MurA Inhibitors Research Articles

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5-cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8–12.2 μM. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin–resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli ΔtolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 μM respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 μg/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 μg/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA.

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

DNA-encoded library screening on two validated enzymes of the peptidoglycan biosynthetic pathway

Screening of DNA-encoded libraries is an emerging technology for discovering hits against protein targets. With the recent launch of the DELopen platform, a facile screening of 4.4 billion compounds is available to accelerate the drug discovery process. Here we report an affinity-based screening of the DELopen library for the first time. The screening was performed against two bacterial enzymes of the peptidoglycan biosynthetic pathway, N-acetylglucosamine-enolpyruvyl transferase (MurA) and d-alanine:d-alanine ligase (DdlB). Several binders were obtained and selected for off-DNA synthesis. Hits with confirmed inhibitory potency were deconstructed into smaller fragments. In this way, two new MurA inhibitors with antibacterial activity were obtained and are available for further optimization.

Discovery of MurA Inhibitors as Novel Antimicrobials through an Integrated Computational and Experimental Approach.

The bacterial cell wall is essential for protecting bacteria from the surrounding environment and maintaining the integrity of bacteria cells. The MurA enzyme, which is an essential enzyme involved in bacterial cell wall synthesis, could be a good drug target for antibiotics. Although fosfomycin is used clinically as a MurA inhibitor, resistance to this antibiotic is a concern. Here we used molecular docking-based virtual screening approaches to identify potential MurA inhibitors from 1.412 million compounds from three databases. Thirty-three top compounds from virtual screening were experimentally tested in Listeria innocua (Gram-positive bacterium) and Escherichia coli (Gram-negative bacterium). Compound 2-Amino-5-bromobenzimidazole (S17) showed growth inhibition effect in both L. innocua and E. coli, with the same Minimum Inhibitory Concentration (MIC) value of 0.5 mg/mL. Compound 2-[4-(dimethylamino)benzylidene]-n-nitrohydrazinecarboximidamide (C1) had growth inhibition effect only in L. innocua, with a MIC value of 0.5 mg/mL. Two FDA-approved drugs, albendazole (S4) and diflunisal (S8), had a growth inhibition effect only in E. coli, with a MIC value of 0.0625 mg/mL. The identified MurA inhibitors could be potential novel antibiotics. Furthermore, they could be potential fosfomycin substitutes for the fosfomycin-resistant strains.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview.

The online version contains supplementary material available at 10.1007/s12088-021-00988-6.

Mode Action Prediction of Butein as Antibacterial Oral Pathogen against Enterococcus faecalis ATCC 29212 and an Inhibitor of MurA Enzyme: In Vitro and In Silico Study

Background: The MurA enzyme, enolpyruvyl UDP-N-acetylglucosamine transferase, is one of the targeted proteins by antibiotics for effective treatment of diseases provided by pathogenic bacteria. It plays a key role in the cell wall biosynthesis of Gram-positive bacteria such as Enterococcus faecalis. Butein is a flavonoid that showed antioxidant and anticancer activities, but recently it is a promising antibacterial agent and reported can inhibit E. faecelis, Eschericia coli, and Mycobacterium tuberculosis. It was isolated from medicinal plants, including Sarang Semut (Myrmecodia pendans). However, the molecular mechanism of butein inhibits bacteria that is no clear. Objective: This study aims to predict the molecular action of the butein against MurA, catalyzing the first step of peptidoglycan biosynthesis. Material and Methods: Butein isolation used a combinational separation technique and characterization using spectroscopic methods. Then, in silico method used was virtual screening using programs including Autodock Vina in PyRx, protein. plus, and ligplots. Butein and UDP-N-acetylglucosamine (UNAG as a positive control) act as ligand were subject binding to 3KQJ MurA as protein. To evaluate in vitro antibacterial activity, we used the Kirby-Bauer method. Results: Butein from M. pendans is a potential compound to inhibit the MurA with binding affinity - 7.6 kcal.mol-1. It is lower than UNAG but higher than Fosfomycin as a MurA inhibitor. Butein attaches to MurA in the same position as UNAG so that it concludes that both is a competitive inhibitor. Meanwhile, in vitro study showed that butein inhibits the E. faecalis growth with inhibit zone of 8.37 mm at 1 mg/ml. Conclusions: Butein as a potent antibacterial agent through blocking MurA enzyme in cell wall formation.

Potential of MurA Enzyme and GBAP in Fsr Quorum Sensing System as Antibacterial Drugs Target: In vitro and In silico Study of Antibacterial Compounds from Myrmecodia pendans.

Background Increasing the resistance issue has become the reason for the development of new antibacterial in crucial condition. Many ways are tracked to determine the most effective antibacterial agent. Some proteins that are a key role in bacteria metabolism are targeted, including MurA in cell wall biosynthesis and gelatinase biosynthesis-activating pheromone (GBAP) in Fsr Quorum Sensing (QS) system.ObjectiveThe objective of this research is the analysis of compounds 1-4 from M. pendans as antibacterial and anti-QS activity trough protein inhibition by in silico study; focus on the structure-activity relationships, to appraise their role as an antibacterial and anti-QS agent in the molecular level.Methods Both activities of M. pendans compounds (1-4) were analyzed by in silico, compared to Fosfomycin, Ambuic acid, Quercetin, and Taxifolin as a standard. Chemical structures of M. pendans compounds were converted using an online program molview. The compounds were docked to MurA, GBAP, gelatinase and serine protease using Autodock Vina in Pyrx 0.8 followed PYMOL to visualization and proteis.plus program to analyze of the complex.Results All compounds from M. pendans bound on MurA, GBAP, gelatinase and serine protease except compound 2. This biflavonoid did not attach to MurA and serine protease yet is the favorable ligand for GBAP and gelatinase with the binding affinity of -6.9 and -9.4 Kcal/mol respectively. Meanwhile, for MurA and serine protease, compound 4 is the highest of bonding energy with values of -8.7 and -6.4 Kcal/mol before quercetin (MurA, -8.9 Kcal/mol) and taxifolin (serine protease, -6.6 Kcal/mol).ConclusionBased on the data, biflavonoid acts better as anti-QS than an inhibitor of MurA enzyme while the others can be acted into both of them either the therapeutic agent of anti-QS or antibacterial agent of MurA inhibitor.

Bromo-Cyclobutenaminones as New Covalent UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Inhibitors.

Drug discovery programs against the antibacterial target UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) have already resulted in covalent inhibitors having small three- and five-membered heterocyclic rings. In the current study, the reactivity of four-membered rings was carefully modulated to obtain a novel family of covalent MurA inhibitors. Screening a small library of cyclobutenone derivatives led to the identification of bromo-cyclobutenaminones as new electrophilic warheads. The electrophilic reactivity and cysteine specificity have been determined in a glutathione (GSH) and an oligopeptide assay, respectively. Investigating the structure-activity relationship for MurA suggests a crucial role for the bromine atom in the ligand. In addition, MS/MS experiments have proven the covalent labelling of MurA at Cys115 and the observed loss of the bromine atom suggests a net nucleophilic substitution as the covalent reaction. This new set of compounds might be considered as a viable chemical starting point for the discovery of new MurA inhibitors.

The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection

Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin. Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent CDI.

Heterocyclic electrophiles as new MurA inhibitors.

An electrophilic fragment library of small heterocycles was developed and characterized in the surrogate GSH-reactivity assay and aqueous stability test that revealed their potential as covalent warheads. Screening the library against MurA from Staphylococcus aureus (MurASA ) and Escherichia coli (MurAEC ) identified heterocyclic fragments with significant inhibitory potency. The validated heterocyclic warhead library might be useful for developing targeted covalent inhibitors for other targets of interest with a new design strategy incorporating heterocyclic electrophiles as warheads.

Discovery of new MurA inhibitors using induced-fit simulation and docking

We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1μM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.

In vitro testing of putative Escherichia coli MurA inhibitors (582.1)

Antibiotic resistance necessitates the continuous development of novel inhibitors targeting a variety of bacterial pathways. The pathway for the synthesis of peptidoglycan, a necessary component of the bacterial cell wall, is an ideal target for antibiotic development for two reasons: 1) the cell wall is a prokaryote‐specific structure, and 2) the cell wall is necessary for bacterial survival. MurA, the target of current study, is the enzyme that catalyzes the first step in peptidoglycan biosynthesis. Our objective is to develop novel inhibitors that competitively bind to the MurA catalytic sight for UDP‐N‐acetylglucosamine. In an effort to study the effects of various putative inhibitors on MurA, the active enzyme was purified in high concentrations. In vitro tests of the known inhibitor fosfomycin and other potential inhibitors were conducted using the purified enzyme. The Malachite green assay as previously described was used to determine the IC50 values for a number of potential inhibitors. Of the compounds assayed, two salts, VS5 and BCB33b, exhibited consistent IC50 values of 160 uM and 300 uM respectively. The IC50 values of the two putative inhibitors are the lowest we have seen out of all the tested compounds; therefore, VS5 and BCB33b will be tested further in vitro, in vivo, and in silico in their salt and acid forms for comparison against the known inhibitor fosfomycin.Grant Funding Source: Faculty Development Committee, Centre College

Differential antibacterial properties of the MurA inhibitors terreic acid and fosfomycin

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus, but its cellular target remains unknown. We recently reported that terreic acid inactivates the bacterial cell wall biosynthetic enzyme MurA in vitro by covalent reaction with residue Cys115 in a similar manner as the MurA-specific antibiotic fosfomycin. To address if terreic acid also targets MurA in vivo, we conducted antibacterial studies using selected E. coli strains in parallel with fosfomycin. While overexpression of MurA conferred resistance to fosfomycin, it did not protect cells treated with terreic acid. Furthermore, flow cytometry revealed that the antibiotic action of terreic acid appears to be primarily bacteriostatic, as opposed to the bactericidal action observed for fosfomycin. Combined, the data suggest that MurA is not the molecular target of terreic acid and that the antibiotic activity of terreic acid proceeds through a different mechanism of action. The methodology applied here provides a reliable and convenient tool to rapidly assess the potential of newly discovered in vitro inhibitors to target residue Cys115 of MurA in the cell.

A(2) expression and assembly regulates lysis in Qβ infections.

The capsids of ssRNA phages comprise a single copy of an ~45 kDa maturation protein that serves to recognize the conjugative pilus as receptor, to protect the ends of the viral RNA and also to escort the genomic RNA into the host cytoplasm. In the Alloleviviridae, represented by the canonical phage Qβ, the maturation protein A(2) also causes lysis. This is achieved by inhibiting the activity of MurA, which catalyses the first committed step of murein biosynthesis. Previously, it was shown that Qβ virions, with a single copy of A(2), inhibit MurA activity. This led to a model for lysis timing in which, during phage infection, A(2) is not active as a MurA inhibitor until assembled into virion particles, thus preventing premature lysis before a sufficient yield of viable progeny has accumulated. Here we report that MurA inactivates purified Qβ particles, casting doubt on the notion that A(2) must assemble into particles prior to MurA inhibition. Furthermore, quantification of A(2) protein induced from a plasmid indicated that lysis is entrained when the amount of the lysis protein is approximately equimolar to that of cellular MurA. Qβ por mutants, isolated as suppressors that overcome a murA(rat) mutation that reduces the affinity of MurA for A(2), were shown to be missense mutations in A(2) that increase the translation of the maturation protein. Because of the increased production of A(2), the por mutants have an attenuated infection cycle and reduced burst size, indicating that a delicate balance between assembled and unassembled A(2) levels regulates lysis timing.

Functional Consequence of Covalent Reaction of Phosphoenolpyruvate with UDP-N-acetylglucosamine 1-Carboxyvinyltransferase (MurA)

The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

Streptococcus mutans (S. mutans) is the primary etiological agent of human dental caries. A gene (SMU_1525) encoding MurA was identified in S. mutans UA159 (ATCC700610). The deduced amino-acid sequence has homology (51% identity) with E. coli MurA protein (AAC76221). In this study, we cloned S. mutans murA (SMU_1525) gene, expressed soluble recombinant MurA protein in E. coli BL21(DE3). The recombinant protein was purified by affinity chromatography. The molecular weight of expressed protein was about 45.6 kD. The activity of the protein was identified by high-pressure liquid chromatography (HPLC) and was shown to have UDP-N-acetylglucosamine enolpyruvyl transferase activity. Then one microtiter plate based colourimetric assay for S. mutans MurA activity was developed. The optimal temperature and pH of the purified enzyme were 37°C and 7.5, respectively. The Km for UDP-GlcNAc was 0.120 mM and for PEP was 0.086 mM. The Vmax for UDP-GlcNAc was 0.048 mM min-1 mg-1 and for PEP was 0.098 mM min-1 mg-1. The activity of S. mutans MurA was inhibited by the fosfomycin, known MurA inhibitor.