A Multitarget Assay for Inhibitors of Membrane-Associated Steps of Peptidoglycan Biosynthesis

Barbosa, Maria D.F.S.; Ross, Harold; Hillman, Milton C.; Meade, Raymond P.; Kurilla, Michael G.; Pompliano, David L.

doi:10.1006/abio.2001.5691

Cited by 22 publications

(8 citation statements)

References 19 publications

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“…The availability of an MraY assay in the HTS format is therefore needed. To date, five HTS assays have been described (3,8,11,17,30). However, these assays involve one or several additional activities (MurG, transglycosylase, or transpeptidase), and they use a radioactive substrate.…”

Section: Validation Of the Hts With Mray Inhibitorsmentioning

confidence: 99%

Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Stachyra¹,

Dini²,

Ferrari³

et al. 2004

Antimicrob Agents Chemother

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We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.The biosynthesis of bacterial peptidoglycan is a complex two-stage process. The first stage consists in the formation of the monomer unit GlcNAc-MurNAc-(pentapeptide), whereas the second one concerns polymerization and maturation reactions (28). In the first stage, two enzymes, MraY and MurG, have in common their attachment to the cytoplasmic membrane and their processing of lipid intermediates: MraY transfers the phospho-MurNAc-pentapeptide motif onto lipid carrier undecaprenyl phosphate, yielding MurNAc-(pentapeptide)-pyrophosphoryl undecaprenol (lipid I), and MurG then adds the GlcNAc residue to yield GlcNAc-MurNAc-(pentapeptide)-pyrophosphoryl undecaprenol (lipid II), which contains the whole monomer unit. In the present study, we have focused our attention to the MraY transferase, which is an integral membrane protein with ten transmembrane segments, five cytoplasmic domains, and six periplasmic domains including the N-and C-terminal ends (4). Besides the synthesis of lipid I, which is fully reversible, MraY catalyzes in vitro an exchange reaction between UMP and UDP-MurNAc-pentapeptide (23). Owing to its exclusive presence in bacteria and to its essential character, which was demonstrated in Escherichia coli (5), MraY is a target of interest for the discovery of novel antibacterial agents. It is inhibited by non-clinically used antibiotics such as tunicamycin, amphomycin, mureidomycin, liposidomycin, and muraymycins (7,9,22). Recently, simplified analogues of liposidomycin, named riburamycins, have been shown to be powerful MraY inhibitors and to possess antibacterial activities against gram-positive organisms (13-16) (J. Biton, K. Braham, C. Dini, O. Krebs, P. Lassaigne, F. Monti, T. Stachyra, V. Steier, and J. Zhang, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-361, 2002). To date, several specific assays for the MraY activity have been published (6,17,18,27,29,30); however, very few can be adapted to the high-throughput format. In the present work, we took advantage of the fluorescent properties of UDP-MurNAc-N ε -dansylpentapeptide and dansylated lipid I (6, 29) to set up a high-throughput screen (HTS) assay for MraY, which was validated with a set of natural and synthetic ...

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Section: Validation Of the Hts With Mray Inhibitorsmentioning

confidence: 99%

Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Stachyra¹,

Dini²,

Ferrari³

et al. 2004

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Those that are amenable to high-throughput screening are either not true enzyme assays (30) or cannot distinguish between inhibitors of the transglycosylase or transpeptidase and those of other enzymes that are assayed along with them (4,5,8,17,24).…”

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confidence: 99%

High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b

Chandrakala

Shandil

Mehra

et al. 2004

Antimicrob Agents Chemother

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Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC 50 s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other ␤-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC 50 s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

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“…Gene knockout experiments have demonstrated conclusively that the mraY gene is essential to cell viability in both E. coli and the gram-positive pathogen Streptococcus pneumoniae and is therefore an attractive target for antimicrobial drug development (6,11,14,48). Several nucleoside natural product antibiotics that contain the uridine moiety of the natural substrate of translocase 1 have been shown to be potent inhibitors of this enzyme, namely, the mureidomycins, the liposidomycins, tunicamycin, and, most recently, the muramycins (12,13,28,29,32,37).…”

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Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

et al. 2004

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Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His 6 ) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His 6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.Enzymes involved in the assembly of the peptidoglycan layer of bacterial cell walls represent important targets for antibacterial chemotherapy (15, 49). The study of this class of enzymes and the search for selective inhibitors are likely to lead to the development of new chemotherapeutic agents, which are urgently needed to combat antimicrobial drug resistance, the threat of which has recently been highlighted by the acquisition of resistance by methicillin-resistant Staphylococcus aureus to vancomycin (43).Peptidoglycan consists of a ␤-1,4-linked N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide (GlcNAc-MurNAc-pentapeptide) polymer, assembled from cytoplasmic precursors UDP-MurNAc-L-Ala-␥-D-Glu-X-D-Ala-D-Ala (UDPMurNAc-pentapeptide; X, L-Lys or meso-diaminopimelic acid [meso-DAP]) and 49

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A Multitarget Assay for Inhibitors of Membrane-Associated Steps of Peptidoglycan Biosynthesis

Cited by 22 publications

References 19 publications

Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

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