Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries

Ahmed El Zoeiby1, François Sanschagrin1, André Darveau2, Jean-Robert Brisson3 and Roger C. Levesque1,*

1 Faculté de Médecine and 2 Département de Biochimie, Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Pavillon Charles-Eugène-Marchand, Faculté des Sciences et Génie, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4; 3 Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6

Received 14 June 2002; returned 13 August 2002; revised 23 September 2002; accepted 24 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 IC50s 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.

Keywords: MurC, peptide inhibitors, phage display


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The alarming increase and spread among pathogens of bacterial resistance to all clinically useful antibacterial agents has been one of the most serious public health problems of the past decade. This critical situation has made it highly imperative to design novel antibacterial agents. Future drugs must be directed towards targets other than those tackled by antibiotics in clinical use, since bacteria have, owing to their amazing adaptive flexibility, succeeded in developing various resistance mechanisms and evading the drug action.

One of the most attractive sites at which to look for novel antibacterial targets is the bacterial peptidoglycan biosynthetic machinery. Peptidoglycan is a heteropolymer composed of alternating units of UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid, cross-linked via short peptide chains.1 This molecule is the cell wall component conferring upon the bacterial cell its shape and rigidity and enabling it to survive in environments of low osmotic pressures. Bacterial cells with defective peptidoglycan structure eventually burst and die. Furthermore, peptidoglycan is a unique bacterial structure, absent from eukaryotic cells. This is why peptidoglycan is one of the most exploited bacterial structures and constitutes the target of many antibacterial compounds, such as ß-lactams, vancomycin and bacitracin, which interfere with the later steps of polymerization of peptidoglycan. However, the early steps of biosynthesis of the peptidoglycan cytoplasmic precursor, N-acetylglucosamine-N-acetylmuramyl pentapeptide, which are also vital for bacterial survival, are not sufficiently exploited as antibacterial targets. There are no antibacterials directed towards these steps, except for fosfomycin, an antibiotic inhibiting MurA,2 the first committed enzyme in the pathway, catalysing the addition of an enolpyruvate residue to UDP-N-acetylglucosamine (Figure 1). Subsequent steps are the reduction of the enolpyruvate moiety to D-lactate by MurB and the sequential addition of the pentapeptide side chain on the D-lactyl group by a series of ATP-dependent amino acid ligases (MurC, MurD, MurE and MurF).3 All of these cytoplasmic essential enzymes remain to be fully characterized, with the aim of developing antibacterials with novel modes of action.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 1. Sequential reactions catalysed by MurA, -B and -C. Abbreviations: MurA, UDP-N-acetylglucosamine enolpyruvyl transferase; MurB, UDP-N-acetylenolpyruvoyl glucosamine reductase; MurC, UDP-N-acetylmuramate:L-alanine ligase; UDP-Glc-NAc, UDP-N-acetylglucosamine; EP-UDP-Glc-NAc, UDP-N-acetylglucosamine enolpyruvate; PEP, phosphoenolpyruvate; UDP-Mur-NAc, UDP-N-acetylmuramic acid; UDP-Mur-NAc-L-Ala, UDP-N-acetylmuramic acid-L-alanine.

 
Phage display is a powerful technology for the selection of short peptide ligands, among a large pool of random peptide permutations, with high binding affinities to proteins of interest.4,5 This approach has proved useful for the detection of various enzyme inhibitors,6 and hence has become a valuable tool for antibacterial drug discovery.

In this paper, we describe the use of two different phage-display libraries for the identification of inhibitory peptides of the bacterial essential enzyme UDP-N-acetylmuramate: L-alanine ligase (MurC). We also describe the enzymic synthesis of the MurC substrate using the MurA and MurB enzymes in combination. Three rounds of the phage-display screening process led to obtaining a clear consensus peptide sequence, and the accordingly synthesized peptides showed inhibition of MurC enzymic activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, DNA manipulations, reagents and techniques

All reagents were purchased from Sigma Aldrich (Oakville, Ontario, Canada) unless otherwise indicated. Buffer D used to maintain enzymes was 20 mM potassium phosphate, 1 mM 2-mercaptoethanol, 0.1 mM MgCl2, 15% (v/v) glycerol (pH 7.0).7 Restriction endonuclease and T4 ligase were obtained from New England Biolabs (Beverly, MA, USA). Agarose gel electrophoresis and plasmid DNA preparations were performed according to published procedures.8 Recombinant plasmids containing Pseudomonas aeruginosa mur genes were propagated in Escherichia coli NovaBlue, endA1 hsdR17(rK12 mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac [F' proA+B+ laclqZ{Delta} M15::Tn10] (Novagen, Madison, WI, USA) prior to protein synthesis in E. coli BL21, F ompT hsdSB(rB mB) gal dcm (DE3) (Novagen). TBS was 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, autoclaved. TBST was TBS + 0.1% (v/v) Tween-20. PEG/NaCl was 20% (w/v) polyethylene glycol-8000, 2.5 M NaCl, autoclaved. Agarose Top (per litre) was: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 1 g MgCl2·6H2O, 7 g agarose, autoclaved. IPTG/Xgal was 1.25 g isopropyl-ß-D-thiogalactoside (Boehringer Mannheim, GmbH, Germany) and 1 g 5-bromo-4-chloro-3-indolyl-ß-D-galactoside dissolved in 25 mL of dimethyl formamide. LB/IPTG/Xgal plates were prepared by autoclaving 1 L of LB broth (EM Science, Gibbstown, NJ, USA) + 15 g agar, the medium was cooled to <70°C, 1 mL of IPTG/Xgal was added, then plates were poured and stored at 4°C in the dark. The host strain used for production, amplification and determination of the titre of bacteriophage was E. coli ER2537, F' laclq{Delta}(lacZ)M15 proA+B+/fhuA2 supE thi {Delta}(lac-proAB) {Delta}(hsdMS-mcrB)5 (rK mK McrBC) (New England Biolabs).

Cloning and overexpression of P. aeruginosa murA, -B and -C

PCR cloning was used to obtain MurA, -B and -C proteins with a His-tag at their C termini. Forward and reverse primers7,9 were designed to contain appropriate restriction sites. Purified PCR products were digested with the restriction enzymes included in upper and lower primers and were cloned into the corresponding sites of the expression vector pET30a (Novagen) under the control of the bacteriophage T7 promoter. The recombinant plasmids pMON3005, pMON3006 and pMON30047,9 were introduced into the E. coli host strain BL21({lambda}DE3) (Novagen) by electroporation for expression of MurA, -B and -C, respectively, with His-tags at their C termini. Recombinant proteins were purified to homogeneity on an affinity nickel column, as described previously.7

Affinity selection of phage-displayed peptides

Phage libraries used were PH.D.-12 and PH.D.-C7C (New England Biolabs). Peptides were fused to a minor coat protein pIII of M13 phage via a flexible linker, Gly–Gly–Gly–Ser. PH.D.-12 and PH.D.-C7C libraries consist of ~2.7 x 109 and ~3.7 x 109 random peptide sequences, respectively. The target protein (purified MurC) was immobilized directly on the plastic surface of a 96-well plate. Two 150 µL aliquots of a 100 µg/mL MurC solution in 0.1 M NaHCO3 (pH 8.6) were incubated in a 96-well plate overnight at 4°C. Protein solution was discarded. The two coated wells were incubated with 200 µL of blocking buffer (New England Biolabs) for 1 h at 4°C then washed six times with TBST. Ten microlitres of each original library was diluted with 100 µL TBST and incubated in the wells for 1 h at room temperature. Non-binding phage were discarded and the plate was washed 10 times with TBST. Bound phage were eluted with 100 µL of 0.2 M glycine–HCl (pH 2.2), 1 mg/mL BSA. The plate was rocked gently for 5 min at room temperature. Each eluate was neutralized with 15 µL of 1 M Tris–HCl (pH 9.1) and 2 µL of each was titrated as described below.

The rest of each eluate was amplified by adding it to 20 mL of E. coli ER2537 culture at early-log phase (OD600 ~0.3) and incubated at 37°C for 4.5 h with shaking at 250 rpm. Each culture was centrifuged at 14 500g for 10 min at 4°C using a Sorvall SA-600 rotor. The supernatant was transferred to a fresh tube and re-centrifuged. The upper 80% of the supernatant (~16 mL) was pipetted to a fresh tube and 1/6 volume of PEG/NaCl was added. Phage were allowed to precipitate at 4°C overnight. Precipitated phage were centrifuged at 14 500g for 15 min at 4°C, the supernatant was decanted and re-centrifuged for 2 min, then residual supernatant was removed with a pipette. The pellet was suspended in 1 mL of TBS, the suspension transferred to a microtube and centrifuged for 5 min at 4°C to pellet residual cells. The supernatant was transferred to a fresh microtube, re-precipitated with 1/6 volume of PEG/NaCl (~167 µL), incubated on ice for 60 min then centrifuged for 10 min at 4°C. The supernatant was discarded, phage were re-centrifuged for 1 min and residual supernatant was removed with a micropipette. The pellet was suspended in 200 µL of TBS, 0.02% NaN3, microcentrifuged for 1 min and the supernatant (the amplified eluate) was transferred to a fresh tube. Ten microlitres of each amplified eluate was titrated as described below and titres were used to calculate phage input for the second round of biopanning (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1.  Phage titres after rounds 1, 2 and 3 of biopanning with the 12-mer and C7C-mer libraries
 
The second round was carried out using each of the first-round amplified eluates as input phage and raising the Tween concentration for the washing steps to 0.5% (v/v). Ten microlitres of each amplified eluate was diluted with 100 µL of TBST, incubated in the wells for 1 h at room temperature. Bound phage were eluted with 100 µL of 0.2 M glycine–HCl (pH 2.2), 1 mg/mL BSA, rocking the plate for 5 min at room temperature. Each eluate was neutralized with 15 µL of 1 M Tris–HCl (pH 9.1) and 2 µL of each was titrated as described below. The rest of each eluate was amplified as described above. Ten microlitres of amplified eluates was titrated and titres were used to calculate phage input for the third round of biopanning (Table 1).

The third round of biopanning was carried out using each of the second-round amplified eluates as input phage, using 0.5% (v/v) Tween in the washing steps. Eight 150 µL aliquots of a 100 µg/mL MurC solution in 0.1 M NaHCO3 (pH 8.6) were used for coating a 96-well plate overnight at 4°C. Four 10 µL aliquots of each amplified eluate were diluted with 100 µL of TBST each, incubated in the wells for 10 min (instead of 1 h) at room temperature. Bound phage from each of the two libraries were eluted using four different methods: 100 µL of 0.2 M glycine–HCl (pH 2.2), 1 mg/mL BSA for 5 min, neutralized with 15 µL of 1 M Tris–HCl (pH 9.1); 100 µL of 1 mM UDP-N-acetylmuramic acid in TBS for 30 min; 100 µL of 1 mM ATP in TBS for 30 min; and 100 µL of 1 mM L-alanine in TBS for 30 min. Two microlitres of the third-round unamplified eluates were titrated as described below.

Phage titration

Ten-fold serial dilutions of phage were prepared in LB broth. For unamplified eluates, 101–104 dilutions were used, and for amplified eluates, 108–1011 dilutions were used. Ten microlitres of each dilution was added to 200 µL of E. coli ER2537 culture at mid-log phase (OD600 ~0.5), quickly vortexed and incubated at room temperature for 5 min. Infected cultures were added one at a time to culture tubes, each containing 3 mL of melted Agarose Top at 45°C, quickly vortexed and immediately poured on to pre-warmed LB/IPTG/Xgal plates. Plates were allowed to cool, inverted and incubated at 37°C overnight.

DNA preparation and sequencing

Eighty culture tubes containing 10 mL of LB broth were inoculated with 100 µL of E. coli ER2537 overnight culture. For each of the two libraries, eluted with each of the four elution methods, 10 plaques from the third-round titration plates were picked randomly with a sterile pipette tip and used to infect the E. coli ER2537 cultures. Culture tubes were incubated at 37°C for 4.5 h with shaking at 250 rpm. Phage DNA was prepared using the Qiaprep spin M13 kit (Qiagen, Chatsworth, CA, USA), then sequenced using the –96 gIII sequencing primer (New England Biolabs). Sequencing was carried out using the dye terminator cycle sequencing technique with AmpliTaq DNA polymerase (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA), and DNA fragments were separated with an ABI Stretch 373 system (Perkin Elmer, Applied Biosystems Division). DNA sequence analyses were carried out on an SGI Origin 2000 computer with Genetics Computer Group software (GCG, version 10.2) of Accelrys.

Synthesis of peptides

Peptides were synthesized on an ABI 433A Peptide Synthesizer using FastMoc chemistry. The activation was carried out with HBTU/DIEA. The N-terminal amino group was protected by Fmoc and side-chain functional groups were protected by t-Bu (Asp, Glu, Ser, Thr and Tyr), Boc (Lys and Trp), Trt (Asn and Gln) and Pmc (Arg). The peptides were cleaved with TFA/thioanisole/water/EDT (90:5:2.5:2.5) for 2–3 h at room temperature, precipitated with ether prior to lyophilization. The peptides were purified on a Vydak 22 x 250 mm C18 reverse-phase HPLC column using a 0.1% TFA/acetonitrile gradient at 10 mL/min.

Synthesis of UDP-N-acetylmuramic acid

A reaction mixture containing TAPS (50 mM, pH 8.0), UDP-N-acetyl glucosamine (2.1 mM), phosphoenolpyruvate (3.9 mM), ß-NADPH (2 mM) and dithiothreitol (DTT; 5 mM) in a final volume of 5 mL was prepared in a 50 mL plastic tube and flushed with nitrogen for 30 min. Fifty microlitres of purified MurA (0.6 mg/mL) and 12.5 µL of purified MurB (2 mg/mL) were added to initiate the reaction. The reaction was flushed with nitrogen for another 10 min and incubated for 5 h at 37°C.10 A control reaction was performed by adding all the substrates and omitting the enzymes.

Purification of UDP-N-acetylmuramic acid

The reaction mixture was filtered through an Amicon YM 10 membrane (Millipore Corporation, Bedford, MA, USA) to remove the enzymes. Separation was performed by FPLC using ÄKTAexplorer (Amersham Pharmacia Biotech, Baie d’Urfé, QC, Canada). The filtered reaction was applied to a 10 µm particle size MonoQ HR 10/10 anion-exchange column (Amersham Pharmacia Biotech) at room temperature; flow rate was 4 mL/min. UV detection was set at 254 nm. Lines A and B were 0.02 M NH4OAc (pH 5.0) and 1.0 M NH4OAc (pH 5.0), respectively; injection volume was 4 mL. The following elution steps were used: an isocratic feed of A for 5 min, a first linear gradient from 0 to 20% B for 20 min, a second linear gradient from 20 to 65% B for 15 min, and finally a third linear gradient from 65 to 100% B for 5 min. The control was incubated for 5 h at 37°C then analysed by FPLC. Fractions of the peak corresponding to UDP-N-acetylmuramic acid were pooled, concentrated and lyophilized. The lyophilized flocculent powder was dissolved in water then re-lyophilized three times to remove the ammonium acetate buffer. Part of the lyophilized flocculent powder was dissolved in D2O and lyophilized three times before nuclear magnetic resonance (NMR) analysis.

Mass spectrometry

The reaction product was analysed on a quadrupole mass spectrometer detector (Agilent Technologies, Mississauga, Ontario, Canada; model HP 1100 LC-MSD). The mobile phase was 1.0 M NH4OAc (pH 5.0). Flow rate was 0.4 mL/min. Injection volume was 30 µL of the FPLC pooled fractions corresponding to UDP-N-acetylmuramic acid. The electrospray ionization (ESI) was set at Vcap = 4500 V, nebulizing gas pressure = 35 psi, drying gas flow rate = 13 L/min, drying gas temperature = 350°C, with the quadrupole scanning from 150 to 850 m/z every 1.03 s with a step size of 0.15 amu. The system control and data evaluation were carried out on an HP ChemStation for LC/MS.

NMR analysis

NMR experiments were acquired on a Varian Inova 600 and 400 MHz spectrometer using standard software. All measurements were made at 25°C on a 2 mg sample dissolved in 0.6 mL of D2O (pH 6.2). The methyl resonance of acetone was used as an internal reference at 2.225 ppm for 1H spectra and 31.07 ppm for 13C spectra. 31P chemical shifts are relative to the resonance of 85% H3PO4 set at 0 ppm. Standard homo- and heteronuclear correlated two-dimensional techniques were used for general assignments: COSY, TOCSY, NOESY, HSQC, and HMBC and 31P HMQC. Selective one-dimensional TOCSY experiments were performed for the determination of accurate coupling constants. As there was strong coupling for the ribose unit, spin simulation was performed with standard Varian software to obtain accurate chemical shifts and coupling constants.

Enzymic assays

The enzymic activity of MurC was measured by detecting production of ADP in an enzyme reaction mixture, as described previously for E. coli and P. aeruginosa MurC assays.9,11 The assay contained, in a final volume of 1 mL, 100 mM Tris–HCl (pH 8.0), 70 µg of pyruvate kinase (Boehringer Mannheim), 70 µg of lactate dehydrogenase (Boehringer Mannheim), 1 mM DTT, 0.38 mM NADH (Boehringer Mannheim), 2 mM phosphoenolpyruvate (Boehringer Mannheim), 20 mM MgCl2 (Fisher Scientific, Fair Lawn, NJ, USA), 25 mM (NH4)2SO4 (Fisher Scientific), 2.5 mM ß-mercaptoethanol, 5 mM ATP, 1 mM L-alanine, 1 mM UDP-N-acetylmuramate and 30 µg of MurC. The mixture without ATP and MurC was incubated at 37°C for 10 min before ATP was added, then incubated for another minute. The reaction was started by the addition of MurC. The decrease in NADH absorbance at 340 nm (extinction coefficient = 6220/cm/M) was monitored on a Cary 1 spectrophotometer.

Lyophilized 12-mer and C7C-mer synthesized peptides were accurately weighed in microtubes, then dissolved in a precise volume of water. Their molar concentrations were calculated according to their molecular weights. The inhibitory capacities of the 12-mer and C7C-mer synthesized peptides were determined by pre-incubating MurC enzyme with various concentrations of peptide solutions for 30 min at room temperature. The reaction rates were calculated from the linear portion of the progress curve after addition of MurC. The 50% inhibitory concentrations (IC50s) for the two peptides were obtained by plotting the percentage residual activity as a function of increasing peptide concentration.

Assay of the inhibition of pyruvate kinase and lactate dehydrogenase by the peptides

A reaction mixture was prepared, containing, in a final volume of 1 mL, 100 mM Tris–HCl (pH 8.0), 7 µg of pyruvate kinase (Boehringer Mannheim), 7 µg of lactate dehydrogenase (Boehringer Mannheim), 1 mM DTT, 0.38 mM NADH (Boehringer Mannheim), 2 mM phosphoenolpyruvate (Boehringer Mannheim), 20 mM MgCl2 (Fisher Scientific), 25 mM (NH4)2SO4 (Fisher Scientific), 2.5 mM ß-mercaptoethanol and 500 µM ADP. The reaction mixture without ADP was incubated at 37°C for 10 min. The reaction was started by the addition of ADP and the decrease in NADH absorbance at 340 nm (extinction coefficient = 6220/cm/M) was monitored immediately.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Purification of P. aeruginosa MurA, -B and -C proteins

P. aeruginosa murA, -B and -C genes were cloned in the pET 30a expression vector, adding six His-tag fusions to their C termini. Overexpressed proteins were purified in milligram quantities to homogeneity via their His-tags by a single chromatographic step on an affinity nickel column. Protein identities were confirmed by N-terminal sequencing of the first 15 amino acid residues.7

Phage display with purified MurC

Microtitre plates were coated with purified MurC. Two phage-display libraries expressing randomized linear 12-mer peptides, and randomized 7-mer peptides flanked by a pair of cysteine residues, were incubated with the immobilized MurC protein to allow the binding of the phage particles displaying peptides with affinity for MurC. Three rounds of biopanning were performed with the two phage libraries. The Tween 20 concentration in the washing steps of the second and third rounds was increased in order to select for tightly binding phage. Elution of bound phage after the first and second rounds was carried out by non-specific disruption of binding interaction using glycine–HCl acidic buffer (Figure 2). The third-round bound phage were eluted by non-specific disruption and by competitive elution with each of the MurC substrates: UDP-N-acetylmuramic acid, ATP and L-alanine (Figure 2). Elution with a known ligand of the target protein will compete the bound phage away from the immobilized protein on the plate, specifically disrupting binding interactions of MurC with its different substrates. This competition would yield phage-displaying peptides with probable binding affinities to each of the MurC substrate-binding sites. The three consecutive rounds of biopanning permitted the enrichment of the phage population with phage having binding capacity to MurC. This is illustrated by the successive increase of the elution percentage from round 1 to round 3 in both libraries (Table 1). In the case of the 12-mer library, elution percentage was 5 x 10–4 and 1 x 10–3 after the first and second round, and 1 x 10–1, 5 x 10–2, 2.5 x 10–2 and 7.5 x 10–3 after the third round. For the C7C-mer library, elution percentage was 5 x 10–5 and 5 x 10–4 after the first and second rounds, and 6.7 x 10–2, 3.3 x 10–2, 2.7 x 10–2 and 3.3 x 10–2 after the third round. This enrichment with MurC-specific phage allowed us to obtain a clear consensus peptide sequence for each library upon sequencing of the DNA of 10 randomly selected phage from each group after the third round of biopanning. The two peptides chosen to represent the obtained consensus sequences with the two libraries were N-Asp– His–Arg–Asn–Pro–Asn–Tyr–Ser–Trp–Leu–Lys–Ser-COOH and N-Cys–Gln–Asp–Thr–Pro–Tyr–Arg–Asn–Cys-COOH (Figure 3). Peptides were synthesized on an ABI 433A Peptide Synthesizer and HPLC purified in order to be tested for their inhibitory effect on MurC.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 2. General scheme for the three rounds of biopanning using the PH.D.-12 and PH.D.-C7C libraries against MurC immobilized on a microtitre plate. The biopanning was carried out using purified MurC, the third round being performed with non-specific elution together with competitive elution with each of the MurC substrates.

 


View larger version (29K):
[in this window]
[in a new window]
 
Figure 3. Consensus peptide sequences of the Ph.D.-12 and Ph.D.-C7C libraries obtained by sequencing of phage eluted after the third round of biopanning. Acidic amino acids (D, E) are in green, polar amino acids (Q, N) are in light green, basic amino acids (K, R, H) are in blue, hydrophobic amino acids (I, L, M, V) are in rose, hydrophobic aromatic amino acids (F, Y, W) are in red, small amino acids (A, S, C, T) are in pink, G (tiny amino acid) is in orange and P (leading to turn formation) is in black (classified according to Venn diagram for the relationships between amino acids).

 
Enzymic synthesis and purification of UDP-N-acetylmuramic acid

Purified MurA and -B proteins were used in combination to synthesize the MurC substrate UDP-N-acetylmuramic acid, starting from the MurA substrate UDP-N-acetylglucosamine. Both control and enzymic reaction were analysed by FPLC, and collected fractions of the peaks shown in Figure 4 (a and b) were analysed by mass spectrometry. The peak eluted at 18 min corresponded to UDP-N-acetylglucosamine, the peak at 22 min corresponded to the oxidized form of ß-NADPH, and the two peaks at 36 and 38 min corresponded to the reduced form of ß-NADPH, as revealed by mass spectrometry results (data not shown). The peak eluted at 32 min, which is the only peak present in the enzymic reaction and absent in the control, was analysed by mass spectrometry and yielded a major peak with m/z 276.1 (Figure 5), corresponding to the calculated mass of the N-acetylmuramic acid moiety after the loss of the UDP moiety and the gain of one proton: C11H18N1O7.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 4. Preparative enzymic synthesis of UDP-N-acetylmuramic acid using purified MurA and -B. Control (a) and enzymic reaction (b) were analysed by FPLC. The peak fractions at 32 min corresponding to UDP-N-acetylmuramic acid were pooled and lyophilized. UNAG, UDP-N-acetylglucosamine; UNAM, UDP-N-acetylmuramic acid.

 


View larger version (13K):
[in this window]
[in a new window]
 
Figure 5. Mass spectrum of purified UDP-N-acetylmuramic acid eluted at 32 min on FPLC and chemical structure of the cleavage product N-acetylmuramic acid. The major peak m/z 276.1 corresponds to the mass of the ionized N-acetylmuramic acid after the gain of one proton.

 
NMR analysis of UDP-N-acetylmuramic acid

NMR methods, as outlined in several reviews,1215 were used for the structural determination of UDP-N-acetylmuramic acid (Figure 6). One-dimensional selective NMR methods were also used to characterize individual components.15,16 From the COSY and TOCSY experiments, the coupled spin systems could be detected. One-dimensional TOCSY experiments were then used to measure accurate coupling constants. As there was strong coupling, a spin simulation of the spectrum was carried out for the resonances of the ribose unit. HSQC was used to assign CH, CH2 and CH3 carbon resonances (Figures 7 and 8). HMBC was used to locate the CO resonances. Location of phosphate groups was confirmed from a 31P HMQC experiment.16 The NMR data are shown in Table 2. The 13C and 1H chemical shifts are similar to those for chemically and enzymically synthesized UDP-N-acetylmuramic acid.10,17 31P chemical shifts and coupling constants for the UDP moiety are similar to those previously reported.16,18



View larger version (12K):
[in this window]
[in a new window]
 
Figure 6. Structure of UDP-N-acetylmuramic acid and labelling for the residues and atoms.

 


View larger version (10K):
[in this window]
[in a new window]
 
Figure 7. Proton and HSQC spectra at 600 MHz of UDP-N-acetylmuramic acid. The HOD resonance is at 4.780 ppm with respect to internal acetone at 2.225 ppm. A large amount of sodium acetate (35x) was present in the sample as judged by the intense acetate resonance at 1.919 ppm, as well as some minor unknown impurity at 1.995 ppm. The resonances are labelled according to the structure shown in Figure 6. F1 and F2 are the 13C and 1H chemical shifts axes, respectively.

 


View larger version (14K):
[in this window]
[in a new window]
 
Figure 8. Expansion of the proton and HSQC spectra at 600 MHz of UDP-N-acetylmuramic acid. The resonances are labelled according to the structure shown in Figure 6. The x resonances are from glycerol present in the sample. F1 and F2 are the 13C and 1H chemical shifts axes, respectively.

 

View this table:
[in this window]
[in a new window]
 
Table 2.  NMR data for UDP-N-acetylmuramic acid
 
Inhibition of MurC catalytic activity by the 12-mer and C7C-mer peptides

A coupled enzymic assay for MurC was used to assess the inhibitory properties of the two synthesized peptides. This assay depends on the hydrolysis of one molecule of ATP into ADP accompanying the L-alanine addition. The release of ADP was monitored by the pyruvate kinase and lactate dehydrogenase coupled system as a decrease in NADH absorbance at 340 nm. The inhibitory capacity of the two peptides was determined by measuring the initial hydrolysis rate of ATP into ADP, assessed in terms of the rate of NADH oxidation. A molar excess of each peptide was pre-incubated with 30 µg of MurC enzyme. Both peptides were able to inhibit the MurC ligase activity. The efficiency of the inhibition by the peptides was estimated from the rate of NADH oxidation in the presence of different peptide concentrations. The percentage residual activity with various peptide concentrations was obtained by comparing the reaction rate in the presence of each concentration with the reaction rate of the uninhibited MurC assay (Figure 9). The IC50s obtained from the measurements were 1.5 and 0.9 mM for the 12-mer and C7C-mer peptides, respectively.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 9. IC50 determinations for the phage-display-derived peptide inhibitors. The residual enzymic activity was measured as a function of the concentration of the 12-mer peptide (a) and the C7C-mer peptide (b).

 
A control experiment was performed to check the inhibition of pyruvate kinase and/or lactate dehydrogenase by the peptides. This assay was achieved by adding ADP directly to the pyruvate kinase and lactate dehydrogenase coupled system in the presence and absence of the peptides. The peptides were tested at the highest concentration used in the inhibition assays of MurC. The concentration of the two enzymes was decreased by 10-fold to reduce the speed of NADH oxidation and allow a valid estimation of the reaction rate. The reaction rate was not reduced by the addition of any of the two peptides (data not shown), which proves that the peptides do not interfere with the pyruvate kinase/lactate dehydrogenase enzyme system and have a specific inhibitory effect on MurC. Finally, the two peptides were tested on P. aeruginosa ATCC 27853, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 (data not shown). They did not exhibit any antibacterial activity against P. aeruginosa or E. coli but showed a very weak inhibition of S. aureus, which might be due to the difference in permeability barriers between Gram-positive and -negative bacteria.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of novel antibacterial agents with completely different modes of action is essential for the treatment of widespread resistant bacterial infections. In the past few years, there has been a growing interest in the Mur pathway, responsible for the synthesis of bacterial peptidoglycan precursor. Recently, inhibitors have been designed for several Mur enzymes. Novel MurA inhibitors representing three new scaffolds were identified; they appear to bind to the enzyme at or near the site where fosfomycin binds.19 4-Thiazolidinones are a new class of MurB inhibitors that are postulated to be diphosphate mimics.20 Phosphinate inhibitors were designed for MurC,21,22 MurD2326 and MurE.27 These compounds mimic the tetrahedral acyl phosphate intermediates formed in the normal pathway of the reactions catalysed by the MurC through MurF ligases, and hence are tightly bound by the enzymes. Various analogues of the amino acid or the N-acetylmuramic acid substrates were also synthesized and investigated as inhibitors of MurD28,29 and MurE.30 One of the factors still hampering the development and mechanistic study of inhibitors to these key enzymes is the lack of their substrates, except for the MurA substrate UDP-N-acetylglucosamine, the only commercially available substrate of the Mur pathway intermediates. Hence the Mur substrates are tools of great importance for the development of this new generation of antibacterials. In this work, we focused on the MurA, -B and -C reactions of the pathway, illustrated in Figure 1. Starting from MurA substrate, we were able to synthesize and purify UDP-N-acetylmuramic acid, the substrate for MurC. The identity of the purified compound was confirmed by mass spectrometry and by NMR analysis results. UDP-N-acetylmuramic acid was used for the inhibition assays of two peptide inhibitors.

It is strongly suggested that peptides which bind to proteins would bind preferentially to functional sites, either substrate-binding sites (active sites) or allosteric regulatory sites, rather than randomly interacting with the protein surface.6 Consequently, these peptides could exert inhibition of the function of the target protein. This fact also explains why the peptide sequences obtained with competitive elutions were also observed with the glycine-eluted phage (Figure 3). We presume that non-specific elution could also select for peptides interfering with the protein active sites. An interesting prospect will be to verify this hypothesis by synthesizing and testing some of the peptides that were competitively eluted, but not eluted by glycine. The powerful technique of phage display has been used by Hyde-DeRuyscher et al.6 to isolate a series of peptide ligands that bound specifically to a broad range of well-characterized enzymes with diverse structures and functions. Thirteen of 17 tested peptides were found to be specific inhibitors of enzyme function. Ki values for the identified peptides ranged from micromolar to nanomolar values. Kinetic analysis of these peptides did not reveal a general trend in the mechanism of inhibition.6

Peptides have also proved useful as screening tools for the detection of small-molecule inhibitors of enzyme function by a competitive binding assay.6 We used two M13 phage display libraries to identify peptide sequences with high binding affinities, and hence with potential biological interactions with the MurC ligase activity essential for bacterial survival. Sequencing the DNA of the phage eluted after the third round of biopanning revealed at least three predominant amino acid sequences for the PH.D.-12 library, all of which have Asp and His residues at positions 1 and 2, a Pro residue at position 3 or 5, and an aromatic amino acid next to Pro, either adjacent to it or separated by another residue (Figure 3). Amino acid sequences obtained from the PH.D.-C7C library possessed a highly predominant Asp–Thr–Pro–Tyr–Arg central motif (Figure 3). The second residue was Gln, Val, Glu, Ile, Leu or His, in decreasing order of the frequency of occurrence. The eighth residue was Asn, Ala, Leu, Thr, Arg or Pro in decreasing order of the frequency of occurrence. The fact that the amino acid sequences obtained from the PH.D.-C7C library showed a more homogeneous consensus than those obtained from the PH.D.-12 library could be attributed to the different nature of each library, the first one possessing a pair of Cys residues flanking the 7-mer peptides and the latter presenting peptides as a linear stretch. In the absence of reducing agents, cysteines spontaneously form a disulphide bond, resulting in peptides being constrained in a disulphide loop. Competitive elution with different MurC substrates didn’t reveal a characteristic amino acid consensus sequence for each substrate, which strongly suggests that the selected peptides interact either with allosteric sites on MurC or with an exceptionally active substrate-binding site. A FASTA search was carried out with all the deduced amino acid sequences from both libraries, but revealed no relevant homologues in databases, due to the fact that the sequences were too short.

A common feature of the two consensus amino acid sequences chosen for peptide synthesis is that both possessed a central Pro residue, followed by a Tyr residue, adjacent to Pro in the case of the C7C-mer peptide and separated by an Asn residue in the case of the 12-mer peptide. Adjacent to this central Pro–(Asn)–Tyr motif, Arg–Asn was present, adjacent to the N terminus in the case of the 12-mer peptide, and to the C terminus in the case of the C7C-mer peptide. The two peptides synthesized were proved to be inhibitors of MurC L-alanine ligase activity. However, the therapeutic use of peptides is not practical due to their enzymic degradation, low permeability and unsuitability for oral administration. Ideally, a major goal of medicinal chemistry is to discover novel structures that circumvent the multiple export and metabolism mechanisms that exist to control levels of active peptides in vivo.31 Therefore, in order to obtain promising lead compounds, these peptide sequences will constitute the core for the synthesis of libraries of peptidomimetic molecules. Those compounds of non-peptide nature would emulate the three-dimensional structure of the original peptides, but possess much more useful pharmacodynamic properties as leads for new antibacterials, being less subject to degradation and of higher bioavailability.32

The Mur enzymes are conserved among various bacterial species and there are common structural motifs,33,34 therefore a potential inhibitor of P. aeruginosa MurC could possess a broad action against other MurC enzymes from different bacterial classes, which validates the choice of this important bacterial enzyme as a target for the development of new inhibitors.


    Acknowledgements
 
We thank Le Service de séquence de peptides de l’Est du Québec. This work was supported by the Canadian Bacterial Diseases Network via the Canadian Centers of Excellence and by a FCAR team grant to R.C.L.—a scholar of exceptional merit from Le Fonds de Recherche en santé du Québec. A.E.Z. obtained a studentship from Université Laval and from La Fondation Marc Bourgie.


    Footnotes
 
* Corresponding author. Tel: +1-418-656-3070; Fax: +1-418-656-7176; E-mail: rclevesq{at}rsvs.ulaval.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Rogers, H. J., Perkins, H. R. & Ward, J. B. (1980). Biosynthesis of peptidoglycan. In Microbial Cell Walls and Membranes (Rogers, H. J., Ed.), pp. 239–97. Chapman & Hall, London, UK.

2 . Christensen, B. G., Leanza, W. J., Beattie, T. R., Patchett, A. A., Arison, B. H., Ormond, R. E. et al. (1969). Phosphonomycin: structure and synthesis. Science 166, 123–5.[ISI][Medline]

3 . Bugg, T. D. & Walsh, C. T. (1992). Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Natural Product Reports 9, 199–215.[ISI][Medline]

4 . Christensen, D. J., Gottlin, E. B., Benson, R. E. & Hamilton, P. T. (2001). Phage display for target-based antibacterial drug discovery. Drug Discovery Today 6, 721–7.[CrossRef][ISI][Medline]

5 . Sidhu, S. S. (2000). Phage display in pharmaceutical biotechnology. Current Opinion in Biotechnology 11, 610–6.[CrossRef][ISI][Medline]

6 . Hyde-DeRuyscher, R., Paige, L. A., Christensen, D. J., Hyde-DeRuyscher, N., Lim, A., Fredericks, Z. L. et al. (2000). Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. Chemistry and Biology 7, 17–25.[CrossRef][ISI][Medline]

7 . El Zoeiby, A., Sanschagrin, F., Havugimana, P. C., Garnier, A. & Levesque, R. C. (2001). In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa. FEMS Microbiology Letters 201, 229–35.[CrossRef][ISI][Medline]

8 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

9 . El Zoeiby, A., Sanschagrin, F., Lamoureux, J., Darveau, A. & Levesque, R. C. (2000). Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase. FEMS Microbiology Letters 183, 281–8.[CrossRef][ISI][Medline]

10 . Reddy, S. G., Waddell, S. T., Kuo, D. W., Wong, K. K. & Pompliano, D. L. (1999). Preparative enzymatic synthesis and characterization of the cytoplasmic intermediates of murein biosynthesis. Journal of the American Chemical Society 121, 1175–8.[CrossRef][ISI]

11 . Jin, H., Emanuele, J. J., Jr, Fairman, R., Robertson, J. G., Hail, M. E., Ho, H. T. et al. (1996). Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase. Biochemistry 35, 1423–31.[CrossRef][ISI][Medline]

12 . Duus, J., Gotfredsen, C. H. & Bock, K. (2000). Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations. Chemical Reviews 100, 4589–614.[CrossRef][ISI][Medline]

13 . Kogan, G. & Uhrin, D. (2000). Current NMR methods in the structural elucidation of polysaccharides. In New Advances in Analytical Chemistry (Atta-ur-Rahman, E., Ed.). Gordon and Breach Science Publisher, Amsterdam, The Netherlands.

14 . Uhrin, D. (1997). Concatenation of polarisation transfer steps in 1D homonuclear chemical shift correlated methods. Application to oligo- and polysaccharides. In Methods for Structure Elucidation by High-Resolution NMR (Batta, G. & Szantay, K. E. K., Jr, Eds). Elsevier Science, Amsterdam, The Netherlands.

15 . Uhrin, D. & Brisson, J. R. (2000). Structure determination of microbial polysaccharides by high resolution NMR spectroscopy. In NMR in Microbiology: Theory and Applications (Barbotin, J. N. and Portais, J. C., Eds). Horizon Scientific Press, Wymondham, UK.

16 . Koplin, R., Brisson, J. R. & Whitfield, C. (1997). UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene. Journal of Biological Chemistry 272, 4121–8.[Abstract/Free Full Text]

17 . Dini, C., Drochon, N., Ferrari, P. & Aszodi, J. (2000). Multi gram synthesis of UDP-N-acetylmuramic acid. Bioorganic and Medicinal Chemistry Letters 10, 143–5.[CrossRef][Medline]

18 . Lee, C. H. & Sarma, R. H. (1976). Nuclear magnetic resonance studies of the solution conformation of nucleoside diphosphohexoses and their components. Biochemistry 15, 697–704.[ISI][Medline]

19 . Baum, E. Z., Montenegro, D. A., Licata, L., Turchi, I., Webb, G. C., Foleno, B. D. et al. (2001). Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrobial Agents and Chemotherapy 45, 3182–8.[Abstract/Free Full Text]

20 . Andres, C. J., Bronson, J. J., D’Andrea, S. V., Deshpande, M. S., Falk, P. J., Grant-Young, K. A. et al. (2000). 4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB. Bioorganic and Medicinal Chemistry Letters 10, 715–7.[CrossRef][Medline]

21 . Marmor, S., Petersen, C. P., Reck, F., Yang, W., Gao, N. & Fisher, S. L. (2001). Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC. Biochemistry 40, 12207–14.[CrossRef][ISI][Medline]

22 . Reck, F., Marmor, S., Fisher, S. & Wuonola, M. A. (2001). Inhibitors of the bacterial cell wall biosynthesis enzyme MurC. Bioorganic and Medicinal Chemistry Letters 11, 1451–4.[CrossRef][Medline]

23 . Gegnas, L. D., Waddell, S. T., Chabin, R. M., Reddy, S. & Wong, K. K. (1998). Inhibitors of the bacterial cell wall biosynthesis enzyme MurD. Bioorganic and Medicinal Chemistry Letters 8, 1643–8.[CrossRef][Medline]

24 . Gobec, S., Urleb, U., Auger, G. & Blanot, D. (2001). Synthesis and biochemical evaluation of some novel N-acyl phosphono- and phosphinoalanine derivatives as potential inhibitors of the D-glutamic acid-adding enzyme. Pharmazie 56, 295–7.[ISI][Medline]

25 . Tanner, M. E., Vaganay, S., van Heijenoort, J. & Blanot, D. (1996). Phosphinate inhibitors of the D-glutamic acid-adding enzyme of peptidoglycan biosynthesis. Journal of Organic Chemistry 61, 1756–60.[CrossRef][ISI][Medline]

26 . Vaganay, S., Tanner, M. E., van Heijenoort, J. & Blanot, D. (1996). Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli. Microbial Drug Resistance 2, 51–4.[ISI][Medline]

27 . Zeng, B., Wong, K. K., Pompliano, D. L., Reddy, S. & Tanner, M. E. (1998). A phosphinate inhibitor of the meso-diaminopimelic acid-adding enzyme (MurE) of peptidoglycan biosynthesis. Journal of Organic Chemistry 63, 10081–6.[CrossRef][ISI]

28 . Pratviel-Sosa, F., Acher, F., Trigalo, F., Blanot, D., Azerad, R. & van Heijenoort, J. (1994). Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichia coli. FEMS Microbiology Letters 115, 223–8.[CrossRef][ISI][Medline]

29 . Auger, G., van Heijenoort, J. & Blanot, D. (1995). Synthesis of N-acetylmuramic acid derivatives as potential inhibitors of the D-glutamic acid-adding enzyme. J Prakt Chem 337, 351–7.[ISI]

30 . Auger, G., van Heijenoort, J., Vederas, J. C. & Blanot, D. (1996). Effect of analogues of diaminopimelic acid on the meso-diaminopimelate-adding enzyme from Escherichia coli. FEBS Letters 391, 171–4.[CrossRef][ISI][Medline]

31 . Bursavich, M. G. & Rich, D. H. (2002). Designing non-peptide peptidomimetics in the 21st century: inhibitors targeting conformational ensembles. Journal of Medicinal Chemistry 45, 541–58.[CrossRef][ISI][Medline]

32 . Nefzi, A., Dooley, C., Ostresh, J. M. & Houghten, R. A. (1998). Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds. Bioorganic and Medicinal Chemistry Letters 8, 2273–8.[CrossRef][Medline]

33 . Bouhss, A., Mengin-Lecreulx, D., Blanot, D., van Heijenoort, J. & Parquet, C. (1997). Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli. Biochemistry 36, 11556–63.[CrossRef][ISI][Medline]

34 . Eveland, S. S., Pompliano, D. L. & Anderson, M. S. (1997). Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily. Biochemistry 36, 6223–9.[CrossRef][ISI][Medline]