Antibiotic Resistance Peptides: Interaction of Peptides Conferring Macrolide and Ketolide Resistance with Staphylococcus aureus Ribosomes. Conformation of Bound Peptides As Determined by Transferred NOE Experiments ² Laurent Verdier, ‡,§ Josyane Gharbi-Benarous, ‡,§ Gildas Bertho, ‡ Pascale Mauvais, | and Jean-Pierre Girault* ,‡ Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UniVersite ´ Rene ´ Descartes-Paris V, UMR 8601 CNRS, 45 rue des Saint-Pe ` res, 75270 Paris Cedex 06, UniVersite ´ Denis Diderot-Paris VII, UFR Chimie, 2 Place Jussieu, F-75251 Paris Cedex 05, and AVentis, 102 route de Noisy, 93235 RomainVille Cedex, France ReceiVed June 25, 2001 ABSTRACT: Two antibiotic resistance peptides, the E-peptide (MRLFV) and the K-peptide (MRFFV) conferring macrolide and ketolide resistance, respectively, were studied in the complex state with bacterial Staphylococcus aureus ribosomes. Interactions of antibiotic resistance peptides with ribosomes were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), suggesting that the peptide-ribosome interaction was associated with the low-affinity binding level. K-Peptide displayed a significantly better response in TRNOEs NMR experiments, in agreement with a better overall antibiotic activity of ketolides. This difference highlights a mimetic effect displayed by the E- and K-peptides. This study shows that conformation plays an essential role for the affinity binding site and, thus, for the resistance mechanism. Specific conformations were preferred in the bound state; their superimposition exhibited a similar cyclic peptidyl chain, while the side chain region varies. The F4 phenyl moiety in E-peptide has moved out of the turn region compared to its folding in the ketolide resistance peptide. In the K-peptide binding surface, the F4 aromatic chain is maintained by stacking with the guanidyl group of the R2 residue providing a particular hydrophobic and globular fragment, which may be important for the ketolide resistance peptide mode of action. Additionally, T 2 (CPMG) measurements were used to characterize equilibrium binding of antibiotic resistance peptides to bacterial ribosomes. The results bring to the fore E- and K-peptide competition with antibiotics for binding to the ribosomes. Their specific interaction and their competitive effects reveal a novel aspect of interaction of resistance peptides with ribosomes and suggest new insights about their mode of action. The resistance mechanism may imply two steps, a competitive effect of the resistance peptide for the macrolide (or ketolide) binding site followed by a “bottle brush” effect in which the drug and the peptide are driven out their binding site on the ribosome. Macrolides and ketolides are important antibacterial antibiotics. The emergence of bacteria resistant to existing antibacterial agents, including macrolide antibiotics, has accelerated the search for newer and more effective anti- bacterial agents (1). The development of the structure- activity relationships is driving the resurgence of interest in macrolides. The new class of antibiotics called ketolides is endowed with remarkable antibacterial activity against mac- rolide-resistant strains. Telithromycin was selected to be the ketolide candidate with a favorable pharmacodynamic profile for the clinical trials, to be put on the market. This class of macrolides, termed “ketolides”, demonstrated significantly improved activity against MLS (macrolide, lincosamide, and streptogramin B) inducibly resistant organisms (2-5). These agents, such as the ketolide telithromycin, have higher in vitro potencies than macrolides, and in vivo infection model results were quite promising (6). Telithromycin has been shown to be active against a variety of bacteria, including macrolide-resistant bacteria and mycobacteria and also to exhibit good intracellular penetration (1). The mechanism of action of ketolides has been observed to be similar to that of the macrolides by means of binding to the 50S ribosomal subunit (7-9) and the subsequent inhibition of bacterial protein synthesis (10, 11). There appears to be two stages to this binding: a weak interaction which can be detected by NMR spectroscopy (12) and a stronger interaction observed by different methods (7-9). The weak binding of antibiotics to Escherichia coli, erythromycin-sensitive and -resistant strains of Streptococcus pneumoniae, and Staphylococcus aureus ribosomes (13) has been characterized extensively by line broadening and transferred NOESY (TRNOESY) 1 experiments and was only observed with the antibiotics belonging to the MLS B classes which have exhibited good activity (12, 14, 15), the 14- and 16-membered macrolides (12, 16-18), the new class of antibiotics called ketolides ² This work was supported by grants from the Romainville Research Center of Medicinal Chemistry of Hoechst Marion Roussel (Aventis). * To whom correspondence should be addressed. Telephone: 01 42 86 21 80. Fax: 01 42 86 83 87. E-mail: giraultj@ biomedicale.univ-paris5.fr. ‡ Universite ´ Rene ´ Descartes-Paris V. § Universite ´ Denis Diderot-Paris VII. | Aventis. 4218 Biochemistry 2002, 41, 4218-4229 10.1021/bi011319e CCC: $22.00 © 2002 American Chemical Society Published on Web 03/08/2002