Modulation of Erm Methyltransferase Activity by Peptides Derived from Phage Display

Abstract

Combinatorial peptide display on phage M13 protein pIII was used to discover peptide sequences that selectively bind to ErmC methyltransferase from Bacillus subtilis. One peptide, Ac-LSGVIAT-NH 2 , inhibited methylation in vitro with a 50% inhibitory concentration of 20 M. Interestingly, the set of six peptides which inhibited ErmC stimulated ErmSF, a homologous methyltransferase from Streptomyces fradiae. Thus, Ac-LSGVIAT-NH 2 may not act directly at the catalytic center of ErmC, but may modulate its activity by binding at a structurally unrelated, but functionally linked, site.The ErmC N-methyltransferase of Staphylococcus aureus and its close relative ErmCЈ from Bacillus subtilis confer resistance to erythromycin and related macrolide antibiotics. These enzymes act by specifically methylating a single adenine residue in the peptidyl transferase center of bacterial 23S rRNA (for reviews, see references 7 and 8). The same methylation confers coresistance to structurally unrelated lincosamide and streptogramin type B antibiotics. The three groups are collectively known as the macrolide-lincosamide-streptogramin B (MLS) antibiotics, and the form of resistance based on the Erm group of methyltransferases is found in a wide range of pathogens. The three-dimensional structures of the ErmAM and ErmCЈ methyltransferases have recently been solved by nuclear magnetic resonance (3, 9) and X-ray crystallography (1, 5), respectively. These developments bring us closer to rationally devising ligands that will selectively bind to Erm enzymes and possibly reduce the efficiency with which they confer MLS resistance.Macrolide antibiotics have served as a mainstay of antimicrobial therapy for approximately the last half century, especially in instances where the recipient of the antibiotic was allergic to beta-lactam antibiotics. Attempts have been made to discover Erm methyltransferase inhibitors that maintain the effectiveness of macrolide antibiotics in the face of the increasing frequency of resistant isolates (2, 3). The latter work (3) was part of a series of major structural studies of Erm methyltransferases (1, 5, 9).Nonpeptide ligands which displace an inhibitory peptide from its binding site on the enzyme might, themselves, have inhibitory activity. The use of inhibitory peptides might thus serve as a platform for the discovery of nonpeptide inhibitors of Erm enzymes. A proposed way to achieve this would be based on the discovery of test ligands to displace an inhibitory peptide from its association with its cognate Erm target. The displacement of an inhibitory ligand would also be easier to measure than methyltransferase activity.Large-scale screening of inhibitory ligands by a direct assay of methyltransferase catalytic activity is cumbersome since it requires the separation of product (methyl-labeled 23S rRNA) from substrate (unreacted S-adenosyl-L-methionine). We report the use of combinatorial phage display to discover peptides that inhibit ErmCЈ methylase activity and which might serve as displaceable ligands in ...

Identification of Essential Residues in the Erm(B) rRNA Methyltransferase ofClostridium perfringens

“…These residues and motifs are generally well conserved among the Erm proteins and may represent potential targets for the development of inhibitors of this family of enzymes. Note that the application of combinatorial phage display technology has led to the discovery of several short peptides that bind to the ErmC protein and inhibit its activity (8). This type of approach provides an alternative method for the identification of Erm inhibitors.…”

Speculative strategies for new antibacterials: all roads should not lead to Rome

“…However, Clancy et al 156 do not state explicitly that such experiments were done.) Though there is no indication of binding exosites for Erms, 158 Giannattasio and Weisblum 159 reported several peptide inhibitors of ErmC 0 (a soluble form of ErmC produced by B. subtilis), which they proposed acted at sites other than the active site of the enzyme.…”

Overcoming antimicrobial resistance by targeting resistance mechanisms

“…Although targeting efflux mechanisms might make sense for S. pneumoniae, the overall predominance of methyltransferase-mediated resistance to macrolides has led researchers to investigate the development of inhibitors for these enzymes. Erm inhibitors (Clancy et al 1995 ;Hajduk et al 1999), including inhibitor peptides identified by phage display (Giannattasio & Weisblum 2000), have been described, in one instance showing potentiation of azithromycin activity in MLS B -resistant Gram-positive and Gram-negative bacteria (Clancy et al 1995). Recently, S-adenosyl--homocysteine mimics have been reported as weak but promising inhibitors of Erm methyltransferases (Hanessian & Sgarbi 2000).…”