Rational Design of a Peptide Agonist That Interacts Selectively with the Orphan Receptor, Bombesin Receptor Subtype 3*

Samuel A. ManteyDagger , David H. Coy§, Tapas K. PradhanDagger , Hisato IgarashiDagger , Ivania M. RizoDagger , Lin ShenDagger , Wei HouDagger , Simon J. Hocart§, and Robert T. JensenDagger

From the Dagger  Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the § Department of Medicine, Peptide Research, Tulane University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, September 25, 2000, and in revised form, November 20, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R)). This receptor is widely distributed in the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiological and pathological processes remain unknown due to lack of selective ligands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R. Pradhan, T. K., Searles, R. P., Spindel, E. R., Battey, J. F., Coy, D. H., and Jensen, R. T. (1997) J. Biol. Chem. 272, 26062-26071) that [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) has high affinity for BRS-3 and using this ligand showed BRS-3 has a unique pharmacology with high affinity for no known natural Bn peptides. However, use of this ligand is limited because it has high affinity for all known Bn receptors. In the present study we have attempted to identify BRS-3 selective ligands using a strategy of rational peptide design with the substitution of conformationally restricted amino acids into the prototype ligand [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) or its D-Phe6 analogue. Each of the 22 peptides synthesized had binding affinities determined for hBRS-3, hGRPR, and hNMBR, and hBRS-3 selective ligands were tested for their ability to activate phospholipase C and increase inositol phosphates ([3H]inositol phosphate). Using this approach we have identified a number of BRS-3 selective ligands. These ligands functioned as receptor agonists and their binding affinities were reflected in their potencies for altering [3H]inositol phosphate. Two peptides with an (R)- or (S)-amino-3-phenylpropionic acid substitution for beta -Ala11 in the prototype ligand had the highest selectivity for the hBRS-3 over the mammalian Bn receptors and did not interact with receptors for other gastrointestinal hormones/neurotransmitters. Molecular modeling demonstrated these two selective BRS-3 ligands had a unique conformation of the position 11 beta -amino acid. This selectivity was of sufficient magnitude that these should be useful in explaining the role of hBRS-3 activation in obesity, glucose homeostasis, hypertension, and other physiological or pathological processes.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 399-amino acid orphan receptor, bombesin receptor subtype 3 (BRS-3),1 shares 51 and 47% amino acid sequence homology with the mammalian bombesin (Bn) receptors (gastrin-releasing peptide receptor (GRP-R) and the neuromedin B receptor (NMB-R), respectively) (1, 2). Studies of the distribution of this orphan receptor show that the BRS-3 receptor is present in the central nervous system and peripheral tissues although the distribution is more limited than the GRP-R and NMB-R (3-6). The BRS-3 receptor has been found on such diverse structures as secondary spermatocytes, pregnant uterus, a number of brain regions, and some human lung, breast, and epidermal cancer cell lines (1, 2)

The role of BRS-3 in physiological or pathological processes is unknown even though BRS-3-deficient mice, produced by targeted disruption, develop obesity, diabetes, and hypertension (7). These results (7) suggest that the BRS-3 receptor may be required for the regulation of glucose metabolism, energy balance, and maintenance of blood pressure. This proposition is yet to be confirmed because the natural ligand of the BRS-3 receptor is still unknown. Results from previous studies (8-10) have demonstrated that the hBRS-3 receptor has a unique pharmacology compared with that of any of the closely related Bn receptor family. BRS-3 does not interact with high affinity with any known natural or synthetic agonist or antagonist of the Bn receptor family (8-10). However, in previous studies we reported the discovery of a synthetic ligand [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and its D-Phe6 analogue that function as high affinity agonists for hBRS-3 for both the native receptor in human small cell cancer cell line, NCI-N417 (1, 11), and hBRS-3 transfected BALB 3T3 cells or hBRS-3 transfected NCI-H1299 nonsmall cell lung cancer cells (8, 10, 12). These studies (1, 11, 13), as well as others (2, 14, 15), demonstrate the BRS-3 receptor is coupled to phospholipase C and its activation causes mobilization of cellular calcium and increases in inositol phosphates. Furthermore, receptor activation stimulates tyrosine phosphorylation of p125 focal adhesion kinase (11). While this high affinity ligand is useful for studying the pharmacology and cell biology of the BRS-3 receptor, its widespread use for physiological studies is limited because the hBRS-3 ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) is not selective for the hBRS-3 receptor (8, 10, 11, 16). It also interacts with high affinity with all the known mammalian and nonmammalian Bn receptor subtypes (GRP-R, NMB-R, and BB4-R) (8, 10, 11, 16).

Therefore, the aim of the present study was to attempt to discover an hBRS-3 selective ligand. The strategy used was to substitute various conformationally restricted amino acids in place of beta -Ala11 in the nonselective BRS-3 ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), and investigate the effects of their substitutions on affinity and potency for altering biological activity on the two mammalian bombesin receptor subtypes, hGRP-R and hNMB-R and the orphan receptor, hBRS-3. Using this approach we have identified a number of hBRS-3 selective ligands which function as fully efficacious agonists and two have sufficient selectivity and potency for the hBRS that they could be useful for investigating its role in physiological and pathological processes.

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Materials-- The following cells and materials were obtained from the sources indicated: BALB 3T3 and AR42J cells were from American Type Culture Collection (ATCC), Rockville, MD; Dulbecco's minimum essential medium and phosphate-buffered saline, from Biofluids, Rockville, MD; G418 sulfate and fetal bovine serum (FBS) from Life Technologies, Inc., Grand Island, NY; Na125I (2200 Ci/mmol) from Amersham Pharmacia Biotech; myo-[2-3H]inositol (20 Ci/mmol), 125I-Gastrin-17 (2000 Ci/mmol), 125I-Bolton-Hunter labeled CCK-8 (2000 Ci/mmol), 125I-somatostatin-14 (2000 Ci/mmol), 125I-VIP (2200 Ci/mmol), and 125I-PACAP (2000 Ci/mmol) were from PerkinElmer Life Sciences; 125I-BH-substance P (2000 Ci/mmol) was from Amersham Pharmacia Biotech; formic acid, ammonium formate, disodium tetraborate, soybean trypsin inhibitor, leupeptin, amastatin, phosphoramidon, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and bacitracin were from Sigma; 1,2,4,6-tetrachloro-3alpha ,6alpha -diphenylglycouril (IODO-GEN) from Pierce Chemical Co., Rockford, IL; AG 1-X8 resin from Bio-Rad, Richmond, CA; Bn, GRP, neuromedin B (NMB), and [Tyr4]Bn were from Bachem, Torrence, CA. Standard protected amino acids and other synthetic reagents were obtained from Bachem Bioscience Inc., King of Prussia, PA. Boc-(R,S)-3-carboxypiperidine (Cpi), Boc-(R,S)-trans-2amino-1-cyclohexane carboxylic acid (Ach), Boc-(R,S)-cis-2-amino-1-cyclohexane carboxylic acid (Achc), Anaspec, Inc., San Jose, CA; and Boc-(R)-3-amino-3-phenylpropionic acid, Boc-(R)-3-amino-3-(4-chlorobenzyl)-propionic acid (Acpb4), and Boc-(R)-3-amino-3-(2-chlorobenzyl)-propionic acid (Acpb2) (Fig. 1) from ChiroTec, Cambridge, United Kingdom. All chemicals were reagent grade.

Cell Culture-- BALB 3T3 cells stably expressing human BRS-3 receptors, human NMB receptors, or human GRP receptors were made as described previously (8, 11, 17). CHO cells stably expressing human vasoactive intestinal peptide receptor subtype 1 (hVIP1-R) and subtype 2 (hVIP2-R) were made as described previously (18). The cells were grown in Dulbecco's modified Eagle's cell media supplemented with 300 mg/liter of G418 sulfate and incubated at 37 °C in a 5% CO2 atmosphere.

Preparation of Peptides-- The peptides were synthesized with solid-phase methods as described previously (14, 19, 20). Briefly, solid-phase syntheses of peptide amides were carried out using Boc chemistry on methylbenzhydrylamine resin (Advanced ChemTech, Louiville, KY) followed by HF-cleavage of free peptide amides. The crude peptides were purified, and in some cases separated into (R)-isomers and (S)-isomers by preparative high liquid chromatography on columns (2.5 × 50 cm) of Vydac C18 silica (10 µm) which was eluted with linear gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid. Homogeneity of the peptides was assessed by analytical reverse-phase high-pressure liquid chromatography and purity was usually 97% or higher. Amino acid analysis (only amino acids with primary amino acid groups were quantitated) gave the expected amino acid ratios. Peptide molecular masses were obtained by matrix-assisted laser desorption mass spectrometry (Thermo Bioanalysis Corp., Hemel, Helmstead, UK) and all corresponded well with calculated values. The unresolved, conformationally restricted, amino acid substitution yielded peptide diastereoisomers, many of which could be resolved during final peptide purification. The optical configurations were tentatively assigned by comparison of their elution behavior with an authentic resolved diastereoisomer (Table I).

Preparation of 125I-[D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), 125I[Tyr4]Bn, 125I-[D-Tyr0]NMB-- These radioligands, with specific activities of 2200 Ci/mmol, were prepared as previously described (8, 17). Briefly, 0.8 µg of IODO-GEN solution (0.01 µg/µl in chloroform) was added to a 5-ml plastic test tube, dried under nitrogen, and washed with 100 µl of 0.5 M potassium phosphate solution (pH 7.4). To this tube 20 µl of potassium phosphate solution (pH 7.4), 8 µg of peptide in 4 µl of water, and 2 mCi (20 µl) of Na125I were added and incubated at room temperature for 6 min. The incubation was stopped by the addition of 100 µl of water and heated with 300 µl of 1.5 M dithiothreitol for 60 min at 80 °C. 125I-[D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) which does not have a COOH-terminal methionine group was not incubated with the dithiothreitol. The radiolabeled peptides were separated using a Sep-Pak (Waters Associates, Milford, MA) and further purified by reverse-phase high performance liquid chromatography, as previously described (8, 17). The fractions with the highest radioactivity and binding were neutralized with 0.2 M Tris buffer (pH 9.5) and stored with 0.5% bovine serum albumin (w/v) at -20 °C.

Preparation of Dispersed Guinea Pig Pancreatic Acini-- Dispersed acini from the guinea pig pancreas was prepared using the modification (21) of the method described previously (22). Dispersed acini were suspended in 10 ml of standard incubation solution containing 0.05% (w/v) bacitracin. Standard incubation solution contained (in mM) 24.5 HEPES (pH 7.45), 98 NaCl, 6 KCl, 2 KH2PO4, 5 sodium pyruvate, 5 sodium fumarate, 5 sodium glutamate, 2 glutamine, 11.5 glucose, 0.5 CaCl2, 1 MgCl2, 1% (w/v) bovine serum albumin, 0.2% (w/v) soybean trypsin inhibitor, 1% (v/v) amino acid mixture, and 1% (v/v) essential vitamin mixture. All incubations were at 37 °C. The incubation solution was equilibrated with 100% O2.

Binding of 125I-Labeled Peptides to BALB 3T3 Cells Transfected with hBRS-3, hGRP-R, or hNMB-R-- The standard binding buffer contained 24.5 mM HEPES (pH 7.4), 98 mM NaCl, 6 mM KCl, 2.5 mM NaH2PO4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM MgCl2, 0.01% (w/v) soybean trypsin inhibitor, 0.2% (w/v) bovine serum albumin, and 0.05% (w/v) bacitracin. BALB 3T3 cells stably expressing hBRS-3 (0.5 × 106/ml), hGRP-R (0.2 × 106/ml), or hNMB-R (0.1 × 106/ml) were incubated with 50 pM 125I-labeled ligand at 22 °C for 60 min. Aliquots (100 µl) were removed and centrifuged through 300 µl of incubation buffer in 400-µl Microfuge tubes at 10,000 × g for 1 min using a Beckman Micro-centrifuge B. The pellets were washed twice with buffer and counted for radioactivity in a gamma -counter. The nonsaturable binding was the amount of radioactivity associated with cells in incubations containing 50 pM radioligand (2200 Ci/mmol) and 1 µM unlabeled ligand. Nonsaturable binding was <10% of total binding in all the experiments. Receptor affinities of ligands were determined using a least-square curve-fitting program (LIGAND) (23) and the Cheng-Prusoff equation (24).

Binding of 125I-Ligands to Dispersed Guinea Pig Pancreatic Acini, AR42J Cells, and hVIP1-R and hVIP2-R-transfected CHO Cells-- Dispersed guinea pig pancreatic acini (1 pancreas/10 ml), AR42J cells (1 × 106/ml), hVIP1-R transfected CHO cells (0.2 × 106/ml), and hVIP2-R transfected CHO cells (1.5 × 106/ml) were suspended in standard incubation solution with 0.05% (w/v) bacitracin. Incubations with 125I-somatostatin and 125I-secretin contained 10 mM phosphoramidon, leupeptin (25 µg/ml), 4-(2-aminoethyl)-benzenesulfonyl fluoride (10 µM), and amastatin (10 nM). 125I-Secretin was prepared using IODO-GEN and purified using high performance liquid chromatography as described previously (25). Incubations with ligands (125I-secretin, 125I-BH-CCK8, 125I-VIP, 125I-PACAP, 125I-gastrin, 125I-BH-substance P, 125I-somatostatin-14, or 125I-[Tyr4]Bn) were for 60 min at 37 °C with pancreatic acini and 23 °C with AR42J cells or hVIP-R-transfected cells. Binding was performed as described (18, 25-29). Briefly, 50 pM ligand was incubated with the cells alone, with unlabeled other peptides at the specified concentration or with 1 µM unlabeled ligand to determine saturable binding. Bound ligand was separated from free ligand by centrifugation. Tubes were washed twice with 2% (w/v) bovine serum albumin in standard incubation solution and radioactivity was counted. Nonsaturable binding was less than 30% of total binding.

Measurement of [3H]IP-- Changes in total [3H]inositol phosphates ([3H]IP) were measured as described previously (11, 30, 31). Briefly, hBRS-3-, hGRP-R-, or hNMB-R-transfected BALB 3T3 cells were subcultured into 24-well plates (5 × 104 cells/well) in regular propagation media and then incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The cells were then incubated with 3 µCi/ml myo-[2-3H]inositol in growth media supplemented with 2% fetal bovine serum for an additional 24 h. Before assay, the 24-well plates were washed by incubating for 30 min at 37 °C with 1 ml/well of phosphate-buffered saline (pH 7.0) containing 20 mM lithium chloride. The wash buffer was aspirated and replaced with 500 µl of IP assay buffer containing 135 mM sodium chloride, 20 mM HEPES (pH 7.4), 2 mM calcium chloride, 1.2 mM magnesium sulfate, 1 mM EGTA, 20 mM lithium chloride, 11.1 mM glucose, 0.05% bovine serum albumin (w/v) and incubated with or without any of the peptides studied. After 60 min of incubation at 37 °C, the experiments were terminated by the addition of 1 ml of ice-cold 1% (v/v) hydrochloric acid in methanol. Total [3H]IP was isolated by anion exchange chromatography as described previously (11, 30, 31). Briefly, samples were loaded onto Dowex AG1-X8 anion exchange resin columns, washed with 5 ml of distilled water to remove free [3H]inositol, then washed with 2 ml of 5 mM disodium tetraborate, 60 mM sodium formate solution to remove [3H]glycerophosphorylinositol. Two ml of 1 mM ammonium formate, 100 mM formic acid solution was added to each of the columns to elute total [3H]IP. Each eluate was mixed with scintillation mixture and measured for radioactivity in a scintillation counter.

Molecular Modeling-- All molecular modeling was performed on a Silicon Graphics Indigo High Impact 10000 computer, using SYBL 6.6 (32) with the standard Kollman all atom force field (33). The initial partial sequence (I), Val10-D-Ala11-His12-Phe13, was built from the predefined amino acids and its conformation set to a beta -sheet. The type II bend was introduced around D-Ala11-His12. The Val was blocked by the addition of an acetyl group and an N-methyl amide group blocked Phe13. Kollman atomic charges were imported for the molecule. The structure was then optimized by energy minimization using the conjugate gradient algorithm to a final root mean square gradient of <= 0.01 Kcal mol Å-1. A distance-dependent dielectric function (34) was employed together with the default settings for all the other minimization options. To facilitate the substitution of the unusual beta -amino acids into this structure, D-Ala11 was first replaced by beta -Ala11 (II). Minimization of the strained beta -Ala11 sequence was carried out by a root mean square fit of the common sequences and the beta -alanine backbone atoms.

Each minimized tetrapeptide was subjected to molecular dynamics at 300 K for >= 20 ps using the Kollman force field with a distance-dependent dielectric function. The molecular trajectories were recorded at 100-fs intervals and the ValCO-PheNH distances, ValCA-PheCA distances, and ValC-XCA-HisCA-PheN torsion angles measured.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Previous studies demonstrated that the novel orphan receptor, hBRS-3, has a unique pharmacology from the other two well studied mammalian bombesin receptor subtypes, the hGRP-R and hNMB-R, with which it has 47-50% amino acid homology (1, 2, 8, 11, 13-16). None of the natural occurring Bn-related peptides have high affinity for the hBRS-3 receptor (1, 8). However, the synthetic Bn analogue, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and its D-Phe6 derivative were found to have high affinities for the hBRS-3 receptor (8). Unfortunately, these two peptides had similar high affinities for all the three known Bn receptor subtypes (i.e. GRP-R, NMB-R, and BB-4) (8, 10, 16). In one previous study (8) it was concluded that the beta -Ala11 substitution in the synthetic peptide [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) is likely responsible for its high affinity for the BRS-3 receptor. The purpose of the present study was to attempt to discover a hBRS-3 selective agonist and the approach used, based on the above proposal (8), was to primarily apply conformational restrictions to position 11 of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) using 8 unusual, conformationally restricted cyclic and alpha - and beta -substituted amino acids (Fig. 1). We investigated the ability of each of these peptides to interact with the hBRS-3 receptor and the other two mammalian Bn-related receptors by determining their binding affinities and biological activities in BALB 3T3 cells transfected with hBRS-3, hGRP-R, and hNMB-R. These transfected cells were used because previous studies have demonstrated that hBRS-3 and other bombesin receptor subtypes transfected into these cells have similar pharmacology and cellular coupling to cells possessing native receptors (8, 13, 30, 31).


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Fig. 1.   Structure of various conformationally restricted amino acids.

The first group of compounds we studied included Bn, NMB, Bn-(6-14), [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and 3 analogues of [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (peptide 1, Table I) with substitutions for the phenylalanine in position 13 (peptides 3-5, Table I) which has been demonstrated previously to be important for determining high affinity interaction with bombesin receptors (35). Bn, NMB, and Bn-(6-14) had very low affinity (Ki >10,000 nM) for the hBRS-3 (Fig. 2, top panel; Table I) which was consistent with results from previous studies in other species (1, 8, 11, 13). The low affinities for hBRS-3 are markedly different from the ability of these peptides to interact with hGRP-R (Fig. 2, middle panel; Table I) or with the hNMB-R (Fig. 2, bottom panel; Table I). Similar to previous results (8, 13, 16, 36) for Bn receptors from other species, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) had high affinities at all the three human Bn receptor subtypes (Fig. 2, Table I). The replacement of phenylalanine in position 13 of [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) by leucine (peptide 3, Table I), 4-chlorophenylalanine (peptide 4, Table I), or beta -naphthylalanine (peptide 5, Table I) did not produce any hBRS-3 selective compounds. However, the selectivity for hGRP-R increased in the case of each of these position 13 substitutions, with the 4-chlorophenylalanine substitution resulting in a highly selective hGRP-R ligand. Specifically, peptide 4 (Table I) with the 4-chlorophenylalanine substitution in position 13 (Table I, Fig. 2) had almost a 200-fold selectivity for the hGRP-R over hBRS-3 and a 9200-fold selectivity for hGRP-R over hNMB-R (Table I, Fig. 2).

                              
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Table I
Affinities of bombesin, neuromedin B, and various synthetic bombesin analogues for human BRS-3, GRP, and NMB receptors
Data are calculated from dose-inhibition curves in Figs. 2-4. The affinities for [D-Phe6, beta -Ala11,Phe13,Nle14]Bn-(6-14), Bn, and NMB were calculated by a least-squares, curve-fitting program (LIGAND) (23). The remaining affinities were calculated using the Cheng-Prusoff equation (24). All values are mean ± S.E. from at least three experiments. The following abbreviations used are: beta -Ala, beta -alanine; Nle, norleucine; Cpa, 4-chlorophenylalanine; Nal, -beta -naphthylalanine, des, deletion of indicated amino acid; Ac, acetyl.


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Fig. 2.   Ability of NMB, Bn, and various analogues of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) altered in positions 6 or 13 to inhibit binding to hBRS-3- (top), hGRP-R- (middle), or hNMB-R- (bottom) transfected BALB 3T3 cells. BALB 3T3 cells stably transfected with hBRS-3 (0.5 × 106 cells/ml), hGRP-R (0.2 × 106 cells/ml), and hNMB-R (0.1 × 106 cells/ml) were incubated for 60 min at 22 °C with 50 pM 125I-[D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), 125I-[Tyr4]Bn, or 125I-[D-Tyr0]NMB, respectively, with or without the indicated concentration of the various peptides added. Results are expressed as the percentage of saturable binding without unlabeled peptide added (percent control). Results are the mean ± S.E. from at least three experiments, and in each experiment the data points were determined in duplicate. Numbers refer to the peptide number in Table I.

We next explored the importance of position 11 by determining the ability of 12 [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) analogues with different conformationally restricted amino acid substitutions (Fig. 1) for the beta -alanine in position 11, to interact with each of the human Bn-related receptors (Fig. 3). Among these 12 peptides were four sets of (S)- and (R)-optical isomers (peptides 6 and 7; 8 and 9; 10 and 11; 14 and 15; Fig. 3, Table I). In general, there were not large differences in the effect on the affinity for the hBRS-3 receptor between the (S)- and (R)-optical isomers of these position 11 substituted analogues as shown by the ratio of the affinities of these peptides to those of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Table I). Except for peptide 10, with a (R)-3-amino butyric acid substitution at position 11 that was equipotent with [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) at both the hBRS-3 and the hGRP-R, the rest of the peptides in this group were about 5-30 times less potent at the hGRP-R and 60-100-times less potent at the hNMB-R than [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14). With two of the four sets of optical isomers (peptides 8 and 9, 14 and 15; Table I) the decrease in affinity with the (R)-isomer was greater for the hGRP-R than for the hBRS-3 receptor and therefore, the (R)-isomer was more selective for the hBRS-3 receptor. Each of the 12-position 11-substituted analogues except for peptide 13 with a (R,S)-cis-2-amino-1-cyclohexane-carboxylic acid substitution and peptides 8 and 9 (Table I, Fig. 3) with an (R)- or (S)-trans-2-amino-1-cyclohexanecarboxylic acid substitution, retained relatively high affinity (Ki <30 nM) for the hBRS-3 receptor (Table I, Fig. 3). In this group of peptides with position 11 substitutions we found eight peptides with varying selectivity for the hBRS-3 over the hGRP-R and the hNMB-R (Table I). Specifically, peptides 8, 9, 10, 11, and 14-17 (Table I, Fig. 3) had selectivities ranging from 2- to 60-fold for the hBRS-3 over the hGRP-R and varying from 10- to 593-fold for the hBRS-3 over the hNMB-R. The two compounds that showed the highest selectivity for the hBRS-3 over the hGRP-R and the hNMB-R were peptides 14 and 15 with an (R)- or (S)-3-amino-3-phenyl-propionic acid replacement at position 11 (Figs. 1 and 3, Table I), respectively. The (R)-isomer (peptide 14; Table I, Fig. 3) had the highest selectivity for the hBRS-3 over the hGRP-R (60-fold) and the hNMB-R (116-fold). The (S)-isomer (peptide 15, Table I, Fig. 3) had the second highest hBRS-3 selectivity of 20-fold over the hGRP-R and 130-fold over the hNMB-R. The most selective hBRS-3 peptide, [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14) (peptide 14) (Table I, Fig. 3), retained high affinity for the hBRS-3, however, it was 10 times less potent than the original peptide, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (peptide 1, Table I) at the hBRS-3 (Ki = 4.1 ± 0.3 nM versus 0.32 ± 0.08, respectively).


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Fig. 3.   Ability of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and various analogues with position 11 conformationally restricted substitutions to inhibit binding to hBRS-3- (top), hGRP-R- (middle), or hNMB-R- (bottom) transfected BALB 3T3 cells. The experimental conditions were similar to those outlined in the legend to Fig. 2. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percent control) and are mean ± S.E. from at least three experiments with each data point determined in duplicate. Numbers refer to peptide numbers in Table I.

Alterations in chain length due to a deletion or addition of amino acids can lead to changes in binding affinities and at times biological properties of peptides interacting with Bn-related or other gastrointestinal hormone receptors, and these changes may have different effects even for closely related receptors (35, 37). Therefore, the third group of peptides we made included five peptides containing alterations in the chain length with amino acid deletions and/or additions at positions 6, 7, 9, or 11 of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Table I, Fig. 4). These types of alterations yielded peptides that were 300- to >1000-fold less potent (Table I, Fig. 4) than the initial peptide (peptide 1, Table I) [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14)), at each of the 3 mammalian Bn-related receptor subtypes, making these peptides (peptides 18, 19, 20, 21 and 22, Table I) generally not useful. An extension of [D-Tyr6,beta -Ala11, Phe13,Nle14]Bn-(6-14) at position 11 by the substitution of an alanine and glycine for the beta -alanine (peptide 20, Table I) did not result in a more selective hBRS-3 ligand. Similarly, deletion of [D-Tyr6] and Gln7 from the amino terminus of [D-Tyr6,Aba11,Phe13,Nle14]Bn-(6-14) (peptides 10 and 11, Table I) resulted in a 967-fold decrease in the affinity for hBRS-3 for the (R)-isomer (peptide 19, Table I, Fig. 4) and a 240-fold decrease for the (S)-isomer (peptide 18, Table I, Fig. 4). However, with the (S)-isomer (peptide 18, Table I, Fig. 4), a greater decrease in affinity occurred with the two mammalian Bn receptors than with the hBRS-3 receptor so that this compound was 6-fold more selective for the hBRS-3 (Table I, Fig. 4). Deletion of the alanine in position 9 and substitution of the constrained amino acid 3-aminobutyric acid in position 10 for valine in the (R)- or (S)-conformation did not result in more hBRS-3-selective ligands (peptides 21 and 22, Table I, Fig. 4).


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Fig. 4.   Comparison of the ability of [D-Tyr6,beta -Ala11, Phe13,Nle14]Bn-(6-14) and various analogues with changes due to amino acid deletion, addition, or substitution at positions 6, 7, 9, and 11 to inhibit binding to the hBRS-3- (top), the hGRP-R- (middle), and the hNMB-R- (bottom) transfected BALB 3T3 cells. The experimental conditions were same as outlined in the legend to Fig. 2. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percent control). Results are the mean ± S.E. of at least three experiments and in each experiment the data points were determined in duplicate. Numbers refer to the peptide number in Table I.

To determine whether the insertion of the conformationally restricted (R)-Apa11 or (S)-Apa11 decreased the specificity of the resultant peptide for the hBRS-3 receptor, its ability to interact with a number of other receptors for gastrointestinal hormones/neurotransmitters was determined (Table II). To investigate their ability to interact with other unrelated receptors, binding to receptors on guinea pig pancreatic acini and AR42J cells was assessed because both possess both GRP-R receptors and receptors for a number of other GI hormones/neurotransmitters (35) as well as to hVIP-R transfected CHO cells (18) (Table II). Neither of the two most selective hBRS-3 peptides ([D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14) and ([D-Tyr6,(S)-Apa11,Phe13,Nle14]Bn-(6-14), peptides 14 and 15, Table I) inhibited binding to receptors for cholecystokinin, gastrin, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP) (VIP1-R and VIP2-R), or substance P (Table II). The peptides in the different assays were biologically active because the assays were performed under the same conditions used to assess binding to bombesin receptors and in the different assays using the same cells, each of the Apa11-substituted peptides inhibited binding to bombesin receptors (Table II). These results demonstrate that the (R)-Apa11 and (S)-Apa11 analogues retain high selectivity for human bombesin receptors.

                              
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Table II
The ability of hBRS-3 selective analogues [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14) (peptide 14) and [D-Tyr6,(S)-Apa11,Phe13,Nle14]Bn-(6-14) (peptide 15), and the nonselective ligand [D-Tyr6, beta -Ala11,Phe13,Nle14]Bn-(6-14) (peptide 1) to interact with various GI hormone/neurotransmitter receptors
The following abbreviations were used: r, rat; h, human. AR42J cells (1 × 106 cells/ml), dispersed guinea pig acini (1 pancreas/10 ml), hVIP1-R (0.2 × 106 cells/ml), and hVIP2-R (3.0 × 106 cells/ml) stably transfected into CHO cells were incubated with 50 pM of the indicated 125I-ligand for 60 min as specified under "Experimental Procedures." Results are the percentage of the total saturable binding with or without 1 µM peptide 1, 14, or 15 (Table I). With 125I-BH-CCK-8 binding to AR42J cells, 0.1 µM gastrin-17 was added to inhibit binding to CCKB-R. All results are mean ± S.E. from at least three experiments and in each experiment each value was performed in duplicate.

Previous studies have demonstrated that hBRS-3 as well as hGRP-R and hNMB-R activation are coupled to stimulation of phospholipase C and activation results in increases in intracellular inositol phosphates and [Ca2+]i (1, 2, 11, 13-15, 31, 38, 39). To determine whether the peptides included in this study had agonist activity, we first studied the ability of these peptides to activate phospholipase C and cause increases in inositol phosphates. All the peptides were agonists, causing increases in total [3H]IP at a concentration of 1 µM in at least one human mammalian Bn-related receptor subtype (Table III). The increases in [3H]IP stimulation for all peptides were highest in hNMB-R containing cells, ranging from 1.7 ± 1.2 to 24.7 ± 0.1-fold over basal. The maximal increases in [3H]IP in hBRS-3 and hGRP-R stimulated by the various peptides were similar, ranging from 1.3 ± 0.7 to 3.5 ± 0.2. Importantly, the two most selective hBRS-3 ligands, the (R)- and (S)-isomers of [D-Tyr6,Apa11,Phe13,Nle14]Bn-(6-14) (peptides 14 and 15, Table II) both stimulated an increase in [3H]IP in the hBRS-3-transfected BALB 3T3 cells at 1 µM to the same fold as that caused by the nonselective high affinity ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) at a 1 µM maximally effective concentration (peptide 1, Table III).

                              
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Table III
Ability of bombesin, neuromedin B, and various synthetic bombesin analogues to stimulate an increase in [3H]inositol phosphates in human BRS-3, GRP, or NMB receptor-containing cells

To determine whether the selectivity detected by binding studies was mirrored in their abilities to activate these receptors, we performed dose-response curves for their abilities to stimulate increases in [3H]IP in hBRS-3 and hGRP-R transfected into BALB 3T3 cells to assess the potencies of the five most hBRS-3-preferring peptides (peptides 11, 14, 15, 17, and 18) and compared them with those of Bn, NMB, and the two initial beta -Ala11 peptides (peptides 1 and 2; Tables I, 3, and 4; Fig. 5). All peptides stimulated increases in [3H]IP in a concentration-dependent manner at the hBRS-3 (Fig. 5) and hGRP-R (Fig. 5). Similar to results in the binding experiments (peptide 1; Table IV, Fig. 2), [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) and its [Phe6] analogue (peptide 2; Table IV, Fig. 5) had similar high potencies for both hBRS-3 and hGRP-R in stimulating [3H]IP increases. Both of these nonselective peptides (peptides 1 and 2, Table IV, Fig. 5) had potencies only 2-3-fold lower than Bn for the hGRP-R and potencies of >300 times greater than Bn for activating the hBRS-3 receptor (Fig. 5, Table IV). The two Apa11 substituted compounds, peptides 14 and 15 (Table I), which were selective for hBRS-3 in binding studies, were full agonists at the hBRS-3 (Table IV) and had selectivity for activating the hBRS-3 over the hGRP-R (Fig. 5, Table IV). Specifically, peptide 14, [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14), caused a half-maximal increase in [3H]IP at 5.8 ± 0.9 nM at the hBRS-3 receptor (Fig. 5, top) compared with 417 ± 10 nM for the hGRP-R (Fig. 5, bottom), a 72-fold selectivity for the hBRS-3. Peptide 15, [D-Tyr6,(S)-Apa11,Phe13,Nle14]Bn-(6-14), caused a half-maximal effect at 3.8 ± 0.3 nM at the hBRS-3 receptor compared with 182 ± 42 nM with the hGRP-R, a selectivity of 48-fold for hBRS-3. The other hBRS-3 preferring compounds identified by binding studies, peptides 11, 17, and 18 (Table IV, Fig. 5) were either slightly hBRS-3 selective or had generally similar potencies for the hBRS-3 and the hGRP-R (Table IV, Fig. 5) in stimulating an increase in [3H]IP.


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Fig. 5.   Ability of NMB, Bn, or various synthetic hBRS-3-selective ligands to stimulate [3H]IP formation in BALB 3T3 cells transfected with hBRS-3 (top) or hGRP-R (bottom). hBRS-3-transfected BALB 3T3 cells or hGRP-R-transfected BALB 3T3 calls were subcultured and preincubated for 24 h at 37 °C with 3 µCi/ml myo-[2-3H]inositol. The cells were then incubated with NMB, Bn, or the various BRS-3-selective ligands at the concentrations indicated for 60 min at 37 °C. Values are expressed as a percentage of total [3H]IP release stimulated by 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), and are mean ± S.E. from at least three experiments. With hBRS-3/BALB 3T3 cells the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) values were 6,137 ± 377 and 36,800 ± 2,560 dpm, respectively. With hGRP-R/BALB 3T3 cells the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) were 3,276 ± 340 dpm and 11,280 ± 955 dpm.

                              
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Table IV
Potency of bombesin-related peptides and various hBRS-3 selective ligands for stimulating phospholipase C activity in hBRS-3, hGRP-R, and hNMB-R containing cells
hBRS-3, hGRP-R, or hNMB-R BALB 3T3 cells were incubated with [3H]inositol and total [3H]IP determined as described in Table I. For each peptide a dose-response curve was performed with concentrations from 0.01 nM to 1 µM. The concentration causing a half-maximal increase, EC50, was calculated for each peptide using Kaleidagraph. Each value is a mean ± S.E. from at least three experiments. For hBRS-3/BALB cells the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) values were 6,140 ± 380 dpm and 36,800 ± 2,560 dpm, respectively. For hGRP-R/BALB 3T3 cells the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) values were 3,300 ± 350 dpm and 11,300 ± 960 dpm, respectively. With the hNMB-R BALB 3T3 cells the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) values were 4,300 ± 340 dpm and 57,600 ± 9,200 dpm, respectively.

To determine whether the substituted peptide's selectivity for hBRS-3 over hNMB-R detected by binding studies was mirrored in their abilities to activate NMB-R receptors, we performed dose-response curves for their abilities to stimulate increases in [3H]IP in hNMB-R transfected BALB 3T3 cells. The potencies of the two most hBRS-3-preferring peptides (peptides 14 and 15) was compared with those of NMB and GRP and the initial beta -Ala11 peptide (peptide 1; Tables I, 3, and 4; Fig. 6, Table IV). All peptides stimulated increases in [3H]IP in a concentration-dependent manner at the hNMB-R (Fig. 6). Similar to results in the binding experiments (peptide 1; Table IV, Fig. 2), [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) had a high affinity for stimulating an increase in [3H]IP at the hNMB-R causing a half-maximal increase at 0.6 ± 0.1 nM. GRP had a low potency causing half-maximal activation at 1300 ± 170 nM, whereas NMB had a high potency for activating the NMB-R causing a half-maximal increase in [3H]IP at 2.5 ± 0.1 nM (Fig. 6, Table IV). The two Apa11 substituted compounds, peptides 15 and 14 (Table I) which were selective for hBRS-3 in binding studies, were 100- and 180-fold less potent than the nonselective [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Fig. 6, Table IV) for activating hNMB-R and increasing [3H]IP demonstrating that the Apa11 substitution in peptide 1 decreased its potency for NMB-R activation similar to reducing its binding affinity (Fig. 6). Therefore, in comparison with their ability to activate hBRS-3 receptors, peptide 15, [D-Tyr6,(S)-Apa11,Phe13, Nle14]Bn-(6-14), caused a half-maximal effect at 3.8 ± 0.3 nM at the hBRS-3 receptor compared with 66 ± 3 nM with the hNMB-R, a selectivity of 17-fold for hBRS-3 (Table IV). Peptide 14, [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14), caused a half-maximal increase in [3H]IP at 5.8 ± 0.9 nM at the hBRS-3 receptor (Fig. 5, top) compared with 110 ± 5 nM for the hNMBP-R (Fig. 6, Table IV), a 19-fold selectivity for the hBRS-3.


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Fig. 6.   Ability of NMB, various synthetic hBRS-3-selective ligands, and GRP to stimulate [3H]IP formation in BALB 3T3 cells transfected with hNMB-R. NMB-R-transfected BALB 3T3 cells were subcultured and preincubated for 24 h at 37 °C with 3 µCi/ml myo-[2-3H]inositol. The cells were then incubated with NMB, GRP, or the various BRS-3-selective ligands at the concentrations indicated for 60 min at 37 °C. Values are expressed as a percentage of total [3H]IP release stimulated by 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), and are mean ± S.E. from at least three experiments. With the control and 1 µM [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) values were 4,261 ± 341 and 57,635 ± 9,178 dpm, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In recent studies we reported the discovery of a high affinity synthetic ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), and its D-Phe6 analogue that function as agonists for the orphan receptor hBRS-3 on both the native hBRS-3 receptor in human small cell cancer cell line, NCI-N417 (8, 11) and for hBRS-3-transfected BALB 3T3 cells or hBRS-3 transfected NCI-H1299 nonsmall cell lung cancer cells (8). Although these two peptides interact with high affinity with the hBRS-3 receptor, unfortunately they lack specificity because they also interact with high affinity with all the known Bn-receptor subtypes (GRP-R, NMB-R, and BB4-R) (8, 10, 11, 16). Previously, in our search for hBRS-3-specific ligands we tested all the naturally occurring bombesin-related peptides (such as Bn, NMB, GRP, phyllolitorin, litorin, and ranatensin) and 26 representative synthetic peptides from six different classes of GRP-R and NMB-R agonists and antagonists (8, 11). Whereas many of these had high affinities and selectivities for the GRP-R, NMB-R, or BB4-R, all were found to interact with the BRS-3 receptor with very low affinities in the micromolar ranges (8, 10, 12). These results demonstrated that the novel orphan receptor, hBRS-3, despite having a 47-55% amino acid homology with the Bn-related receptors (GRP-R, NMB-R, or BB4-R) (1, 2, 40), has a unique pharmacological profile which is quite different from that of the members of the structurally related bombesin receptor family.

In the present study, to attempt to develop a selective BRS-3 ligand we, therefore, used as a starting point the synthetic Bn analogue that functions as a high affinity but nonselective hBRS-3 ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (10, 11). The primary strategy used to attempt to identify a BRS-3 selective ligand was to substitute different conformationally restricted amino acids in the nonselective BRS-3 ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), and investigate the effects of their substitution on affinity and biological activity of the two mammalian bombesin receptor subtypes, hGRP-R and hNMB-R, and the orphan receptor hBRS-3. In a previous study (8) we suggested that the high affinity interaction with the BRS-3 receptor seen with [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), but not seen with bombesin, was likely principally due to the substitution of the beta -alanine in position 11 of bombesin for glycine and perhaps, to a lesser degree, from the substitution of the penultimate phenylalanine in position 13 for leucine in native bombesin. Therefore in this present study, most of the amino acid alterations were in positions 11 or 13 of the nonselective ligand, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14). The 20 peptides synthesized could be placed in three general groups depending on the position of amino acid change in the prototype compound, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14). One group was comprised of the natural occurring peptides, Bn and NMB; the synthetic Bn analogue, Bn-(6-14); and three synthetic analogues of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) with changes in the penultimate Phe13 position. The second group consisted of 12 peptides with changes in the beta -Ala11 position and the third group consisted of five peptides that had alterations in the chain length with amino acid deletions and/or additions at positions 6, 7, 8, 9, or 11.

The results from the first group of peptides demonstrate with the human BRS-3 receptor and the two human mammalian Bn receptors, our findings were consistent with previous findings in other species (8, 11, 16, 37) which showed that the naturally occurring bombesin-related peptides such as bombesin and neuromedin B have very low affinities (i.e. Ki >10 µM) for the hBRS-3 receptor compared with their high affinities for the hGRP-R or the hNMB-R, respectively. Alterations of the position 13 phenylalanine by adding an electron withdrawing chloro group to the phenyl ring, increasing the hydrophobicity by substituting a beta -naphthylalanine, or replacing the phenyl ring by an amino acid without an aromatic group (i.e. leucine) did not make the peptides more hBRS-3-selective. In contrast, each of the three position 13 substitutions increased the affinity for the hGRP-R with the greatest increase in hGRP-R selectivity seen with the 4-chlorophenylalanine substitution, which resulted in a peptide that had 200-fold selectivity for hGRP-R over hBRS-3 and 9200-fold selectivity for hGRP-R over the hNMB-R. This is in agreement with a previous study (37) which suggested that the presence of the penultimate phenylalanine is particularly important for high affinity for the NMB-preferring bombesin receptor. The present study also aimed to elucidate the structural features of position 11 (beta -Ala) of [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-13) which was previously shown to be uniquely responsible for high BRS-3 affinity (8) and thus a series of peptides were synthesized in which the beta -Ala structure was constrained either via amino acid backbone cyclization strategies or substitution of various groups either in the alpha - or beta -position of this amino acid. Of these, analogues containing (R)- and (S)-isomers of 3-carboxypiperidine (Cpi11) (peptides 6 and 7, Table I) retained binding affinity for the hBRS-3, but both were ~100-fold less potent than the parent peptide (peptide 1, Table I). Little difference in binding affinity between Cpi11 isomers was observed. Analogues 8 and 9 (Table I) containing an amino group and carboxyl group locked into the 1,2-trans position on a cyclohexane ring (amino acid iii, Fig. 1) were actually selective for the BRS-3 receptor in that they retained some affinity for it, albeit around 1000 times less than for analogue 1, at the expense of very little affinity for the GRP receptor and virtually none for NMB receptors. When the amino and carboxyl group were in the cis-configuration on the cyclohexane ring (structure iv, Fig. 1), the unresolved (R,S)-mixture of isomers exhibited almost no affinity for any of the 3 receptors, perhaps not surprisingly given the increased steric crowding that this configuration would produce.

More interesting effects on binding affinity were produced by substitutions present on either the alpha - or beta  -position of beta -Ala. Replacement of beta -Ala with its alpha -methyl-substituted analogue 3-aminoisobutyric acid (Aia, structure vi, Fig. 1) resulted only in about a 100-fold loss of both BRS-3 and GRP receptor binding when the unresolved (R,S)-mixture was screened. The (R)-isomer of 3-amino butyric acid (Aba, structure v, Fig. 1) which contains a methyl group in the beta -position of beta -Ala essentially retained full affinity for both BRS-3 and GRP receptors and although the (S)-isomer had less affinity for both receptors, a small preference for BRS-3 over GRP was evident (peptides 10 and 1, Table I). Thus, the beta -substitution approach was extended to use of a larger phenyl moiety via (R)- and (S)-3-amino-3-phenylpropionic acid (Apa, structure vii, Fig. 1) which finally yielded the desired dissociation of affinity between BRS-3 and GRP receptors. Although [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14) lost about 10-fold affinity for BRS-3, it lost 1210-fold affinity for GRP to give a 59-fold selectivity for BRS-3 receptors (peptide 14, Table I). Its (S)-isomer lost a little BRS-3 affinity but had slightly higher affinity for GRP, thus making it less selective (peptide 15, Table I). Although the structural optimization of the beta -substituent must await future synthetic work, it appears that a phenyl group is close to optimum since use of slightly larger and more flexible substituted benzyl groups as in amino acids (R)-Acpb4 and (R)-Acpb2 (structures viii and ix, Fig. 1) resulted in loss of affinity for both BRS-3 and GRP receptors as well as loss of selectivity (peptides 16 and 17, Table I).

In the absence of crystallographic or NMR structural data, it is of course difficult to assign the observed results to specific three-dimensional structural features of the various analogues. We have suggested previously (41) based on structure-activity studies on cyclized analogues that the bioactive, receptor-bound conformation of bombesin agonists consists of a type II beta -bend conformation encompassing residues Val10-Gly-His-Phe13. Additionally, it is known that beta -amino acids can adopt a variety of often more stable conformations relative to alpha -amino acids, including beta -turns (42). With this in mind, the tetrapeptide sequence of the (R)- and (S)-isomers of analogues 6-15 (Table I) were subjected to computer molecular modeling in a beta -bend template with the results shown in Fig. 7. From these initial molecular modeling studies, each beta -amino acid substituent was readily able to adopt a beta -bend conformation which overlapped well with the conformation of the Ala and beta -alanine analogues. These conformations were maintained during the short molecular dynamics simulation of each analogue. Thus, the major differentiation between these nonpolar, substituted beta -amino acids rests in the orientation of the substituent alkyl rings and methyl or phenyl groups in both the (R)- and (S)-isomers of the selective Apa11 analogue into an area of space unique to these peptides, possibly accounting for the loss of affinity of both of these peptides for GRP receptors and the quite similar affinities of both isomers for BRS-3 and GRP receptors. Generally, although the carboxypiperidine and cyclohexane-derived cyclic amino acids seem quite capable of adopting beta -turns, parts of the ring methylene groups occupy areas of space not filled in more potent, linear amino acid-containing structures, thus perhaps introducing adverse receptor interactions and accounting for their lower affinities (Fig. 7).


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Fig. 7.   Molecular modeling of the tetrapeptide sequence, Val10-X11-His12-Phe13, in the beta -turn conformation. All structures were aligned by an root mean square fit to the common residues and the beta -alanine backbone atoms of the sequence Val10-X11-His12-Phe13 as described under "Experimental Procedures." In the figure the energy minimized Bn tetrapeptide sequences in the beta -turn conformation are superimposed. The beta -amino acid residues are colored as follows: beta -Ala, white; Aba, blue; Aia, green; Apa, orange; Cpi, yellow; and trans-Ach, magenta (cis-Ach not shown for clarity) (see Fig. 1 for structures). The (i-i+3) hydrogen bonds characteristic of the beta -bend turn are shown in dotted yellow lines.

Changes in the peptide backbone length due to deletion or addition of amino acids may lead to changes in receptor affinities and biological activity of peptides that interact with some gastrointestinal hormone receptors and frequently different effects may be seen with even closely related receptors (35). To examine the effects of such changes, the third group of Bn analogues that we investigated contained alterations in the chain length with amino acid removal and/or addition at positions 6, 7, 9, or 11 (Table I, Fig. 4). These peptides had affinities 300- to >1000-fold less than the prototype peptide, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (peptide 1, Table I), at each of the three receptors. These alterations, in general, did not yield hBRS-3 selective compounds as were seen with conformationally restricted beta -Ala11 substitutions in the group 2 peptides. Specifically, from this group of compounds, Ac-[(S)-Aba11,Phe13,Nle14]Bn-(8-14) (peptide 18, Tables I and III) was the only one that showed some BRS-3 selectivity. This peptide (peptide 18, Table I) had a 6-fold higher affinity for the hBRS-3 over the hGRP-R but its low affinity (Ki 460 nM) for the hBRS-3 makes it only marginally useful as a potential selective ligand for the hBRS-3 receptor.

Activation of the hBRS-3 receptor as well as the two mammalian Bn receptors results in the elevation of phospholipase C activation (1, 2, 8, 11, 13, 14, 31, 39, 43, 44). To examine whether the above alterations in the prototype peptide, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), altered the potency for receptor activation in a similar fashion to changes in receptor binding affinity, their ability to activate phospholipase C and increase the generation of inositol phosphates was assessed. All the substituted peptides included in the three groups were agonists, causing increases in total [3H]IP stimulation at micromolar concentrations in at least one of the three receptors. Similar to the binding affinities, the prototype peptide, [D-Tyr6,beta -Ala11,Phe13,Nle14]Bn-(6-14), had high potencies for stimulating increases in [3H]IP at both the hBRS-3 receptor and the two mammalian Bn receptors. Similar to the binding affinity studies, the peptides that show the greatest selectivity for the hBRS-3 receptor over hGRP-R and hNMB-R in increasing [3H]IP stimulation were [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14) (peptide 14, Table IV) and [D-Tyr6,(S)-Apa11,Phe13,Nle14]Bn-(6-14) (peptide 15, Table IV). Specifically, peptide 14, [D-Tyr6,(R)-Apa11,Phe13,Nle14]Bn-(6-14), had a 72- and 19-fold higher potency for activating the BRS-3 receptor than the GRP-R or NMB-R, respectively, and peptide 15, [D-Tyr6,(S)-Apa11,Phe13,Nle14]Bn-(6-14), had a 48- and 17-fold greater potency for activating the BRS-3 than the hGRP-R and hNMB-R. These results demonstrated that each of these selective hBRS-3 ligands functioned as fully efficacious selective agonist at the hBRS-3 receptor.

In conclusion, using constrained amino acid substitution in the prototype nonselective ligand, [D-Tyr6,beta -Ala11,Phe13, Nle14]Bn-(6-14), at least two peptides were identified with sufficient selectivity for the BRS-3 over the hGRP-R or hNMB-R that they likely will be generally useful for investigating the possible role of BRS-3 activation in physiological or pathological processes.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprints should be addressed: National Institutes of Health, NIDDK, DDB, Bldg. 10, Room 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600; E-mail: robertj@bdg10.niddk.nih.gov.

Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M008737200

    ABBREVIATIONS

The abbreviations used are: BRS-3, bombesin receptor subtype 3; NMB-R, neuromedin B receptor; GPR, gastrin-releasing peptide receptor; CCK-8, COOH-terminal octapeptide of cholecystokinin; PACAP, pituitary adenylate cyclase activating peptide; VIP, vasoactive intestinal peptide; Bn, bombesin; CHO, Chinese hamster ovary; IP, inositol phosphate; GI, gastrointestinal; [Ca2+]i, intracellular Ca2+; Ac, acetyl; Cpi, Boc-(R,S)-3-carboxypiperidine; Ach, Boc-(R,S)-trans-2-amino-1-cyclohexane carboxylic acid; Achc, Boc-(R,S)-cis-2-amino-1-cyclohexane carboxylic acid; Acpb4, Boc-(R)-3-amino-3-(4-chlorobenzyl)-propionic acid; Acpb2, Boc-(R)-3-amino-3-(2-chlorobenzyl)-propionic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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