mur Enzymes. The PCA, DCCM, and vector movements and binding free energy results provided further evidence for the stability of gallomyricitrin’s interactions inside the binding sites by forming hydrogen bonds. The cross-correlation analysis reveals that Mur enzymes exhibit a positive and negative correlated motion between residues in different protein domains. The computational results contribute in several ways to our understanding of inhibition activity and provide a basic insight into the binding activity of gallomyricitrin as a multi-target drug for tuberculosis. Communicated by Ramaswamy H. Sarma

Glucosamine-6-phosphate synthase de Mycobacterium tuberculosis um estudo in silico para predição de um modelo tridimensional refinado

Tuberculosis is one of the main causes of death by an infectious agent in the world, according to the World Health Organization. Studies indicate that enzymes involved in the biosynthesis of uridine diphosphate-N-acetylglucosamine are essential for the life cycle of the bacterium. One of these enzymes is glucosamine-6-phosphate synthase (GlmS), which does not have a three-dimensional structure available in the protein database on the internet. In this work, structural bioinformatics methods (comparative modeling and molecular refinement) were used to build a refined three-dimensional model for the GlmS enzyme of Mycobacterium tuberculosis. The model was generated using four templatestructures (crystallographic). The results obtained for the stereochemical and general parameters of the refined model were better than the original model and similarto those templatestructures, validating the refined model.

Covalent Inactivation of Mycobacterium tuberculosis Isocitrate Lyase by cis-2,3-Epoxy-Succinic Acid.

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.

Structure-based virtual screening of essential Mycobacterium tuberculosis enzymes AspS and KatG for potential inhibitors.

Mycobacterium tuberculosis (TB) is a leading global cause of disease-related death. Recent works have studied metabolic pathways of the mycobacterium, highlighting essential enzymes to target via competitive inhibition through computational molecular modeling to suppress the organism's life cycle. We used the Protein Databank (PDB), the UniProt Knowledgebase and the iDock server in this study. In vitro toxicity screening and pharmacokinetic properties were assessed to determine potential ligand safety and drug properties. Our results have revealed five and nine potential ligands for the enzymes AspS and KatG respectively. The KatG active site has displayed binding affinities of -13.443 to -12.895 kcal/mol, while AspS ligands range from -6.580 to -6.490kcal/mol. The intermolecular forces responsible for the differing binding affinities of each enzyme are primarily Coulombic interactions for AspS, versus Coulombic and extensive hydrogen bonding interactions in KatG.

Acetylation of Isoniazid Is a Novel Mechanism of Isoniazid Resistance in Mycobacterium tuberculosis.

Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.

Lignans and Neolignans Anti-tuberculosis Identified by QSAR and Molecular Modeling

Tuberculosis is a disease with high incidence and high mortality rate, especially in Brazil. Although there are several medications available for treatment, in cases of resistance, there is a need to use more than one medication. Therefore, cases of toxicity increase and reports of resistance have been worrying the population. In addition, some medications have a short period of effectiveness. To achieve the goal, ligand-based and structure-based approaches were used. Thus, in an attempt to discover potent inhibitors against Mycobacterium tuberculosis enzymes, we sought to identify natural products with high therapeutic potential for the treatment of Tuberculosis through QSAR, Molecular Modeling and ADMET studies. The results showed that the models generated from two sets of molecules with known activity against M. tuberculosis enzymes InhA and PS were able to select 11 and 8 compounds, respectively, between Lignans and Neolignans with 50 to 60% activity probability. In addition, molecular docking contributed to confirm the mechanism of action of compounds and increase the accuracy of methodologies. All molecules showed higher binding energy values for the drug Isoniazid. We conclude that compounds 33, 34, 110, 114 and 133 are promising for InhA target and compounds 07, 08, 19, 21, 42, 48, 75 and 141 for target PS. In addition, most molecules did not show any toxicity according to the evaluated parameters. Therefore, Lignans and Neolignans may be an alternative for the treatment of Tuberculosis.

In silico targeting methylerythritol phosphate pathway IspD enzyme of Mycobacterium tuberculosis for novel anti-mycobacterial drug discovery

Pharmacokinetic concerns in the management of drug induced vomiting in co-morbid tuberculosis patient: A case report from MalaysiaAtif M, Sulaiman SAS, Shafie AA, Muttalif AR4, Ali I, Saleem F

A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Arabinosyltransferase C enzyme of Mycobacterium tuberculosis, a potential drug target: An insight from molecular docking study

Multi-drug resistant in Mycobacterium tuberculosis (M.tb) is considered as major bottleneck in the treatment and cure of tuberculosis (TB). Several anti-tubercular drugs fail in its efficacy due to drug-resistant M.tb developed mechanism for resistance. So, research across globe has been carried out to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug development methods have been proved unsuccessful as it fails to develop a broad-spectrum drug due to lack of structure based approach. Several studies have been conducted in this regard and identified several drug target sites that influence drug-resistant M.tb strains. In this study, the attempt was to study the interaction between the protein Arabinosyltransferase C with the two existing drugs (Ethambutol and Isoniazid) and five modified molecules derived from Ethambutol by calculating their binding affinity and mode of binding through molecular docking study using AutoDock 4. From the comparison study of the existing drug (EMB and INH) and the five proposed modified molecules (Emb1, Emb2, Emb3, Emb4 and Emb5), it is analysed that Emb1 and Emb3 with binding affinities -5.77 kcal/mol and -5.13 kcal/mol respectively can be considered as potential inhibitors of Arabinosyltransferase C in Mycobacterium tuberculosis which is responsible for cell wall synthesis. The facts provided may be further verified experimentally for future drug discovery process to make a stand against tuberculosis and contribute an advance research for worthy antimycobacterial strategies.

Mycobacterium tuberculosis Decaprenylphosphoryl-β-d-ribose Oxidase Inhibitors: Expeditious Reconstruction of Suboptimal Hits into a Series with Potent in Vivo Activity.

Decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) is an essential enzyme in Mycobacterium tuberculosis and has recently been studied as a potential drug target, with inhibitors progressing to clinical studies. Here we describe the identification of a novel series of morpholino-pyrimidine DprE1 inhibitors. These were derived from a phenotypic high-throughput screening (HTS) hit with suboptimal physicochemical properties. Optimization strategies included scaffold-hopping, synthesis, and evaluation of fragments of the lead compounds and property-focused optimization. The resulting optimized compounds had much improved physicochemical properties and maintained enzyme and cellular potency. These molecules demonstrated potent efficacy in an in vivo tuberculosis murine infection model.

In silico Design, ADME Prediction, Molecular Docking, Synthesis of Novel Triazoles, Indazoles & Aminopyridines and in vitro Evaluation of Antitubercular Activity

To design and synthesize novel triazoles, indazoles and aminopyridines from various (thiophene-2-yl)prop-2-en-1-one derivatives as antitubercular leads by in silico and in vitro methods. in silco Drug design, ADME prediction and molecular docking studies were performed to assess drug likeliness and antitubercular potential of all 30 novel triazoles, indazoles and aminopyridines. in silico Drug design studies revealed that the synthetic routes applied were appropriate according to the calculations of Swiss-ADME that measure synthetic accessibility. Most of the synthesized compounds found to have considerable binding score with enoyl ACP reductase enzyme of Mycobacterium tuberculosis. All the synthesized compounds were evaluated for antitubercular potential against Drug Resistant Mycobacterium tuberculosis H37Rv strain by Luciferase reporter assay method. Most of the synthesized compounds exhibited remarkable antitubercular potential against resistant strain.

Identification of peptidomimetic compounds as potential inhibitors against MurA enzyme of Mycobacterium tuberculosis

Increasing prevalence of resistance to anti-tubercular drugs has become the foremost challenge now. According to WHO, over half a million of multidrug resistance cases (rifampicin, isoniazid, etc.) were reported in 2017, mostly emerging from countries such as China, India, and Russia. Therefore, developing new drugs or repurposing existing ones is need of the hour. The Mycobacterium cell wall biogenesis pathway offers many attractive targets for drug discovery against Tuberculosis (TB). MurA, a transferase enzyme that catalyzes the initial step of peptidoglycan (PG) biosynthesis, is one among them. A peptidoglycan layer resides over the plasma membrane and is an integral component of the bacterial cell wall. Therefore, disruption of their formation through inhibition of MurA enzyme should lead to deficiency in Mycobacterium cell synthesis. Based on this strategy, we have designed this study where two libraries of peptidomimetic compounds (Asinex & ChemDiv) were first screened against our modeled MurA structure and then validated through molecular dynamic simulations. From our virtual screening, top four compounds (ChemDiv: D675-0102, D675-0217; Asinex: BDE25373574, BDE 26717803) were selected based on their docking scores, binding energies, and interactions with catalytic site residues, for further evaluation. Results revealed stable ligand-MurA interactions throughout 50 ns of MD simulation and also druggability acceptable pharmacokinetic profile for all four compounds. Thus, based on our findings, these compounds could be considered as potential inhibitors of Mycobacterium MurA enzyme and hence be further tested for in vitro experimental validation as TB therapeutic drug candidate.Communicated by Ramaswamy H. Sarma

Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA.

Pyrazinamide (PZA) is a key drug for tuberculosis treatment. The active form of PZA, pyrazinoic acid (POA), appears to inhibit multiple targets in M. tuberculosis. Recently, the bifunctional enzyme Rv2783 was reported as a new target of POA. However, the mechanism by which POA inhibits Rv2783 is not yet clear. Here, we report how a new A2104C substitution in Rv2783c, identified in PZA-resistant clinical isolates, conferred resistance to PZA in M. tuberculosis. Expression of the mutant allele recapitulated the PZA resistance. All catalytic activities of Rv2783, but not the mutant, were inhibited by POA. Additionally, POA competed with transfer-messenger RNA (tmRNA) for binding to Rv2783, other than the mutant. These results provide new insight into the molecular mechanism of the antitubercular activity of PZA.

Predictive Binding Affinity of Plant-Derived Natural Products Towards the Protein Kinase G Enzyme of Mycobacterium tuberculosis (MtPknG).

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a growing public health concern worldwide, especially with the emerging challenge of drug resistance to the current drugs. Efforts to discover and develop novel, more effective, and safer anti-TB drugs are urgently needed. Products from natural sources, such as medicinal plants, have played an important role in traditional medicine and continue to provide some inspiring templates for the design of new drugs. Protein kinase G, produced by M. tuberculosis (MtPKnG), is a serine/threonine kinase, that has been reported to prevent phagosome-lysosome fusion and help prolong M. tuberculosis survival within the host’s macrophages. Here, we used an in silico, target-based approach (docking) to predict the interactions between MtPknG and 84 chemical constituents from two medicinal plants (Pelargonium reniforme and Pelargonium sidoides) that have a well-documented historical use as natural remedies for TB. Docking scores for ligands towards the target protein were calculated using AutoDock Vina as the predicted binding free energies. Ten flavonoids present in the aerial parts of P. reniforme and/or P. sidoides showed docking scores ranging from −11.1 to −13.2 kcal/mol. Upon calculation of all ligand efficiency indices, we observed that the (−ΔG/MW) ligand efficiency index for flavonoids (4), (5) and (7) was similar to the one obtained for the AX20017 control. When taking all compounds into account, we observed that the best (−ΔG/MW) efficiency index was obtained for coumaric acid, coumaraldehyde, p-hydroxyphenyl acetic acid and p-hydroxybenzyl alcohol. We found that methyl gallate and myricetin had ligand efficiency indices superior and equal to the AX20017 control efficiency, respectively. It remains to be seen if any of the compounds screened in this study exert an effect in M. tuberculosis-infected macrophages.

Structure-Activity Relationships of cyclo(l-Tyrosyl-l-tyrosine) Derivatives Binding to Mycobacterium tuberculosis CYP121: Iodinated Analogues Promote Shift to High-Spin Adduct.

A series of analogues of cyclo(l-tyrosyl-l-tyrosine), the substrate of the Mycobacterium tuberculosis enzyme CYP121, have been synthesized and analyzed by UV-vis and electron paramagnetic resonance spectroscopy and by X-ray crystallography. The introduction of iodine substituents onto cyclo(l-tyrosyl-l-tyrosine) results in sub-μM binding affinity for the CYP121 enzyme and a complete shift to the high-spin state of the heme FeIII. The introduction of halogens that are able to interact with heme groups is thus a feasible approach to the development of next-generation, tight binding inhibitors of the CYP121 enzyme, in the search for novel antitubercular compounds.

Novel Benzylidenehydrazide-1,2,3-Triazole Conjugates as Antitubercular Agents: Synthesis and Molecular Docking.

Novel 1,2,3-triazole based benzylidenehydrazide derivatives were synthesized and evaluated for antitubercular activity against Mycobacterium tuberculosis (MTB) H37Ra, M. bovis BCG and cytotoxic activity. Most of the derivatives exhibited promising in vitro potency against MTB characterized by lower MIC values. Among all the synthesized derivatives, compound 6a and 6j were the most active against active and dormant MTB H37Ra, respectively. Compound 6d was significantly active against dormant and active M. bovis BCG. The structure activity relationship has been explored on the basis of anti-tubercular activity data. The active compounds were also tested against THP-1, A549 and Panc-1 cell lines and showed no significant cytotoxicity. Further, the synthesized compounds were found to have potential antioxidant with IC50 range = 11.19-56.64 µg/mL. The molecular docking study of synthesized compounds was performed against DprE1 enzyme of MTB to understand the binding interactions. Furthermore, synthesized compounds were also analysed for ADME properties and the potency of compounds indicated that, this series can be considered as a starting point for the developement of novel and more potent anti-tubercular agents in future.

An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

A structural and functional perspective on the enzymes of Mycobacterium tuberculosis involved in the L-rhamnose biosynthesis pathway

Tuberculosis is one of the leading causes of death from bacterial infections. The multi-drug resistant strain has warranted the development of new drug molecules which can inhibit the growth of Mycobacterium tuberculosis (M.tb). Most of the known drugs inhibit the enzymes in the cell wall biosynthesis pathway. One such pathway is L-rhamnose, which involves four druggable enzymes RmlA, B, C and D. The 3D structure analyses of these protein models (RmlA, B and D) and crystal structure (RmlC) has been carried out. Multiple sequence alignments of homologs from distant species of 32 taxa and analyses of available structures were performed in order to study the conservation of sequence and structural motifs, and catalytically important residues. Based on these results and reported mechanism in other organisms, we have predicted putative catalytic mechanism of M.tb enzymes involved in the L-rhamnose biosynthesis pathway.

N-[2-(1H-Indol-3-yl)-1-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)ethyl]-4-methylbenzenesulfonamide

N-[1-Hydrazinyl-3-(1H-indol-3-yl)-1-oxopropan-2-yl]-4-methylbenzenesulfonamide (1) on cyclization with carbon disulfide in ethanolic potassium hydroxide affords N-[2-(1H-indol-3-yl)-1-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)ethyl]-4-methylbenzenesulfonamide (2) in 84% yield. The structure of compound 2 was supported by mass spectrometry, FT-IR and 1H- and 13C-NMR spectroscopy. To investigate the potential of compound 2 to act as antitubercular agent, it was docked against the enoyl reductase (InhA) enzyme of Mycobacterium tuberculosis. The docking pose and non-covalent interactions gave insights on its plausible inhibitory action.