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• W1986701157 abstract "We have screened a synthetic peptide combinatorial library composed of 2 × 107β-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors μ, δ, and κ and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human κ-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [35S]guanosine 5′-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for κ-receptors. To fine-tune the potency and selectivity of these κ-peptides we replaced their turn-forming template by other turn mimetic molecules. This “turn-scan” process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs. We have screened a synthetic peptide combinatorial library composed of 2 × 107β-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors μ, δ, and κ and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human κ-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [35S]guanosine 5′-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for κ-receptors. To fine-tune the potency and selectivity of these κ-peptides we replaced their turn-forming template by other turn mimetic molecules. This “turn-scan” process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs. Ligands for κ-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.Journal of Biological ChemistryVol. 274Issue 46PreviewPages 27518 and 27521, Figs. 3 and 8: The sterochemistry of the BTD scaffold (Fig. 3) and the BTD-III peptide (Fig. 8) as drawn in our paper is wrong; the two amino acid arms should be above the plane of the paper (where the BTD lies) and not below. The correct figures are shown below. Full-Text PDF Open Access human opioid receptor-like 3-amino-N-1-carboxymethyl-2-oxo-5-phenyl-1,4-benzodiazepine 8-aminomethyl-5,6,7,8-tetrahydro-2-naphthoic acid 8amino-5,6,7,8-tetrahydro-2-naphthoic acid (3S,6S,9R)-2-oxo-3amino-7-thia-1-aza-bicyclo[4.3.0]nonane-9-carboxylic acid (±)-4-[(αR *)-α-[(2S *,5R*)-4-alkyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide 3-amino-1-carboxymethyl-2,3,4,5-tetrahydro-1H-[1]-benzazepine-2-one [5R-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzo[b]furan-4acetamide d-cyclohexylalanine l-cyclohexylalanine p-chloro-d-phenylalanine p-chloro-l-phenylalanine [d-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin 5-amino-1,2,4,5,6,7,-tetrahydroazepino[3,2,1-hi]indole-4-one-2-carboxylic acid human δ-opioid receptor human κ-opioid receptor human μ-opioid receptor N,N-diallyl-Tyr-αaminobutyric acid-α-aminobutyric acid-Phe-Leu synthetic peptide combinatorial library turn forming template guanosine 5′-3-O-(thio)triphosphate high performance liquid chromatography Chinese hamster ovary p- nitro-l-phenylalanine Opiates exert their pharmacological actions through three receptor types (1Browstein M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5391-5393Crossref PubMed Scopus (333) Google Scholar, 2Goldstein A. Naidu A. Mol. Pharmacol. 1989; 36: 265-272PubMed Google Scholar), μ, δ, and κ. Their genes have been cloned (see Ref.3Kieffer B.L. Cell. Mol. Neurobiol. 1995; 15: 615-635Crossref PubMed Scopus (355) Google Scholar), and the analysis of their amino acid sequence indicated that they belong to the G-protein-coupled receptor family and display a high degree of homology. The cloning of opioid receptors led to the discovery of an additional member for this receptor family referred to as opioid receptor like (ORL11; see Ref. 4Kieffer B.L. Dickenson A. Besson J.M. The Pharmacology of Pain. Springer-Verlag, Berlin1997: 281-303Google Scholar). Although ORL1 shares high sequence similarities with opioid receptors, it does not bind opioid ligands with high affinity. Opiate drugs, the prototype of which is morphine, are largely used in medicine for the treatment of pain, but their administration is associated with severe side effects, including high abuse potential (see Ref. 5Schug S.A. Zech D. Grond S. Drug Safety. 1992; 7: 200-213Crossref PubMed Scopus (129) Google Scholar). Most nonanalgesic actions of opiates have been associated with the activation of μ-receptors (6Kieffer B.L. Trends Pharmacol. Sci. 1999; 20: 537-544Abstract Full Text Full Text PDF Scopus (425) Google Scholar), and the development of δ- and κ-compounds both as pharmacological tools and therapeutic agents is an extremely active research field. Unlike opioid receptors, there is only a small number of available ligands for ORL1 including the endogenous heptadecapeptide nociceptin/orphanin FQ (7Meunier J.-C. Mollereau C. Toll L. Suaudeau C. Moisand C. Alvinerie P. Butour J.-L. Guillemot J.-C. Ferrara P. Monserrat B. Mazarguil H. Vassart G. Parmentier M. Costentin J. Nature. 1995; 377: 532-535Crossref PubMed Scopus (1818) Google Scholar, 8Reinscheid R.K. Nothacker H.-P. Bourson A. Ardati A. Henningsen R.A. Bunzow J.R. Grandy D.K. Langen H. Monsma F.J. Civelli O. Science. 1995; 270: 792-794Crossref PubMed Scopus (1769) Google Scholar) hexapeptides, recently identified by Dooley et al. (9Dooley C.T. Spaeth C.G. Berzetei-Gurske I.P. Craymer K. Adapa I.D. Brandt S.R. Houghten R.A. Toll L. J. Pharmacol. Exp. Ther. 1997; 283: 735-741PubMed Google Scholar) using combinatorial chemistry techniques, and naloxone benzoylhydrazone (10Noda Y. Mamiya T. Nabeshima T. Nishi M. Higashioka M. Takeshima H. J. Biol. Chem. 1998; 273: 18047-18051Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), previously described as a μ- and κ-ligand (11Pasternak G.W. Clin. Pharmacol. 1993; 16: 1-18Google Scholar). This recently discovered neurotransmitter system is likely to participate in a broad range of physiological and behavioral functions, with possible interactions with the opioid system (see Ref. 12Darland T. Heinricher M.M. Grandy D.K. Trends Neurosci. 1998; 21: 215-221Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). At present our comprehension of thein vivo functions of the ORL1 receptor is severely limited by the lack of ligands, agonists as well as antagonists, with high selectivity and bioavailability. Combinatorial strategies are important new approaches to drug discovery, and synthetic peptide combinatorial libraries (SPCL) have repeatedly shown their usefulness as a source of new drug leads; in particular, when SPCL have been applied to the search for new ligands of the opioid receptors, potent hexapeptides (13Dooley C.T. Chung N.N. Wikes B.C. Schiller P.W. Houghten R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10811-10815Crossref PubMed Scopus (85) Google Scholar, 14Dooley C.T. Chung N.N. Wikes B.C. Schiller P.W. Bidblack J.M. Pasternak G.W. Houghten R.A. Science. 1994; 266: 2019-2022Crossref PubMed Scopus (156) Google Scholar, 15Dooley C.T. Kaplan R.A. Chung N.N. Schiller P.W. Bidlack J.M. Hougthen R.A. Peptide Res. 1995; 8: 124-137PubMed Google Scholar) and tetrapeptides (15Dooley C.T. Kaplan R.A. Chung N.N. Schiller P.W. Bidlack J.M. Hougthen R.A. Peptide Res. 1995; 8: 124-137PubMed Google Scholar) were identified. In the latter work for example, a single library in positional scanning format (PS-SPCL (16Pinilla C. Appel J.R. Blanc P. Houghten R.A. Biotechniques. 1992; 13: 901-905PubMed Google Scholar)) was screened with the μ-, δ- and κ-opioid receptors, and potent, selective ligands were found for each of the three receptors. Peptides, however, generally display unfavorable pharmacological properties, like poor bioavailability, short duration of action, and lack of oral activity (17Farmer P.S. Ariens E.J. Drug Design. Academic Press, Inc., New York1980: 119-143Crossref Google Scholar), prompting the effort to evolve them into peptidomimetics (18Hruby V.J. Al-Obeidi F. Kazmierski W. Biochem. J. 1990; 268: 249-262Crossref PubMed Scopus (552) Google Scholar, 19Olson G.L. Bolin D.R. Bonner M.P. Bos M. Cook C.M. Fry D.C. Graves B.J. Hatada M. Hill D.E. Kahn M. Madison V.S. Rusiecki V.K. Sarabu R. Sepinwall J. Vincent G.P. Voss M.E. J. Med. Chem. 1993; 36: 3039-3049Crossref PubMed Scopus (299) Google Scholar). Moore (20Moore G.J. Trends Pharmacol. Sci. 1994; 2: 253-258Google Scholar) has divided the peptide-to-peptidomimetic transition into three logical steps: (a) identification of the amino acid side chains responsible for activity (“pharmacophoric groups”); (b) establishment of the spatial relationship between these groups (“pharmacophore model”); (c) selection of an organic template suitable for reproducing the geometry of the pharmacophore model. The most difficult and usually rate-limiting step is the second one, since only rarely can the biologically relevant peptide topology be deduced from direct observation of the receptor-ligand complex. Although, as noted above, SPCL are very effective to carry out stepa, their use in steps b and c is still in its infancy (21Nikolaiev V. Stierandova A. Krchnak V. Seligmann B. Lam K.S. Salmon S.E. Lebl M. Pept. Res. 1993; 6: 161-170PubMed Google Scholar, 22Lee J. Barret R.E. Bovy P.R. Lett. Pept. Sci. 1995; 2: 253-258Crossref Scopus (2) Google Scholar). We have recently proposed the concept of selection-driven design of peptidomimetics (23Bianchi E. Folgori A. Wallace A. Nicotra M. Acali S. Phalipon A. Barbato G. Bazzo R. Cortese R. Felici F. Pessi A. J. Mol. Biol. 1995; 247: 154-160Crossref PubMed Scopus (82) Google Scholar, 24Sollazzo M. Bianchi E. Felici F. Cortese R. Pessi A. Cortese R. Combinatorial Libraries: Synthesis, Screening, and Application Potential. Walter de Gruyter & Co., Berlin1995: 127-143Google Scholar, 25Bianchi E. Barbato G. Wallace A. Cortese R. Felici F. Bazzo R. Pessi A. Epton R. Innovation and Perspectives in Solid Phase Synthesis: Peptides, Proteins, and Nucleic Acids. Mayflower Worldwide Ltd., Birmingham, UK1996: 159-162Google Scholar), a process whereby a first generation peptide pharmacophore is rapidly derived from screening of a panel of libraries with predetermined ligand geometry. Our first example on the application of this strategy was the development of a conformationally homogeneous library of α-helical peptides and the concurrent selection of a peptide mimicking the lipopolysaccharide antigen of the human pathogen Shigella flexneri (23Bianchi E. Folgori A. Wallace A. Nicotra M. Acali S. Phalipon A. Barbato G. Bazzo R. Cortese R. Felici F. Pessi A. J. Mol. Biol. 1995; 247: 154-160Crossref PubMed Scopus (82) Google Scholar). Here we report the results of the screening of a β-turn SPCL on human μ-, δ- and κ-opioid receptors (hMOR, hDOR, hKOR) and ORL1 receptor. Naloxone, [d-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin (DAMGO), GDP, and GTPγS were purchased from Sigma. BW373U86 was kindly provided by Dr. K. J. Chang (Burroughs Wellcome Co., Research Triangle Park, NC). CI-977 was a gift from John Hughes (Parke-Davis Neuroscience Research Center, Cambridge, UK). [3H]Diprenorphine (37 Ci/mmol; 1 Cu = 37 GBq) and [leucyl-3H]nociceptin (172 Ci/mmol) were obtained from Amersham Pharmacia Biotech, and [35S]GTPγS (1156 Ci/mmol) was from NEN Life Science Products. The hMOR cDNA was a gift from Lei Yu (Department of Medical and Molecular Genetics, Indianapolis, IN). The carrier plasmid used in the electroporation procedure (pBluescript) was from Stratagene (La Jolla, USA). All the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl alcohol-protected amino acids were obtained from Novabiochem, Bachem (Bubendorf, Germany), or Neosystem (Strasbourg, Germany). The SPCL and the individual peptides were synthesized as described previously (23Bianchi E. Folgori A. Wallace A. Nicotra M. Acali S. Phalipon A. Barbato G. Bazzo R. Cortese R. Felici F. Pessi A. J. Mol. Biol. 1995; 247: 154-160Crossref PubMed Scopus (82) Google Scholar,26Wallace A. Altamura S. Toniatti C. Vitelli A. Bianchi E. Delmastro P. Ciliberto G. Pessi A. Peptide Res. 1994; 7: 27-31PubMed Google Scholar, 27Wallace A. Koblan K.S. Hamilton K. Marquis-Omer D.J. Miller P.J. Mosser S.D. Omer C.A. Schaber M.D. Cortese R. Oliff A. Gibbs J.B. Pessi A. J. Biol. Chem. 1996; 271: 31306-31311Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) using PyBOP®/HOBt/DIPEA (1:1:2) activation, 5-fold excess, and a coupling time of 20 min to 2 h as judged by the standard ninhydrin and TNBS color tests. The undefined or “mixed” (X) positions were incorporated by coupling a mixture of activated amino acids, with the relative ratios suitably adjusted to yield close to equimolar incorporation. 8-Amino-5,6,7,8-tetrahydro-2-naphthoic acid (ATA) and 8-aminomethyl-5,6,7,8-tetrahydro-2-naphthoic acid (AMTA) were synthesized following the procedure of Ernest et al. (28Ernest I. Kalvoda J. Rihs G. Mutter M. Tetrahedron Lett. 1990; 31: 4011-4014Crossref Scopus (32) Google Scholar). All the other turn-forming templates, i.e.3-amino-1-carboxymethyl-2,3,4,5-tetrahydro-1H-(1)-benzazepine-2-one (BZA (29Parsons W.H. Davidson J.L. Taub D. Aster S.D. Thorsett E.D. Patchett A.A. Ulm E.H. Lamont B.I. Biochem. Biophys. Res. Commun. 1983; 117: 108-113Crossref PubMed Scopus (26) Google Scholar)), 3-amino-N-1-carboxymethyl-2-oxo-5-phenyl-1,4-benzodiazepine (4BZD) (30James G.L. Goldstein J.L. Brown M.S. Rawson T.E. Somers T.C. McDowell R.S. Crowley C.W. Lucas B.K. Levinson A.D. Marsters J.C.J. Science. 1993; 260: 1937-1942Crossref PubMed Scopus (607) Google Scholar)), 3-amino-1-carboxymethylcaprolactame (31Robl J.A. Cimarusti M.P. Simpkins L.M. Weller H.N. Pan Y.Y. Malley M. DiMarco J.D. J. Am. Chem. Soc. 1994; 116: 2348-2355Crossref Scopus (68) Google Scholar), 5-amino-1,2,4,5,6,7-tetrahydro-azepino[3,2,1-hi]indole-4-one-2-carboxylic acid (Haic (32De Lombeart S. Blanchard L. Stamford L.B. Sperbeck D.M. Grim M.D. Jenson T.M. Rodriguez H.R. Tetrahedron Lett. 1994; 35: 7513-7516Crossref Scopus (36) Google Scholar)), and (3S,6S,9R)-2-oxo-3-amino-7-thia-1-aza-bicyclo[4.3.0]nonane-9-carboxylic acid (BTD (33Nagai U. Sato K. Tetrahedron Lett. 1985; 26: 647-650Crossref Scopus (164) Google Scholar)) were obtained from Neosystem (Strasbourg). Purification of individual peptides and separation of diastereoisomers was carried out by reversed phase HPLC on a Nucleosyl C-18, 250 × 21-mm, 100-Å, 7-mm column using H2O, 0.1% trifluoroacetic acid and acetonitrile, 0.1% trifluoroacetic acid as eluents. Analytical HPLC was performed on a Ultrasphere C-18, 250 × 4.6-mm, 80-Å, 5-mm column (Beckman). Purified (≥95%) peptides were characterized by mass spectrometry and amino acid analysis. All cell lines were from ATCC and maintained in the presence of 5% fetal calf serum and 5% CO2. COS-1 cells were grown in Dulbecco's modified Eagle's medium (Eurobio, Les Ulis, France), and CHO cells were grown in Dulbecco's modified Eagle's-F-12 medium (Eurobio). CHO stably transfected with pCDNA3/Neo (Invitrogene, Nu Leek, Netherlands) or hORL1 and hKOR were gifts from Lawrence Toll (Torrey Pines Institute for Molecular Biology, San Diego, CA) and C. Mollereau (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France), respectively. Cells were electroporated essentially as described (34Befort K. Tabbara L. Kieffer B.L. Neurochem. Res. 1996; 21: 1301-1307Crossref PubMed Scopus (41) Google Scholar). Briefly, 2 × 107 COS-1 cells were seeded the night before transfection at a density of 107cells/140-mm dish. Cells were washed two times with phosphate-buffered saline and detached by applying trypsin/EDTA (Eurobio). Cells were collected by centrifugation for 10 min at 400 × g and resuspended at a density of 108 cells/ml in EP 1× buffer (50 mm K2HPO4, 20 mmCH3CO3K, 20 mm KOH, pH 7.4). hMOR, hDOR, or hKOR plasmidic DNA, prepared using Nucleobond columns (Macherey Nagel, Düren, Germany) and consisting of variable amounts of receptor-encoding plasmid and a carrier plasmid (pBluescript) up to a final 20-μg DNA quantity was diluted into EP 1× buffer to a total volume of 300 μl. The DNA mix was then supplemented with 13 μl of 1 m MgSO4 and incubated with 200 μl of cell suspension for 20 min at room temperature. The cell/DNA mixture was then transferred to a 0.4-cm cuvette and electroporated using a Gene Pulser apparatus (Bio-Rad) at a capacitance setting of 2000 microfarads and voltage setting of 240 volts. Cells were then immediately transferred into 50 ml of Dulbecco's modified Eagle's medium with 10% fetal calf serum and seeded into 2 140-mm dishes. After 72 h of growth, the cells were harvested, and membranes were then prepared as described previously (34Befort K. Tabbara L. Kieffer B.L. Neurochem. Res. 1996; 21: 1301-1307Crossref PubMed Scopus (41) Google Scholar). Transfected cells (4 140-mm dishes at a 50 to 100% confluency) were washed with 2× phosphate-buffered saline, scrapped off the plates in phosphate-buffered saline, pelleted by centrifugation at 400 ×g for 10 min at 4 °C, frozen at −80 °C for 30 min at least, and thawed in 30 ml of cold 50 mm Tris-HCl, pH 7, when membranes were prepared for ligand binding experiments, and 30 ml of cold 50 mm Tris-HCl, pH 7, 2.5 mm EDTA, and 0.1 mm phenylmethylsulfonyl fluoride (added extemporaneously) was added for [35S]GTPγS binding experiments. All the following steps were performed at 4 °C. The cell lysate was Dounce-homogenized and spun at 400 × gfor 10 min. The pellet was resuspended in 15 ml of buffer, Dounce-homogenized, and spun again at 400 × g for 10 min. Both supernatants were pooled and centrifuged at 100,000 ×g for 30 min. The pellet was then resuspended in 4 ml of 50 mm Tris HCl, pH 7, and the protein concentration was measured using the Bradford assay. Membranes were then aliquoted at a 1-mg protein/ml concentration and stored at −80 °C. When membranes were prepared for [35S]GTPγS binding experiments, the pellet was resuspended in 25 ml of 50 mm Tris-HCl, pH 7, Dounce-homogenized, and spun again at 100,000 × g for 30 min. The pellet was then resuspended in 4 ml of 50 mmTris-HCl, pH 7, 0.32 m sucrose, and the protein concentration was measured as described above. Binding experiments were done as described previously (35Simonin F. Befort K. Gaveriaux-Ruff C. Matthes H. Nappey V. Lannes B. Micheletti G. Kieffer B. Mol. Pharmacol. 1994; 46: 1015-1021PubMed Google Scholar). For saturation experiments, various concentrations (from 5 × 10−11 to 6.4 × 10−9m) of [3H]diprenorphine (hMOR, hDOR, hKOR) or [leucyl-3H]nociceptin (hORL1) were used. For competition experiments, membrane proteins were diluted in 50 mm Tris-HCl, pH 7.4, and incubated with [3H]diprenorphine (0.2 nm for hMOR and hDOR and 0.4 nm for hKOR) or 0.1 nm[leucyl-3H]nociceptin (for hORL1), and variable concentrations of competitor peptide (7.8 × 10−11 to 5 × 10−5m) in a total volume of 0.2 ml for 1 h at 25 °C. Nonspecific binding was determined in the presence of 1 μm naloxone (hMOR, hDOR, hKOR) or 1 μm nociceptin/orphanin FQ (hORL1). K iand K d values were determined using the EBDA/Ligand program (G. A. McPherson, Biosoft, Cambridge, UK).K d values were in good agreement with those described in the literature (7Meunier J.-C. Mollereau C. Toll L. Suaudeau C. Moisand C. Alvinerie P. Butour J.-L. Guillemot J.-C. Ferrara P. Monserrat B. Mazarguil H. Vassart G. Parmentier M. Costentin J. Nature. 1995; 377: 532-535Crossref PubMed Scopus (1818) Google Scholar, 35Simonin F. Befort K. Gaveriaux-Ruff C. Matthes H. Nappey V. Lannes B. Micheletti G. Kieffer B. Mol. Pharmacol. 1994; 46: 1015-1021PubMed Google Scholar, 36Mestek A. Hurley J.H. Bye L.S. Campbell A.D. Chen Y. Tian M. Liu J. Schulman H. Yu L. J. Neurosci. 1995; 15: 501-527Crossref Google Scholar, 37Simonin F. Gavériaux-Ruff C. Befort K. Matthes H.W.D. Lannes B. Micheletti G. Mattei M.-G. Charron G. Bloch B. Kieffer B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7006-7010Crossref PubMed Scopus (192) Google Scholar). For the opioid receptors, 5 μg of hMOR, hKOR, and hDOR membrane proteins were incubated 1 h at 30 °C in 50 mm Hepes, pH 7.6, 5 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1% bovine serum albumin, GDP (3 μm for hKOR, and 30 μm for hMOR and hDOR), 0.2 nm[35S]GTPγS, and ligands (1.8 × 10−11to 1 × 10−5m for the opioid ligand, and 2.8 × 10−10 to 5 × 10−5m for the competitor peptides) in a final volume of 0.2 ml (34Befort K. Tabbara L. Kieffer B.L. Neurochem. Res. 1996; 21: 1301-1307Crossref PubMed Scopus (41) Google Scholar). For hORL1, 5 μg of membrane proteins were incubated 1 h at 37 °C in 50 mm Tris, pH 7.4, 5 mmMgCl2, 1 mm EGTA, 100 mm NaCl, 0.1% bovine serum albumin, 40 μm GDP, 0.2 nm[35S]GTPγS, and ligands (1.8 × 10−11to 1 × 10−5m for nociceptin/orphanin FQ, and 2.8 × 10−10 to 5 × 10−5m for the competitor peptides) in a final volume of 0.2 ml. Nonspecific binding was determined in the presence of 10 μm GTPγS. Incubation mixtures were rapidly washed using a cell harvester (Brandell, Gaithersburg, MD) with cold 50 mm Tris-HCl, pH 7, 5 mm MgCl2, 50 mm NaCl on H2O-presoaked GF/B filters. Bound radioactivity was determined by scintillation counting. EC50 values were determined using the Prism software (GraphPad, San Diego, USA). To find new ligands for the opioid and ORL1 receptors, we have screened a reverse-turn peptide SPCL composed of 2 × 107 N-terminal-acetylated and C-terminal-amidated peptides in a so-called positional scanning format (26Wallace A. Altamura S. Toniatti C. Vitelli A. Bianchi E. Delmastro P. Ciliberto G. Pessi A. Peptide Res. 1994; 7: 27-31PubMed Google Scholar). Each peptide of this library is constrained in a β-turn conformation by a rigid turn-forming mimetic block (ATA) in its center (see Fig.1). The library is composed of four sublibraries: Ac-O1X-ATA-XX-NH2, Ac-XO2-ATA-XX-NH2, Ac-XX-ATA-O3X-NH2, Ac-XX-ATA-XO4-NH2 (Ac, acetyl). Each sublibrary is composed of 68 peptide mixtures, in which the position labeled (On) is defined by the amino acids indicated in the legend of Fig. 2. We used an expanded combinatorial set, including many noncoded amino acids, and most of the residues were present both with l and d chirality.Figure 2Screening of the β-turn mimetic SPCL . A, hMOR and hDOR membranes were labeled using a nonselective opioid antagonist [3H]diprenorphine (0.2 nm for hMOR and 0.4 nm for hDOR). B, hKOR and hORL1 membranes were labeled using 0.4 nm [3H]diprenorphine and 0.1 nm [leucyl-3H]nociceptin, respectively. Assays were carried out using target receptor either transiently expressed in COS-1 cells (hMOR, hDOR, hKOR) or stably expressed in CHO cells (hORL1). Each panel represents the screening of one of the four SPCL on one receptor. Each bar within apanel represents percent inhibition of binding by a peptide mixture (each individual peptide was at a concentration of 1.6 nm for hMOR, hDOR, hORL1 and 0.16 nm for hKOR) defined in the O position with one of the 68 amino acids indicated below. Arrows indicated the selected amino acids for individual peptide synthesis. 1, l-Val;2, l-Ile; 3, l-Trp;4, l-Gln; 5, l-Asn;6, l-Arg; 7, l-His;8, l-Tyr; 9, l-Pro;10, l-Phe; 11, l-Met;12, l-Glu; 13, l-Asp;14, l-Lys; 15, l-Thr;16, l-Ser; 17, l-Leu;18, l-Ala; 19, l-Gly;20, l-α-aminobutyric; 21, aminoisobutyric; 22, β-alanine; 23, γ-aminobutyric; 24, 6-aminohexanoic; 25, β-cyclohexyl-l-alanine; 26, 3,4-dehydro-l-proline; 27, γ-carboxyglutamic;28, homo-l-phenylalanine; 29, hydroxy-l-proline; 30, l-norleucine;31, l-norvaline; 32,l-ornithine; 33,p-chloro-l-phenylalanine; 34,p-nitro-l-phenylalanine; 35,l-phenylglycine; 36, sarcosine; 37,d-Val; 38, d-Ile; 39,d-Trp; 40, d-Gln; 41,d-Asn; 42, d-Arg; 43,d-His; 44, d-Tyr; 45,d-Pro; 46, d-Phe; 47,d-Met; 48, d-Glu; 49,d-Asp; 50, d-Lys; 51,d-Thr; 52, d-Ser; 53,d-Leu; 54, d-Ala; 55, β-cyclohexyl-d-alanine; 56,d-norleucine; 57, d-norvaline;58, p-chloro-d-phenylalanine;59, (3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanoic;60, (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic;61, 5-aminovaleric; 62, 8-aminooctanoic;63, 2,3-diamino-α-l-propionic; 64,d-phenylglycine; 65, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic;66, 1,2,3,4-tetrahydroisoquinoline-3-l-carboxylic;67, 1,2,3,4-tetrahydroisoquinoline-3-l-carboxylic;68, 2,3-diamino-β-l-propionic.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Screening of the β-turn mimetic SPCL . A, hMOR and hDOR membranes were labeled using a nonselective opioid antagonist [3H]diprenorphine (0.2 nm for hMOR and 0.4 nm for hDOR). B, hKOR and hORL1 membranes were labeled using 0.4 nm [3H]diprenorphine and 0.1 nm [leucyl-3H]nociceptin, respectively. Assays were carried out using target receptor either transiently expressed in COS-1 cells (hMOR, hDOR, hKOR) or stably expressed in CHO cells (hORL1). Each panel represents the screening of one of the four SPCL on one receptor. Each bar within apanel represents percent inhibition of binding by a peptide mixture (each individual peptide was at a concentration of 1.6 nm for hMOR, hDOR, hORL1 and 0.16 nm for hKOR) defined in the O position with one of the 68 amino acids indicated below. Arrows indicated the selected amino acids for individual peptide synthesis. 1, l-Val;2, l-Ile; 3, l-Trp;4, l-Gln; 5, l-Asn;6, l-Arg; 7, l-His;8, l-Tyr; 9, l-Pro;10, l-Phe; 11, l-Met;12, l-Glu; 13, l-Asp;14, l-Lys; 15, l-Thr;16, l-Ser; 17, l-Leu;18, l-Ala; 19, l-Gly;20, l-α-aminobutyric; 21, aminoisobutyric; 22, β-alanine; 23, γ-aminobutyric; 24, 6-aminohexanoic; 25, β-cyclohexyl-l-alanine; 26, 3,4-dehydro-l-proline; 27, γ-carboxyglutamic;28, homo-l-phenylalanine; 29, hydroxy-l-proline; 30, l-norleucine;31, l-norvaline; 32,l-ornithine; 33,p-chloro-l-phenylalanine; 34,p-nitro-l-phenylalanine; 35,l-phenylglycine; 36, sarcosine; 37,d-Val; 38, d-Ile; 39,d-Trp; 40, d-Gln; 41,d-Asn; 42, d-Arg; 43,d-His; 44, d-Tyr; 45,d-Pro; 46, d-Phe; 47,d-Met; 48, d-Glu; 49,d-Asp; 50, d-Lys; 51,d-Thr; 52, d-Ser; 53,d-Leu; 54, d-Ala; 55, β-cyclohexyl-d-alanine; 56,d-norleucine; 57, d-norvaline;58, p-chloro-d-phenylalanine;59, (3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanoic;60, (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic;61, 5-aminovaleric; 62, 8-aminooctanoic;63, 2,3-diamino-α-l-propionic; 64,d-phenylglycine; 65, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic;66, 1,2,3,4-tetrahydroisoquinoline-3-l-carboxylic;67, 1,2,3,4-tetrahydroisoquinoline-3-l-carboxylic;68, 2,3-diamino-β-l-propionic.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The β-turn mimetic library was used in conjunction with a deconvolution selection process to identify individual peptides capable of inhibiting the binding of radioligands to membrane homogenates of COS-1 or CHO cells expressing recombinant human μ-, δ-, and κ-opioid receptors (hMOR, hDOR, and hKOR) and the human opioid-like receptor (hORL1, see “Experimental Procedures”). The sublibraries were screened at a fixed concentration of 500 μm (1.6 nm for each individual peptide). For hKOR, all the mixtures inhibited >90% of radioligand binding in this initial screening; the library was therefore screened again at a 10-fold lower concentration (50 μm). Results of the screening of the four sublibraries with the four receptors are shown in Fig. 2. A lot of peptide mixtures in each sublibrary were found to be active (>75% inhibition) on either one or several receptors, particularly hKOR and hMOR. We therefore selected the most active and/or selective consensus sequences to synthesize individual peptides. For the first sublibrary (position O1), l-Arg, d-Trp, andl-Cha were the most active residues on the four receptors and were then selected (excepted for hDOR for which we selected onlyl-Cha and d-Trp). More selective residues were also selected for hMOR (homo-l-phenylalanine andl-Arg) and for hKOR (l-Fno). The second sublibrary (position O2) was the most active one on the four receptors, especially on hMOR and hKOR (all the peptide mixtures showed >75% inhibition of the binding). For these two receptors we" @default.
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• W1986701157 date "1999-09-01" @default.
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• W1986701157 title "Ligands for κ-Opioid and ORL1 Receptors Identified from a Conformationally Constrained Peptide Combinatorial Library" @default.
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• W1986701157 doi "https://doi.org/10.1074/jbc.274.39.27513" @default.
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