Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries
| Autor: | André Darveau, Ahmed El Zoeiby, Jean-Robert Brisson, Roger C. Levesque, François Sanschagrin |
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| Jazyk: | angličtina |
| Rok vydání: | 2003 |
| Předmět: |
Phage display
magnetic synthesis Peptidomimetic specificity Peptide pseudomonas aeruginosa chemistry.chemical_compound Drug Delivery Systems Sequence Analysis Protein libraries Pharmacology (medical) Enzyme Inhibitors Peptide Synthases Peptide sequence Antibacterial agent mass spectrometry chemistry.chemical_classification magnetic-resonance peptide inhibitor Infectious Diseases Biochemistry resonance acid Microbiology (medical) Escherichia Canada Molecular Sequence Data selection Biopanning Biology peptide library target expression Escherichia coli bacterial Amino Acid Sequence structure Peptide library gene substrates Pharmacology molecule ligands DNA sequence Molecular biology pseudomonas ATP enzyme nuclear nuclear magnetic resonance chemistry identification Peptidoglycan biosynthesis protein Bacteriophage M13 |
| Popis: | Objectives The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. Methods We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Results Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. Conclusion The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules. |
| Databáze: | OpenAIRE |
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