A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase: the first committed step in peptidoglycan biosynthesis

The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10⁹ C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC₅₀ value of 200 μM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.     

5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design,synthesis, molecular docking and evaluation

In continuation of our efforts to develop new compounds with antimicrobial properties we describe design, synthesis, molecular docking study and evaluation of antimicrobial activity of seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones. All compounds showed antibacterial activity against eight Gram positive and Gram negative bacterial species. Twelve out of seventeen compounds were more potent than streptomycin and all compounds exhibited higher potency than ampicillin. Compounds were also tested against three resistant bacterial strains: MRSA, P. aeruginosa and E. coli. The best antibacterial potential against ATCC and resistant strains was observed for compound 8 (2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-nitrobenzylidene)-1,3thiazolidin-4-one). The most sensitive bacterium appeared to be S. typhimirium, followed by B. cereus while L. monocitogenes and M. flavus were the most resistant. Compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited antifungal activity better than the reference drugs bifonazole and ketokonazole (3-115 times). It was found that compound 8 appeared again to be the most potent. Molecular docking studies on E. coli MurB, MurA as well as C. albicans CYP 51 and dihydrofolate reductase were used for the prediction of mechanism of antibacterial and antifungal activities confirming the experimental results.     

MurA as a Primary Target of Tulipalin B and 6-Tuliposide B

(?)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.     

Screening of potential lead molecules against prioritised targets of multi-drug-resistant-Acinetobacter baumannii – insights from molecular docking,molecular dynamic simulations and in vitro assays

Acinetobacter baumannii, an opportunistic pathogen, has become multi-drug resistant (MDR) to major classes of antibacterial and poses grave threat to public health. The current study focused to screen novel phytotherapeutics against prioritised targets of Acinetobacter baumannii by computational investigation. Fourteen potential drug targets were screened based on their functional role in various biosynthetic pathways and the 3D structures of 9 targets were retrieved from Protein Data Bank and others were computationally predicted. By extensive literature survey, 104 molecules from 48 herbal sources were screened and subjected to virtual screening. Ten clinical isolates of A. baumannii were tested for antibiotic susceptibility towards clinafloxacin, imipenem and polymyxin-E. Computational screening suggested that Ajmalicine ((19α)-16, 17-didehydro-19-methyloxayohimban-16-carboxylic acid methyl ester from Rauwolfia serpentina), Strictamin (Akuammilan-17-oic acid methyl ester from Alstonia scholaris) and Limonin (7, 16-dioxo-7, 16-dideoxylimondiol from Citrus sps) exhibited promising binding towards multiple drug targets of A. baumannii in comparison with the binding between standard drugs and their targets. Limonin displayed promising binding potential (binding energy ?9.8 kcal/mol) towards diaminopimelate epimerase (DapF) and UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA). Ajmalicine and Strictamin demonstrated good binding potential (?9.5, ?8.5 kcal/mol, respectively) towards MurA and shikimate dehydrogenase (?7.8 kcal/mol). Molecular dynamic simulations further validated the docking results. In vitro assay suggested that the tested isolates exhibited resistance to clinafloxacin, imipenem and polymyxin-E and the herbal preparations (crude extract) demonstrated a significant antibacterial potential (p ≤ .05). The study suggests that the aforementioned lead candidates and targets can be used for structure-based drug screening towards MDR A. baumannii.     

Characterization and inhibition study of MurA enzyme by capillary electrophoresis

A capillary electrophoresis-based enzyme assay for UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is described. This method, based on UV detection, provides baseline separation of one of the reaction products, enolpyruvyluridine 5'-diphospho-N-acetylglucosamine (EP-UDP-GlcNAc), from substrates phosphoenolpyruvate (PEP) and uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, can be accomplished based on the separated peaks. This methodology was used to determine the Michaelis constant, Km, and product formation rate constant, Kcat, for MurA. Additionally, the CE method was used to evaluate the inhibition effects on MurA using one specific compound as an example. By following similar procedures, the apparent Km values in the presence of different inhibitor concentrations were determined. The inhibition constant, Ki, can be determined from these apparent Km values. In addition, this CE method can be used to study the inhibition mechanism. The principle of this approach is generally applicable to other enzyme studies.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

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

Abstract

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     

Identification of a novel UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine enolpyruvyl transferase (MurA) from <Emphasis Type="Italic">Vibrio fischeri</Emphasis> that confers high fosfomycin resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.     

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline,a macrolactone isolated from Streptomyces maizeus

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline’s antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA—the enzyme that catalyzes the first step of peptidoglycan biosynthesis—over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC₅₀ of 480?μM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.