Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries: J.Antimicrob.Chemother.

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 moleculesNRC publication: Ye

Virtual screening for potential inhibitors of homology modeled Leptospira interrogans MurD ligase

The life-threatening infections caused by Leptospira serovars remain a global challenge since long time. Prevention of infection by controlling environmental factors being difficult to practice in developing countries, there is a need for designing potent anti-leptospirosis drugs. ATP-dependent MurD involved in biosynthesis of peptidoglycan was identified as common drug target among pathogenic Leptospira serovars through subtractive genomic approach. Peptidoglycan biosynthesis pathway being unique to bacteria and absent in host represents promising target for antimicrobial drug discovery. Thus, MurD 3D models were generated using crystal structures of 1EEH and 2JFF as templates in Modeller9v7. Structural refinement and energy minimization of the model was carried out in Maestro 9.0 applying OPLS-AA 2001 force field and was evaluated through Procheck, ProSA, PROQ, and Profile 3D. The active site residues were confirmed from the models in complex with substrate and inhibitor. Four published MurD inhibitors (two phosphinics, one sulfonamide, and one benzene 1,3-dicarbixylic acid derivative) were queried against more than one million entries of Ligand.Info Meta-Database to generate in-house library of 1,496 MurD inhibitor analogs. Our approach of virtual screening of the best-ranked compounds with pharmacokinetics property prediction has provided 17 novel MurD inhibitors for developing anti-leptospirosis drug targeting peptidoglycan biosynthesis pathway

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF–inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target