Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN Design of a potent endozepine antagonist Je ´ ro ˆ me Leprince 1 , Hassan Oulyadi 2 , David Vaudry 1 , Olfa Masmoudi 1 , Pierrick Gandolfo 1 , Christine Patte 1 , Jean Costentin 3 , Jean-Luc Fauche ` re 4 , Daniel Davoust 2 , Hubert Vaudry 1 and Marie-Christine Tonon 1 1 Institut Fe ´de ´ratif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Laboratoire de Neuroendocrinologie Cellulaire et Mole ´culaire, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 413, CNRS, Universite ´ de Rouen, Mont-Saint-Aignan, France; 2 IFRMP 23, Laboratoire de Re ´sonance Magne ´tique Nucle ´aire, Institut de Recherche en Chimie Organique Fine, CNRS Unite ´ Mixte de Recherches 6014, Universite ´ de Rouen, Mont-Saint-Aignan, France; 3 IFRMP 23, Laboratoire de Neuropsychopharmacologie, CNRS Unite ´ Mixte de Recherches 6036, Universite ´ de Rouen, Rouen, France; 4 Institut de Recherches SERVIER, Suresnes, France The octadecaneuropeptide (ODN; QATVGDVNTDRPG LLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca 21 ] i ) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [D-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [D-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo 128 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca 21 ] i in cultured rat astrocytes. Cyclo 128 OP also mimicked the effect of both OP and ODN on polyphos- phoinositide turnover. Conversely, the cyclo 128 [D-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca 21 ] i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimen- sional 1 H-NMR and molecular dynamics. Cyclo 128 OP exhibited a single conformation characterized by a g turn comprising residues Pro2 – Leu4 and a type III b turn encompassing residues Leu5–Lys8. Cyclo 128 [D-Leu 5 ]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg– Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs. Keywords: solid-phase peptide synthesis; cyclic peptides; structure-activity relationship; astrocytes; cytosolic calcium concentration. Diazepam-binding inhibitor (DBI) is an 86-amino-acid polypeptide that has been originally isolated from rat brain extracts as an endogenous ligand of benzodiazepine (BZ) receptors [1]. Proteolytic cleavage of DBI generates several biologically active peptides including the triakontatetra- neuropeptide TTN (DBI 17250 ) [2] and the octadecaneuro- peptide ODN (DBI 33250 ) [3] which are collectively designated by the term endozepines [4]. Intracerebroven- tricular injection of endozepines provokes anxiogenic effects [5], induces proconflict behavior [1,6], reverses the anti- conflict action of diazepam [1] and inhibits food intake [7]. The mechanism of action of endozepines is not fully understood. It has been initially proposed that these peptides act as inverse agonists of central-type benzodiazepine receptors [6] thus inhibiting the activity of the GABA A - chloride channel complex [8]. Subsequently, endozepines were found to interact with peripheral-type BZ receptors and to stimulate cholesterol transport into mitochondria [9]. More recently, it has been shown that, in rat astrocytes, ODN activates a metabotropic receptor positively coupled to phospholipase C, leading to an increase in cytosolic calcium concentration [10,11]. Structure-activity relation- ship studies have shown that the C-terminal octapeptide of ODN (OP; ODN 11218 ) is the minimum sequence retaining full calcium-mobilizing activity [12]. The Ala-scan of OP has revealed that replacement of the Leu6 residue suppresses the activity of the peptide. It has also been found that the [D-Leu5]OP analog exhibits a weak antagonistic activity [12]. Correspondence to H. Vaudry, European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 413, Unite ´ Associe ´e au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. Fax: 1 33 235 14 6946, Tel.: 1 33 235 14 6624, E-mail: hubert.vaudry@univ-rouen.fr Note: all optically active amino acids are of the L configuration unless otherwise noted. (Received 17 April 2001, revised 30 July 2001, accepted 21 September 2001) Abbreviations: ODN, octadecaneuropeptide; OP, octapeptide; DBI, diazepam-binding inhibitor; BZ, benzodiazepine; TTN, triakontatetraneuropeptide; HMP, 4-hydroxymethyl-phenoxymethyl- copolystyrene-1%-divinylbenzene resin; HBTU, O-benzotriazol-1-yl- N,N,N 0 ,N 0 -tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; PEG-PS, poly(ethylene glycol)–polystyrene resin; NMP, N-methylpyrrolidin- 2-one; DMF, N,N-dimethylformamide; [Ph 3 P] 4 Pd, tetrakis(triphenylphosphine)palladium(0); NMM, N-methylmorpholine; DMEM, Dulbecco’s modified Eagle’s medium; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; IP, inositol phosphate; PIP, polyphosphoinositide. Eur. J. Biochem. 268, 6045–6057 (2001) q FEBS 2001