Structural and pharmacological characteristics of chimeric peptides derived from peptide E and b-endorphin reveal the crucial role of the C-terminal YGGFL and YKKGE motifs in their analgesic properties Eric Condamine a,b, *, Karine Courchay a,b , Jean-Claude Do Rego a,c , Je ´ro ˆme Leprince a,d , Catherine Mayer a,b , Daniel Davoust a,b , Jean Costentin a,c , Hubert Vaudry a,d a European Institute for Peptide Research (IFRMP 23), University of Rouen, France b CNRS UMR 6014, Laboratory of Organic Chemistry and Structural Biology, University of Rouen, 76821 Mont-Saint-Aignan, France c CNRS FRE 2735, Laboratory of Experimental Neuropsychopharmacology, University of Rouen, 22 Boulevard Gambetta, 76183 Rouen, France d INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, Regional Platform for Cell Imaging (PRIMACEN), University of Rouen, 76821 Mont-Saint-Aignan, France 1. Introduction Mammalian opioid peptides are a family of neuropeptides that are processed from at least four large precursor proteins known as proopiomelanocortin, proenkephalin A, prodynorphin and prono- ciceptin/orphanin-FQ [15,25]. Except for those derived from pronociceptin/orphanin-FQ, all endogenous opioid peptides de- rived from the three other precursors share a common N-terminal sequence (Leu-enkephalin YGGFL or Met-enkephalin YGGFM), which interacts with specific membrane receptors termed m, d and k opioid receptors [29,59]. Peptide E is a 25-amino acid opioid peptide derived from proenkephalin A that was originally isolated from the bovine adrenal medulla [30]. Bovine peptide E (BPE) has the particularity to possess a Met-enkephalin and a Leu-enkepha- lin sequence at its N- and C-terminus, respectively (Fig. 1). Since its discovery, several in vivo and in vitro pharmacological studies suggested that BPE is a potent opioid peptide, which produces substantial analgesia after intracerebroventricular injection [16,17,24,30]. Fifteen years after the first description of BPE, its amphibian homologue (FPE) has been isolated from the brain of the European green frog Rana ridibunda [14]. Considering their primary structures (Fig. 1), FPE was expected to be as analgesic as BPE. Indeed, in addition to their very similar primary structures (FBE differs from BPE by only two substitutions, M 15 ! Q 15 and L 25 ! M 25 ), these peptides possess at their N-terminus the same sequence (Met-enkephalin) that is known to be necessary for interaction with the receptor-binding pocket [1,19,26]. Thus, using as a reference b-endorphin, an endogenous opioid peptide known for its potent and sustained analgesic effects [21,40], a structure– activity relationship study has been carried out on BPE and FPE [13]. Paradoxically, the hot-plate and acetic acid-induced writhing tests showed that, comparatively to b-endorphin, peptides E are Peptides 31 (2010) 962–972 ARTICLE INFO Article history: Received 3 December 2009 Received in revised form 27 January 2010 Accepted 27 January 2010 Available online 4 February 2010 Keywords: Bovine peptide E b-Endorphin Opioid peptides Chimeric peptides NMR structure Molecular modeling Structure–activity relationships Analgesic activity ABSTRACT Peptide E (a 25-amino acid peptide derived from proenkephalin A) and b-endorphin (a 31-amino acid peptide derived from proopiomelanocortin) bind with high affinity to opioid receptors and share structural similarities but induce analgesic effects of very different intensity. Indeed, whereas they possess the same N-terminus Met-enkephalin message sequence linked to a helix by a flexible spacer and a C-terminal part in random coil conformation, in contrast with peptide E, b-endorphin produces a profound analgesia. To determine the key structural elements explaining this very divergent opioid activity, we have compared the structural and pharmacological characteristics of several chimeric peptides derived from peptide E and b- endorphin. Structures were obtained under the same experimental conditions using circular dichroism, computational estimation of helical content and/or nuclear magnetic resonance spectroscopy (NMR) and NMR-restrained molecular modeling. The hot-plate and writhing tests were used in mice to evaluate the antinociceptive effects of the peptides. Our results indicate that neither the length nor the physicochemical profile of the spacer plays a fundamental role in analgesia. On the other hand, while the functional importance of the helix cannot be excluded, the last 5 residues in the C-terminal part seem to be crucial for the expression or absence of the analgesic activity of these peptides. These data raise the question of the true function of peptides E in opioidergic systems. ß 2010 Elsevier Inc. All rights reserved. * Corresponding author at: Laboratoire de Re ´ sonance Magne ´ tique Nucle ´ aire, Institut de Biologie Structurale CEA-CNRS-UJF «J.-P. Ebel» (UMR CNRS 5075), 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. Tel.: +33 438 782606; fax: +33 438 785494. E-mail address: eric.condamine@ibs.fr (E. Condamine). Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.01.012