In situ assay for identifying inhibitors of bacterial transglycosylase

Branstrom, Arthur; Midha, Sunita; Goldman, Robert C.

doi:10.1111/j.1574-6968.2000.tb09338.x

Cited by 25 publications

(19 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In reactions with the UDPMurNAc-pentapeptide substrate, the potent transglycosylase inhibitor moenomycin inhibited peptidoglycan polymerization with an IC 50 of 0.02 M, consistent with published values (11). Under these assay conditions, telavancin inhibited transglycosylase activity with a potency (IC 50 ϭ 0.6 M) comparable to that observed for inhibition of peptidoglycan biosynthesis in intact cells ( Fig.…”

Section: Inhibition Of Macromolecular Biosynthesis In Intact Cellssupporting

confidence: 73%

“…The transglycosylase (polymerization) reaction was assayed by the method of Branstrom et al (11) by using membranes prepared from E. coli BAS849/pUG18, UDP-MurNAc-pentapeptide, or UDP-MurNAc-tetrapeptide substrate purified from enterococci by the method of Kohlrausch and Holtje (32) and [ 14 C]UDP-N-acetylglucosamine (1 Ci/ml). Reaction mixtures were incubated for 2 h to allow accumulation of lipid II.…”

Section: Methodsmentioning

confidence: 99%

“…S. aureus ATCC 33591 (MRSA) was purchased from the American Type Culture Collection (Manassas, Va.). Escherichia coli BAS849/ pUG18 was constructed at Theravance by transforming the pUG18 plasmid (11) for murG overexpression into E. coli BAS849, which has an outer membrane permeability defect (47).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

Higgins

Chang

Debabov

et al. 2005

Antimicrob Agents Chemother

409

323

View full text Add to dashboard Cite

The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited latestage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K ؉ . The timing of these changes correlated with rapid, concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.The emergence and spread of bacterial resistance to vancomycin, an important antibiotic used to treat serious infections caused by gram-positive bacteria, has prompted active research to discover new glycopeptides and semisynthetic analogs with improved antimicrobial properties. Vancomycin and related glycopeptide antibiotics inhibit cell wall synthesis in susceptible bacteria by binding with high specificity to peptidoglycan precursors containing the C-terminal D-alanyl-D-alanine (D-Ala-DAla) motif (8). The peptide portion of glycopeptide antibiotics forms a carboxylate binding pocket that imparts, through a combination of five hydrogen bonds plus favorable hydrophobic interactions, strong affinity for the D-Ala-D-Ala-containing terminus of lipid II (8,46,54). Rational approaches toward the design of glycopeptides with improved antimicrobial activities have been described previously (for reviews, see references 35 and 36). One promising approach has been the discovery of lipoglycopeptides, analogs containing hydrophobic groups substituted at the amine position of the disaccharide moiety (20,39,40,45).Telavancin, a semisynthetic derivative of vancomycin possessing a hydrophobic (decylaminoethyl) side chain appended to the vancosamine sugar and a hydrophilic [(phosphonomethyl)aminomethyl] group on the resorcinol-like 4Ј position of amino acid 7 (33), is in late-stage clinical development for the treatment of serious gram-positive infections. Telavancin and other lipoglycopeptides exhibit superior in vitro activity compa...

show abstract

Section: Inhibition Of Macromolecular Biosynthesis In Intact Cellssupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

Higgins

Chang

Debabov

et al. 2005

Antimicrob Agents Chemother

409

323

View full text Add to dashboard Cite

show abstract

“…Polymerization of lipid II into peptidoglycan was monitored in situ (Branstrom et al, 2000a). The first step of this assay allowed synthesis and accumulation of lipid II in a reaction mixture containing Esc.…”

Section: Inhibition Of Transglycosylation and Accumulation Of Lipidmentioning

confidence: 99%

Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase

et al. 2000

View full text Add to dashboard Cite

Moenomycin is a natural product glycolipid that inhibits the growth of a broad spectrum of Gram-positive bacteria. In Escherichia coli, moenomycin inhibits peptidoglycan synthesis at the transglycosylation stage, causes accumulation of cell-wall intermediates, and leads to lysis and cell death. However, unlike Esc. coli, where 5-6 log units of killing are observed, 0-2 log units of killing occurred when Gram-positive bacteria were treated with similar multiples of the MIC. In addition, bulk peptidoglycan synthesis in intact Gram-positive cells was resistant to the effects of moenomycin. In contrast, synthetic disaccharides based on the moenomycin disaccharide core structure were identified that were bactericidal to Gram-positive bacteria, inhibited cell-wall synthesis in intact cells, and were active on both sensitive and vancomycinresistant enterococci. These disaccharide analogues do not inhibit the formation of N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphorylundecaprenol (lipid II), but do inhibit the polymerization of lipid II into peptidoglycan in Esc. coli. In addition, cell growth was required for bactericidal activity. The data indicate that synthetic disaccharide analogues of moenomycin inhibit cell-wall synthesis at the transglycosylation stage, and that their activity on Gram-positive bacteria differs from moenomycin due to differential targeting of the transglycosylation process. Inhibition of the transglycosylation process represents a promising approach to the design of new antibacterial agents active on drug-resistant bacteria.

show abstract

“…An alternative to isolating the substrate is allowing lipid II to accumulate in situ in membranes by using detergent to inhibit transglycosylation and subsequently removing the detergent to assay transglycosylation (5,18). However, the product peptidoglycan is typically separated from lipid II by paper chromatography (5,10).…”

mentioning

confidence: 99%

Screen for Inhibitors of the Coupled Transglycosylase-Transpeptidase of Peptidoglycan Biosynthesis in Escherichia coli

Ramachandran

Chandrakala

Kumar

et al. 2006

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spc r strain. Using this assay, we show that PBP1a is ϳ10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC 50 ) of moenomycin, a TG inhibitor, is ϳ10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC 50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spc r strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.

show abstract

In situ assay for identifying inhibitors of bacterial transglycosylase

Cited by 25 publications

References 12 publications

Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase

Screen for Inhibitors of the Coupled Transglycosylase-Transpeptidase of Peptidoglycan Biosynthesis in Escherichia coli

Contact Info

Product

Resources

About