MurE Ligase Research Articles

Novel hybrid thiazoles, bis-thiazoles linked to azo-sulfamethoxazole: Synthesis, docking, and antimicrobial activity

The reaction of sulfamethoxazolehydrazonoyl chloride with thiosemicarbazones, bis-thiosemicarbazones, or 4-amino-3-mercapto-1,2,4-triazole in dioxane in the presence of triethylamine as a basic catalyst at reflux resulted in the regioselective synthesis of thiazoles and bis-thiazoles linked to azo-sulfamethoxazole as novel hybrid molecules. The structures of the new compounds were confirmed using a range of spectra. Each compound's antibacterial properties were evaluated using the agar well-diffusion technique, and most of them demonstrated significant potency. In silico investigations revealed that the described compounds had strong interactions with the binding sites of MurE ligase, tyrosyl-tRNA synthetase, and dihydropteroate synthase, demonstrating inhibitory activity.

Anticancer behaviour of 2,2′-(pyridin-2-ylmethylene)bis(5,5-dimethylcyclohexane-1,3-dione)-based palladium(II) complex and its DNA, BSA binding propensity and DFT study

Herein, we report the synthesis and biological evaluation of [Pd(L)(OH2)Cl] complex (where L = 2,2′-(pyridin-2-ylmethylene)bis(5,5-dimethylcyclohexane-1,3-dione) as a novel promising anticancer candidate. The complex was characterized by single-crystal X-ray diffraction and other various spectroscopic techniques. Besides, the optimized structure was determined through DFT calculations revealing that the coordination geometry of [Pd(L)(OH2)Cl] complex is square planar. The binding propensity of [Pd(L)(OH2)Cl] complex with DNA and BSA was assessed by the spectrophotometric method. The antimicrobial profile of the ligand and its [Pd(L)(OH2)Cl] complex was screened against clinically important bacterial strains. [Pd(L)(OH2)Cl] complex showed promising activity against these microorganisms. Pd(L)(OH2)Cl] complex exhibited a potent antiproliferative potential compared to its ligand against different human cancer cells (A549, HCT116, MDA-MB-231, and HepG2) with less toxic effect against normal cells (WI-38). Additionally, [Pd(L)(OH2)Cl] complex exerted its anticancer effects against the most responsive cells (HCT116 cells; IC50 = 11 ± 1 μM) through suppressing their colony-forming capabilities and triggering apoptosis and cell cycle arrest at S phase. Quantitative PCR analysis revealed a remarkable upregulation of the mRNA expression level of p53 and caspase-3 by 4.8- and 5.9-fold, respectively, relative to control. Remarkable binding properties and non-covalent interactions between L and its [Pd(L)(OH2)Cl] complex with the binding sites of different receptors including CDK2, MurE ligase, DNA, and BSA were established using molecular docking. Based on our results, [Pd(L)(OH2)Cl] complex is an intriguing candidate for future investigations as a potential anticancer drug for the treatment of colon cancer.

Unveiling MurE ligase potential inhibitors for treating multi-drug resistant Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen with ability to cause serious infection such as bacteremia, ventilator associated pneumonia, and wound infections. As strains of A. baumannii are resistant to almost all clinically used antibiotics and with the emergence of carbapenems resistant phenotypes warrants the search for novel antibiotics. Considering this, herein, a series of computer aided drug designing approach was utilized to search novel chemical scaffolds that bind stronger to MurE ligase enzyme of A. baumannii, which is involved peptidoglycan synthesis. The work identified LAS_22461675, LAS_34000090 and LAS_51177972 compounds as promising binding molecules with MurE enzyme having binding energy score of −10.5 kcal/mol, −9.3 kcal/mol and −8.6 kcal/mol, respectively. The compounds were found to achieve docked inside the MurE substrate binding pocket and established close distance chemical interactions. The interaction energies were dominated by van der Waals and less contributions were seen from hydrogen bonding energy. The dynamic simulation assay predicted the complexes stable with no major global and local changes noticed. The docked stability was also validated by MM/PBSA and MM/GBSA binding free energy methods. The net MM/GBSA binding free energy of LAS_22461675 complex, LAS_34000090 complex and LAS_51177972 complex is −26.25 kcal/mol, −27.23 kcal/mol and −29.64 kcal/mol, respectively. Similarly in case of MM-PBSA, the net energy value was in following order; LAS_22461675 complex (−27.67 kcal/mol), LAS_34000090 complex (−29.94 kcal/mol) and LAS_51177972 complex (−27.32 kcal/mol). The AMBER entropy and WaterSwap methods also confirmed stable complexes formation. Further, molecular features of the compounds were determined that predicted compounds to have good druglike properties and pharmacokinetic favorable. The study concluded the compounds to good candidates to be tested by in vivo and in vitro experimental assays. Communicated by Ramaswamy H. Sarma

Antituberculosis and Antiproliferative Activities of the Extracts and Tetrahydrobisbenzylisoquinoline Alkaloids from Phaeanthus ophthalmicus: In Vitro and In Silico Investigations

Tuberculosis (TB) and cancer are among the maladies with high morbidity and mortality rates to date. This prompted the utilization of natural products in the discovery and development of new anti-TB and anticancer derivatives. In this study, we explored the antitubercular and antiproliferative activities of extracts and tetrahydrobisbenzylisoquinoline alkaloids tetrandrine (1) and limacusine (2) from the Philippine medicinal plant Phaeanthus ophthalmicus. Antitubercular evaluation using colorimetric MABA (microplate Alamar blue assay) assays revealed antitubercular activities of the extracts and fractions [minimum inhibitory concentration (MIC) = 15.6 to < 64 μg/mL]. Among the two isolated alkaloids, limacusine (2) exhibited inhibitory activity against Mycobacterium tuberculosis (MIC = 42.6 μg/ml). In addition, CellTiter-Blue cell viability assay showed antiproliferative activity for limacusine (2) against K-562. Both tetrahydrobisbenzylisoquinoline alkaloids exhibited cytotoxicity on HeLa cells. To probe the binding mechanisms of 1 and 2 against putative protein targets, molecular docking simulations were carried out. Limacusine (2) showed high binding propensities to mycobacterial enzymes ATP-dependent murE ligase and enoyl acyl carrier protein reductase, thus illustrating possibilities of cell wall and mycolic biosynthesis inhibitory mechanisms, respectively. High binding affinity was also observed in 2 vs. FLT3, a protein target implicated in myeloid leukemia cell proliferation. Additionally, the limacusine-InhA and limacusine-FLT3 complexes were found to be dynamically stable as per molecular dynamics (MD) simulations. The results of in vitro experiments and molecular docking studies suggest tetrahydrobisbenzylisoquinoline alkaloids from P. ophthalmicus, especially limacusine (2), as promising natural products against TB and myeloid leukemia.

CLONING AND EXPRESSION OF A MURE LIGASE ENZYME AS A POTENTIAL TARGET AGAINST BACTERIA XANTHOMONAS ORYZAE PV. ORYZAE

Xanthomonas oryzae pv. oryzae (Xoo) is causal agent of bacterial blight (BB) in rice. Many genes in Xoo have been identified in recently years. One of these genes, a gene coded for uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan by coupled to the hydrolysis of ATP has more popular interest. However, there are no experimental data to confirm hypothesis of this enzyme in Xoo. A significant overview at the ATP binding site of most the MurE ligases demonstrated much more variable with amino acid sequence identities in this part, variable percentage around 22 to 26%. Besides, a refined homology structural feature between EcMurE and XooMurE will very important for determining possible involvement of the MurE ligase activity in Xoo. Therefore, a new recombinant protein named XooMurE from Xoo was purified with the N-terminal His-tagged form through a Ni-NTA column in this study. After purification, the Histag was removed then out of the N-terminal His-tagged XooMurE by TEV protease. Purification effectiveness of XooMurE over 95% in this study could produce an essential material for e studies about mechanism of XooMurE and consequently available direction for discovering novel anti-bacterial compounds against Xanthomonas oryzae pv. oryzae (xoo).

In Silico Molecular Analysis and Docking of Potent Antimicrobial Peptides Against MurE Enzyme of Methicillin Resistant Staphylococcus Aureus

During the last decade there has been an alarming increase in the appearance of antibiotic-resistant bacteria. The drug-resistant microorganisms dubbed superbugs are projected to kill 10 million people a year by 2050. The annual frequency of deaths from Methicillin-resistant Staphylococcus aureus (MRSA) is rapidly increasing and surpassing those caused by human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Muramyl ligase E (MurE), an enzyme involved in the peptidoglycan biosynthesis of the bacterial cell wall, is a highly druggable target in MRSA. Six anti Methicillin-resistant Staphylococcus Aureus peptides were selected for deep bioinformatic analysis. The six anti-Methicillin-resistant Staphylococcus Aureus peptides were modeled and then docked to MurE ligase and their binding interactions were studied. The findings suggested that the interactions of three antimicrobial peptides (Flowlicidin-2, Ostricacin-2 and Ostricacin-3) with MurE could be essential for their inhibitory activity.

Wide-open conformation of UDP-MurNc-tripeptide ligase revealed by the substrate-free structure of MurE from Acinetobacterbaumannii.

MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc-l -Ala-d -Glu using ATP and producing UDP-MurNAc-l -Ala-d -Glu-meso-A2 pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacterbaumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.

Design and Synthesis of Various 5'-Deoxy-5'-(4-Substituted-1,2,3-Triazol-1-yl)-Uridine Analogues as Inhibitors of Mycobacterium tuberculosis Mur Ligases.

The synthesis of hitherto unknown 5′-deoxy-5′-(4-substituted-1,2,3-triazol-1-yl)-uridine and its evaluation, through an one-pot screening assay, against MurA-F enzymes involved in Mycobacterium tuberculosis (Mtb), are described. Starting from UDP-N-acetylmuramic acid (UDP-MurNAc), the natural substrate involved in the peptidoglycan biosynthesis, our strategy was to substitute the diphosphate group of UDP-MurNAc by a 1,2,3-triazolo spacer under copper-catalyzed azide-alkyne cycloaddition conditions. The structure-activity relationship was discussed and among the 23 novel compounds developed, N-acetylglucosamine analogues 11c and 11e emerged as the best inhibitors against the Mtb MurA-F enzymes reconstruction pathway with an inhibitory effect of 56% and 50%, respectively, at 100 μM. Both compounds are selective inhibitors of Mtb MurE, the molecular docking and molecular dynamic simulation suggesting that 11c and 11e are occupying the active site of Mtb MurE ligase.

MurE inhibitors as antibacterial agents: a review

Peptidoglycan, an essential component of the bacterial cell wall plays a critical role in protecting bacteria against osmotic lysis. The ATP-dependent MurC-F ligases are crucial for the early stages of peptidoglycan biosynthesis. MurE ligase is third in the series and catalyzes the addition of l-Lysine (l-Lys) in Gram-positive bacteria or meso-diaminopimelic acid (meso-A2pm) in most Gram-negative bacteria to form UDP-N-acetylmuramoyl-l-Ala-d-Glu-l-Lys/A2pm. The high substrate specific for l-Lys or meso-A2pm makes this enzyme an attractive target for the development of antibacterial agents. Several MurE inhibitors have been reported including phosphinates, peptidosulfonamides, napthylfuran-2-ones, benzene-1,3-dicarboxylic acids, phosphorylatedhydroxyethylamines, natural compounds, 5-benzylidenethiazolidin-4-ones, N-alkyl-2-alkynyl-4(1H)-quinolones, rhodanine substituted d-glutamic acids, 2,5-dimethyl pyrroles, 2,5-disubstitued furans, tetrahydroisoquinolines etc. In the present review we present an update status and structural information of MurE enzyme inhibitors which may be utilized for the design of potent inhibitors against this enzyme.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

Estimation of structure and stability of MurE ligase from Salmonella enterica serovar Typhi

MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0 = 3.13 kcal mol−1, m = 1.52 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.05 M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0 = 3.04 kcal mol−1, m = 1.20 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.53 M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.

RETRACTED: Targeting Mycobacterial Enzymes with Natural Products

Tuberculosis (TB) is a recurring threat to contemporary civilization. It affects not only those within developing countries, but has also appeared again in places where it was once considered eradicated. TB co-infection in patients infected by HIV is, at the time of writing, the most common cause of death. In the field of searching for new antimycobacterial drug leads, compounds of natural origin still remain a promising source. The review is intended to gather information about natural products (metabolites of plants, fungi, bacteria, and marine sponges) that show activity against mycobacterial enzymes. Here, natural metabolites are presented as being inhibitors/activators of the mycobacterial enzymes involved in mycobacterial growth invitro (ClpC1, ClpP, MurE ligase, mycothiol S-conjugate amidase, β-ketoacyl-ACP synthase, InhA) and invivo, as regards the host cell (PtpB). Each enzyme is briefly described so as to generate an understanding of its role in mycobacterial growth and engender a perception of the mechanism of action of the studied natural compounds. Furthermore, after the introduction of the enzyme, its inhibitors are listed and exactly characterized.

Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase.

Objectives(S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action.MethodsBiomimetic Pictet–Spengler or Bischler–Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds.ResultsIn this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis.ConclusionsAs the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis.

A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus

An in-depth evaluation was undertaken of a new antibacterial natural product (1) recently isolated and characterised from the plant Hypericum olympicum L. cf. uniflorum. Minimum inhibitory concentrations (MICs) were determined for a panel of bacteria, including: meticillin-resistant and -susceptible strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus; vancomycin-resistant and -susceptible Enterococcus faecalis and Enterococcus faecium; penicillin-resistant and -susceptible Streptococcus pneumoniae; group A streptococci (Streptococcus pyogenes); and Clostridium difficile. MICs were 2–8mg/L for most staphylococci and all enterococci, but were ≥16mg/L for S. haemolyticus and were >32mg/L for all species in the presence of blood. Compound 1 was also tested against Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium but was inactive. The MIC for Mycobacterium bovis BCG was 60mg/L, and compound 1 inhibited the ATP-dependent Mycobacterium tuberculosis MurE ligase [50% inhibitory concentration (IC50)=75μM]. In a radiometric accumulation assay with a strain of S. aureus overexpressing the NorA multidrug efflux pump, the presence of compound 1 increased accumulation of 14C-enoxacin in a concentration-dependent manner, implying inhibition of efflux. Only moderate cytotoxicity was observed, with IC50 values of 12.5, 10.5 and 8.9μM against human breast, lung and fibroblast cell lines, respectively, highlighting the potential value of this chemotype as a new antibacterial agent and efflux pump inhibitor.

Identification of potential drug targets in Yersinia pestis using metabolic pathway analysis: MurE ligase as a case study

Sporadic outbreaks of plague, lack of a vaccine, emergence of multidrug-resistant strains of Yersinia pestis, and its potential use in bioterrorism, call for an urgent need to develop new drugs for plague. We have used comparative metabolic pathway analysis to identify 245 drug-target candidate enzymes in Y. pestis CO92 which are non-homologous to host Homo sapiens and likely to be essential for the pathogen's survival. Further analysis revealed that 25 of these are potential choke point enzymes. As a case study, structure of a choke point enzyme, MurE ligase, was modeled and docking studies performed against a library of compounds leading to identification of a potential inhibitor. This approach enables rapid potential drug-target identification, thereby facilitating search for new antimicrobials.

Synthesis and antibacterial evaluation of a new series of N-Alkyl-2-alkynyl/(E)-alkenyl-4-(1H)-quinolones.

To gain further insight into the structural requirements of the aliphatic group at position 2 for their antimycobacterial activity, some N-alkyl-4-(1H)-quinolones bearing position 2 alkynyls with various chain length and triple bond positions were prepared and tested for in vitro antibacterial activity against rapidly-growing strains of mycobacteria, the vaccine strain Mycobacterium bovis BCG, and methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and -16. The compounds were also evaluated for inhibition of ATP-dependent MurE ligase of Mycobacterium tuberculosis. The lowest MIC value of 0.5 mg/L (1.2–1.5 µM) was found against M. fortuitum and M. smegmatis. These compounds displayed no or only weak toxicity to the human lung fibroblast cell line MRC-5 at 100 µM concentration. The quinolone derivatives exhibited pronounced activity against the epidemic MRSA strains (EMRSA-15 and -16) with MIC values of 2–128 mg/L (5.3–364.7 µM), and M. bovis BCG with an MIC value of 25 mg/L (66.0–77.4 µM). In addition, the compounds inhibited the MurE ligase of M. tuberculosis with moderate to weak activity showing IC50 values of 200–774 µM. The increased selectivity towards mycobacterial bacilli with reference to MRC-5 cells observed for 2-alkynyl quinolones compared to their corresponding 2-alkenyl analogues serves to highlight the mycobacterial specific effect of the triple bond. Exploration of a terminal bromine atom at the side chain of N-alkyl-2-(E)-alkenyl-4-(1H)-quinolones showed improved antimycobacterial activity whereas a cyclopropyl residue at N-1 was suggested to be detrimental to antibacterial activity.

An antibacterial from Hypericum acmosepalum inhibits ATP-dependent MurE ligase from Mycobacterium tuberculosis

In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of Hypericum acmosepalum. The structures of both compounds were characterised by extensive one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and were confirmed by mass spectrometry. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration ranges of 2–128mg/L and 0.5–128mg/L, respectively. Hyperenone A also showed growth-inhibitory activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG at 75mg/L and 100mg/L. Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and both were non-toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the ATP-dependent MurE ligase of M. tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively, whereas hypercalin B did not have any effect on enzyme activity.

Interaction of N-methyl-2-alkenyl-4-quinolones with ATP-dependent MurE ligase of Mycobacterium tuberculosis: antibacterial activity, molecular docking and inhibition kinetics.

ObjectivesThe aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria.MethodsUsing the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc2155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase.ResultsThe novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC50 ∼40–200 μM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones.ConclusionsN-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors.

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K(m) values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.

Antibacterial from Hypericum acmosepalum showing inhibition of ATP dependent MurE ligase from Mycobacterium tuberculosis

In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of H. acmosepalum. The structure of compounds were characterised by extensive 1- and 2D NMR spectroscopy and confirmed by MS spectrometry. Both hyperenone A and hypercalin B exhibited anti-bacterial activity against multidrug-resistant strains of Staphylococcus aureus, with MIC values ranging from 2–128µg/mL to 0.5–128µg/mL, respectively. Hyperenone A also showed growth inhibition against Mycobacterium tuberculosis H37Rv and M. bovis BCG at 50µg/mL (H37Rv) and 75µg/mL (BCG). The hyperenone A did not inhibit growth of Escherichia coli and was not toxic to cultured mammalian macrophages cells at the concentration at which anti-mycobacterial activity was determined. Both the compounds were also tested for their ability to inhibit MurE and MurF enzymes of M. tuberculosis which are crucial enzymes in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively whereas hypercalin B did not have any effect on both the enzymes. This is first report of a natural product from plant source showing inhibition of ATP dependent MurE ligase from M. tuberculosis. These studies represent promising starting points for further development. These highly active antibacterial compounds encourage further studies on the Hypericum genus.