Minor Modifications of a Cholecystokinin-B/Gastrin Receptor Non-peptide Antagonist Confer a Broad Spectrum of Functional Properties

Abstract

The development of non-peptide agonists for peptide hormone receptors would markedly expand the treatment options for a large number of diseases. However, difficulty in identifying non-peptide molecules which possess intrinsic activity has been a major obstacle in achieving this goal. At present, most of the known non-peptide ligands for peptide hormone receptors appear in standard functional assays to be antagonists. Here, we report that a constitutively active mutant of the human cholecystokinin-B/gastrin receptor, Leu325 → Glu, offers the potential to detect even trace agonist activity of ligands which, at the wild type receptor isoform, appear to lack efficacy. The enhanced functional sensitivity of the mutant receptor enabled us to detect intrinsic activity of L-365,260, an established non-peptide antagonist for the cholecystokinin-B/gastrin receptor. Extending from this observation, we were able to demonstrate that minor structural modifications could convert L-365,260 into either: (i) an agonist or (ii) an inverse agonist (attenuates ligand-independent signaling). The ability to confer functional activity to small non-peptide ligands suggests that the properties of endogenous peptide hormones can be mimicked, and even extended, by considerably less complex molecules. The development of non-peptide agonists for peptide hormone receptors would markedly expand the treatment options for a large number of diseases. However, difficulty in identifying non-peptide molecules which possess intrinsic activity has been a major obstacle in achieving this goal. At present, most of the known non-peptide ligands for peptide hormone receptors appear in standard functional assays to be antagonists. Here, we report that a constitutively active mutant of the human cholecystokinin-B/gastrin receptor, Leu325 → Glu, offers the potential to detect even trace agonist activity of ligands which, at the wild type receptor isoform, appear to lack efficacy. The enhanced functional sensitivity of the mutant receptor enabled us to detect intrinsic activity of L-365,260, an established non-peptide antagonist for the cholecystokinin-B/gastrin receptor. Extending from this observation, we were able to demonstrate that minor structural modifications could convert L-365,260 into either: (i) an agonist or (ii) an inverse agonist (attenuates ligand-independent signaling). The ability to confer functional activity to small non-peptide ligands suggests that the properties of endogenous peptide hormones can be mimicked, and even extended, by considerably less complex molecules. Current understanding of G-protein-coupled receptor activation has in large part been based on the study of biogenic amine receptors (1Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (992) Google Scholar). The corresponding endogenous ligands, together with synthetic derivatives of these small molecules, cover a spectrum of functional activities ranging from full agonists to antagonists. With the discovery of constitutively active receptor mutants, this range has been further extended to include inverse agonists, distinguished by their ability to attenuate ligand-independent signaling (2Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (466) Google Scholar). Another major group of G-protein-coupled receptors is activated by endogenous peptide molecules. Compared with biogenic amines, these peptide agonists are significantly larger and structurally more complex. Since endogenous peptides exert important hormone and neurotransmitter functions, there is considerable interest in whether their function can be mimicked by non-peptide drugs (3Schwartz T.W. Gether U. Schambye H.T. Hjorth S.A. Curr. Pharmaceut. Design. 1995; 1: 325-342Google Scholar). This possibility is suggested by the opioid receptor system. Naturally occurring opioid receptor non-peptide agonists (e.g.morphine) as well as synthetic derivatives have been utilized since the early 19th century (4Reisine T. Pasternak G. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996: 521-555Google Scholar). Over the last 10 years, numerous non-peptide compounds have been identified which recognize specific peptide hormone receptor subtypes with high affinity. Unlike the corresponding endogenous peptide agonists, the vast majority of these new ligands appear to lack intrinsic activity and have been pharmacologically classified as antagonists (5Alexander S.P.H. Peters J.A. Trends Pharmacol. Sci. 1997; 18: 3-75PubMed Google Scholar). The difficulty in generating non-peptide agonists is exemplified by the extensive efforts which have focused on the identification of ligands for the human cholecystokinin-B/gastrin receptor (CCK-BR). 1The abbreviations used are: CCK-BR, cholecystokinin-B/gastrin receptor; G-17, gastrin heptadecapeptide (unsulfated form); CCK-8, cholecystokinin octapeptide (sulfated form); CCK-8US, cholecystokinin octapeptide (unsulfated form). 1The abbreviations used are: CCK-BR, cholecystokinin-B/gastrin receptor; G-17, gastrin heptadecapeptide (unsulfated form); CCK-8, cholecystokinin octapeptide (sulfated form); CCK-8US, cholecystokinin octapeptide (unsulfated form). This receptor has been implicated in modulating memory, anxiety, and pain perception, as well as in regulating gastrointestinal mucosal growth and secretion (6Crawley J.N. Corwin R.L. Peptides. 1994; 15: 731-755Crossref PubMed Scopus (609) Google Scholar, 7Walsh J.H. Walsh J.H. Dockray G.J. Gastrin. Raven Press, New York1994: 75-121Google Scholar, 8Wank S.A. Am. J. Physiol. Gastrointest. Liver Physiol. 1995; 269: G628-G646Crossref PubMed Google Scholar). With the exception of some peptide-derived compounds (9Schmassmann A. Garner A. Flogerzi B. Hasan M.Y. Sanner M. Varga L. Halter F. Gut. 1994; 35: 270-274Crossref PubMed Scopus (34) Google Scholar, 10Ding X.Q. Chen D. Håkanson R. Gastroenterology. 1995; 109: 1181-1187Abstract Full Text PDF PubMed Scopus (33) Google Scholar), all of the synthetic CCK-BR non-peptide ligands which have been discovered to date are reported to be antagonists and thus appear unable to satisfy the structural requirements for receptor activation. The prototype of such non-peptide, selective CCK-BR antagonists is L-365,260, a benzodiazepine-based ligand which was discovered in 1989 (11Lotti V.J. Chang R.S.L. Eur. J. Pharmacol. 1989; 162: 273-280Crossref PubMed Scopus (392) Google Scholar). Widely tested both in vivo and in vitro, this compound has become a cornerstone in the characterization and pharmacological classification of CCK receptors. We now report that L-365,260 has unexpected residual intrinsic activity, which we were able to detect using a constitutively active CCK-BR mutant. Extending from this finding, we demonstrate that slight structural modifications of L-365,260 convert this ligand into either: (i) a non-peptide agonist for the wild type CCK-BR, or (ii) an inverse agonist. These findings illustrate that existing non-peptide ligands may have considerably higher potential to activate peptide hormone receptors than was previously appreciated, and suggest that constitutively active receptor mutants provide promising tools to detect and optimize such functional properties. Cell culture media and fetal calf serum were obtained from Life Technologies, Inc. (Gaithersburg, MD) and from Intergen (Purchase, NY), respectively. 125I-CCK-8 (2,200 Ci/mmol) and myo-[3H]inositol (45–80 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). Unlabeled gastrin heptadecapeptide (G-17, unsulfated form), CCK-8 (sulfated form), CCK-8US (unsulfated form), and CCK-4 were obtained from Peninsula Laboratories (Belmont, CA). L-365,260, L-740,093 (R and S forms), YM022, and L-364,718 were generously provided by Wyeth Research Ltd. (Taplow, United Kingdom). The “peptoid” compounds PD-135,158 and PD-136,450 were a gift from Parke-Davis Research Center (Cambridge, UK). COS-7 cells (1 × 106) were plated onto 10-cm culture dishes (Costar, Cambridge, MA) and grown overnight in Dulbecco's modified Eagle's medium, 10% fetal calf serum at 37 °C. The cells were transfected with 5 μg of wild type or mutant CCK-BR cDNA (12Lee Y.-M. Beinborn M. McBride E.W. Lu M. Kolakowski Jr., L.F. Kopin A.S. J. Biol. Chem. 1993; 268: 8164-8169Abstract Full Text PDF PubMed Google Scholar), subcloned into the expression vector pcDNAI (Invitrogen). The following day, cells were split into 6-well plates (3 × 105/well) (Nunc). The cells were then prelabeled overnight with 3 μCi/ml myo-[3H]inositol in serum-free Dulbecco's modified Eagle's medium. To assess ligand-induced inositol phosphate production, the medium was replaced with phosphate-buffered saline containing 10 mm LiCl, and cells were incubated with the respective ligands for 30 min at 37 °C. Each ligand was tested at a concentration which was at least 25-fold higher than the corresponding dissociation constant (K i value) determined in radioligand binding experiments (see below, Table I). According to the simple Langmuir isotherm (fractional receptor occupancy = ligand concentration/[Ligand concentration +K i]) the ligand concentrations which were utilized result in >95% receptor occupancy (13Hulme E.C. Birdsall N.J.M. Hulme E.C. Receptor-Ligand Interactions. Oxford University Press, New York1992: 63-176Google Scholar), and will thus induce near maximal receptor stimulation and inositol phosphate production. After incubation, cells were lysed and extracted with methanol/chloroform; the upper phase was analyzed for inositol phosphates by strong anion exchange chromatography (14Seidler U. Pfeiffer A. Am. J. Physiol. Gastrointest. Liver Physiol. 1991; 260: G133-G141Crossref PubMed Google Scholar). Inositol phosphate production was expressed as a percentage of the total cellular tritium content which was incorporated during an overnight exposure tomyo-[3H]inositol. Concentration-response curves were calculated using the GraphPad Prizm software (GraphPad, San Diego, CA).Table IBinding affinities of peptide, peptoid, and non-peptide ligands at the wild type and the constitutively active CCK-BRWild type receptor, K iLeu325 → Glu mutant,K inMPeptidesGastrin-171.35 ± 0.280.78 ± 0.16CCK-80.12 ± 0.010.07 ± 0.01CCK-8US2.20 ± 0.781.16 ± 0.51CCK-411.6 ± 4.07.97 ± 2.08PeptoidsPD-135,1582.25 ± 0.611.01 ± 0.19PD-136,4500.99 ± 0.100.59 ± 0.12Non-peptidesL-740,093 (R)0.19 ± 0.020.18 ± 0.04YM022 (R)0.07 ± 0.010.08 ± 0.01L-364,718 (S)150 ± 42170 ± 34L-365,260 (R)7.2 ± 0.97.8 ± 1.5L-740,093 (S)19.5 ± 1.516 ± 1.4K i values were averaged from three to five independent experiments (mean ± S.E.). Open table in a new tab K i values were averaged from three to five independent experiments (mean ± S.E.). Twenty-four hours after transfection, COS-7 cells were seeded into 24-well dishes (1 × 104/well) (Costar). After an additional 24 h, competition binding experiments were performed in Hank's balanced salt solution supplemented with 25 mm HEPES, pH 7.3, 0.2% bovine serum albumin, and 0.15 mm phenylmethylsulfonyl fluoride, using 20 pm125I-CCK-8 as the radioligand. After an 80-min incubation in the absence or presence of unlabeled ligands, cell monolayers were washed three times with Hank's balanced salt solution and then hydrolyzed in 1 n NaOH for γ-counting (15Beinborn M. Lee Y.-M. McBride E.W. Quinn S.M. Kopin A.S. Nature. 1993; 362: 348-350Crossref PubMed Scopus (194) Google Scholar). All binding affinities were calculated by nonlinear curve fitting using the LIGAND computer program (16McPherson G.A. J. Pharmacol. Methods. 1985; 14: 213-228Crossref PubMed Scopus (1187) Google Scholar). Receptor densities in transfected COS-7 cells were calculated from125I-CCK-8 binding experiments with increasing concentrations of unlabeled CCK-8 as the homologous competitor. Molecular characterization of the third intracellular loop of the human CCK-BR led to the identification of a point mutation (Leu325 → Glu) which results in constitutive receptor activity (17Beinborn M. Wu M.-J. Quinn S.M. Goeke E.-K. Kolakowski Jr., L.F. Kopin A.S. Gastroenterology. 1996; 110A1059Google Scholar). When transiently expressed in COS-7 cells, the Leu325 → Glu mutant triggers agonist independent production of inositol phosphates, to levels exceeding cells expressing the wild type receptor at similar densities (Fig.1 A). Despite this difference in ligand-independent signaling, the wild type and the mutant receptors increase inositol phosphate production to comparable levels when stimulated with saturating concentrations of either CCK-8 or G-17, two of the principal endogenous agonists for the CCK-BR (6Crawley J.N. Corwin R.L. Peptides. 1994; 15: 731-755Crossref PubMed Scopus (609) Google Scholar, 7Walsh J.H. Walsh J.H. Dockray G.J. Gastrin. Raven Press, New York1994: 75-121Google Scholar, 18Liddle R.A. Walsh J.H. Dockray G.J. Cholecystokinin. Raven Press, New York1994: 175-216Google Scholar). In addition, both CCK-8 and G-17 have similar potencies and binding affinities when respective values at the wild type and the mutant receptor isoforms are compared (Fig. 1 B, TableI). Contrary to expectation, we found that the non-peptide ligand, L-365,260, has significant partial agonist activity when stimulating the mutant (versus the wild type) receptor (Fig.2 A). L-365,260, previously considered a prototype CCK-BR antagonist (5Alexander S.P.H. Peters J.A. Trends Pharmacol. Sci. 1997; 18: 3-75PubMed Google Scholar, 11Lotti V.J. Chang R.S.L. Eur. J. Pharmacol. 1989; 162: 273-280Crossref PubMed Scopus (392) Google Scholar), is able to activate the Leu325 → Glu receptor mutant with approximately half the efficacy of the full agonist, G-17. On closer examination, it became apparent that L-365,260 also induces a small yet significant (p < 0.01) increase of inositol phosphate production in cells expressing the wild type CCK-BR (Fig. 2 A, left panel). These findings prompted us to investigate the functional properties of compounds which are structurally related to L-365,260, including YM022 (19Nishida A. Miyata K. Tsutsumi R. Yuki H. Akuzawa S. Kobayashi A. Kamato T. Ito H. Yamano M. Katuyama Y. Satoh M. Ohta M. Honda K. J. Pharmacol. Exp. Ther. 1994; 269: 725-731PubMed Google Scholar) and (R)-L-740,093, as well as its stereoisomer, G.A. S. J.G. R. J.A. S. J. Chem. 1994; PubMed Scopus Google Scholar). All of these molecules a (Fig. 2 and are reported to function as antagonists. However, when with the constitutively active receptor mutant (Fig. 2 it became apparent that these compounds cover a range of intrinsic The of signaling with the Leu325 → Glu receptor us to demonstrate that minor structural of L-365,260 result in considerable functional the of the with an with the L-365,260 into G.A. S. J.G. R. J.A. S. J. Chem. 1994; PubMed Scopus Google Scholar), the most agonist of the tested its strong signaling also as a partial agonist at the wild type CCK-BR (Fig. 2 A). the agonist activity of stimulation of the wild type CCK-BR with this compound triggers a increase in signaling (Fig. the of a non-peptide agonist for the human is that the of the non-peptide agonist has the functional properties. signaling of the constitutively active receptor to that of the wild type (Fig. 2 and the of an inverse agonist. inverse agonists have been identified for two of peptide hormone and the receptors M. H. M. Y. 1995; Google Scholar, T. B. R. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). (R)-L-740,093, of these non-peptide inverse agonists to have intrinsic activity at the respective wild type receptors and was classified as an these illustrate that inverse is a of ligands which are considered To more classification of these further functional characterization using constitutively active receptors will be The discovery of non-peptide inverse agonists a for a new of at constitutively active peptide hormone receptors which result in human G. R.A. Lee M. Trends Pharmacol. Sci. 1995; 16: Full Text PDF PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). the of has been to constitutively active stimulating hormone receptors J. L. J. P. J. J. G. Nature. 1993; PubMed Scopus Google drugs which these could be utilized to inverse agonists could the of in with constitutively active hormone receptors A. L. S. T. Nature. 1993; PubMed Scopus Google Scholar). The activity of at the wild type and mutant the pharmacological classification of these compounds as agonist and inverse agonist, 3 A). is that the of these compounds can their which findings with the L-740,093 was reported for synthetic derivatives of the cholecystokinin of these ligands, and which are distinguished by their were found to as agonist and at the receptor J. Dockray G.J. D. L. G. 1996; PubMed Scopus Google Scholar). to be established whether of ligands for peptide hormone receptors will also have functional properties. Unlike the L-740,093 YM022 has on the activity of either the wild type or the mutant receptors (Fig. 2 A). This lack of intrinsic activity, and the ability of YM022 to CCK-8 inositol phosphate (17Beinborn M. Wu M.-J. Quinn S.M. Goeke E.-K. Kolakowski Jr., L.F. Kopin A.S. Gastroenterology. 1996; 110A1059Google Scholar), are with the properties of an To further the functional classification of the CCK-BR non-peptide ligands, the of YM022 with was inositol phosphate production was by YM022 in a B, further that as a non-peptide agonist. In addition, YM022 was able to attenuate the of on the constitutively active CCK-BR mutant (Fig. B, This is in with the that the activity of inverse agonists also be to by antagonists (2Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (466) Google Scholar). To whether the enhanced signaling with the Leu325 → Glu mutant to a structurally of molecules, we compared activation of the wild type and the mutant by two ligands, PD-135,158 and of these compounds have in in vivo been to be partial CCK-BR agonists (9Schmassmann A. Garner A. Flogerzi B. Hasan M.Y. Sanner M. Varga L. Halter F. Gut. 1994; 35: 270-274Crossref PubMed Scopus (34) Google Scholar, 10Ding X.Q. Chen D. Håkanson R. 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This the identification and development of new for the treatment of a spectrum of diseases. We for ligands, and Parke-Davis for We also M. A. J. S. A. and F. for of the and B. of the for