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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Identification of Bombesin Receptor Subtype-Specific Ligands: Effect of N-Methyl Scanning, Truncation, Substitution, and Evaluation of Putative Reported Selective Ligands

From the Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland (S.A.M., N.G., M.S., T.K.P., L.S., R.T.J.); and the Department of Medicine, Peptide Research, Tulane University Health Science Center, New Orleans, Louisiana (D.H.C.)

Received April 27, 2006; accepted August 28, 2006.

	Abstract

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Mammalian bombesin (Bn) receptors include the gastrin-releasing peptide receptor, neuromedin B receptor, and bombesin receptor subtype 3 (BRS-3). These receptors are involved in a variety of physiological/pathologic processes, including thermoregulation, secretion, motility, chemotaxis, and mitogenic effects on both normal and malignant cells. Tumors frequently overexpress these receptors, and their presence is now used for imaging and receptor-mediated cytotoxicity. For these reasons, there is an increased need to develop synthetic, selective receptor subtype-specific ligands, especially agonists for these receptors. In this study, we used a number of strategies to identify useful receptor subtype-selective ligands, including synthesizing new analogs (N-methyl-substituted constrained analogs, truncations, and substitutions) in [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14), which has high affinity for all Bn receptors and is metabolically stable, as well as completely pharmacologically characterized analogs recently reported to be selective for these receptors in [Ca²⁺]_i assays. Affinities and potencies of each analog were determined for each human Bn receptor subtype. N-Methyl substitutions in positions 14, 12, 11, 10, 9, and 8 did not result in selective analogs, with the exception of position 11, which markedly decreased affinity/potency. N-Terminal truncations or position 12 substitutions did not increase selectivity as previously reported by others. Of the four shortened analogs of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) reported to be potent selective BRS-3 ligands on [Ca²⁺]_i assays, only AcPhe,Trp,Ala,His(Bzl),Nip,Gly,Arg-NH₂ had moderate selectivity for hBRS-3; however, it was less selective than previously reported Apa¹¹ analogs, demonstrating these are still the most selective BRS-3 analogs available. However, both of these analogs should be useful templates to develop more selective BRS-3 ligands.

Studies suggest that Bn receptors are involved in a wide variety of physiological and pathological processes, including in the CNS (satiety, thermo-regulation, and regulation of blood pressure), regulating metabolism (glucose metabolism and energy balances), regulating normal growth and development, regulating immunologic responses, and mediating various numerous gastrointestinal responses (motility and secretion) (Bunnett, 1994; Ohki-Hamazaki et al., 2005). Bn peptides have potent growth effects on numerous human tumors and function as autocrine growth factors (Jensen and Moody, 2006). Furthermore, because so many human neoplasms overexpress Bn receptor subtypes, their presence is currently being extensively evaluated, both to allow localization of these tumors by imaging methods as well as to deliver cytotoxic agents to these tumors (Moody et al., 2004).

Because of their widespread roles in both normal and neoplastic tissues, the development of synthetic ligands, which are selective for one Bn receptor subtype and function as selective agonists or antagonists, is important. For imaging studies and the use of Bn receptor overexpression or ectopic expression to deliver receptor-mediated cytotoxic agents, the development of selective agonists, particularly, if metabolically stable, is important because they are rapidly internalized (Benya et al., 1992; Mantey et al., 1993). There are relatively few selective synthetic Bn analogs that are metabolically stable for the various Bn receptor subtypes. This has been a particular problem with the BRS-3 receptor because its natural ligand is unknown and only a few synthetic ligands have been described that interact with this receptor with moderate to high affinity (Mantey et al., 1997; Pradhan et al., 1998; Ryan et al., 1998b; Weber et al., 2002, 2003; Boyle et al., 2005). This is important because recent studies using BRS-3-deficient mice, produced by targeted disruption, develop hypertension, obesity, and diabetes (Ohki-Hamazaki et al., 1997); however, little is known about the role of BRS-3 in these processes because of the lack of selective ligands.

In this study, in an attempt to identify selective ligands for Bn receptor subtypes, we used a number of different strategies. First, we have synthesized N-methyl-substituted constrained analogs, such as those of [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14), because this analog has been shown to be metabolically stable and to have high affinity and potency for activating all Bn receptor subtypes (Mantey et al., 1997; Pradhan et al., 1998; Ryan et al., 1998a,b; Reubi et al., 2002; Moody et al., 2004). This approach was used because it has successfully yielded highly selective peptide ligands with a number of other receptors (Reissmann et al., 1996; Pradhan et al., 1998; Rajeswaran et al., 2001). The second approach involved synthesizing analogs of [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) that were N-terminally truncated or had selective substitutions, because this was reported to result in increasing selectivity for one type of Bn receptor in a recent study (Darker et al., 2001). The final approach used was to synthesize various shortened [D-Tyr⁶, Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) analogs recently reported to be selective for a Bn receptor subtype and fully characterize them pharmacologically by binding studies and assays assessing receptor activation at each of the three human Bn receptor subtypes. This latter strategy was included because these agents were not fully characterized at each receptor pharmacologically in these studies, with only calcium or calcium FLIPR assay results available in most cases. Finally, we compared the selectivity for the BRS-3 receptor of the most potent and selective shortened [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) analogs to two full-length analogs we described recently (Mantey et al., 2001, 2004).

	Materials and Methods

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Materials. The following cells and materials were obtained from the sources indicated. BALB 3T3 (mouse fibroblast) cells were from American Type Culture Collection (ATCC, Manassas, VA); Dulbecco's minimum essential medium, phosphate-buffered saline, RPMI 1640 medium, trypsin-EDTA, and fetal bovine serum were from Biofluids (Rockville, MD); G418 sulfate was from Life Technologies, Inc. (Grand Island, NY); Na¹²⁵I (2200 Ci/mmol) and myo-[2-³H]inositol (20 Ci/mmol) were from Amersham Pharmacia Biotech (Piscataway, NJ); formic acid, ammonium formate, disodium tetraborate, soybean trypsin inhibitor, and bacitracin were from Sigma-Aldrich (St. Louis, MO); Dowex AG 1-X8 anion-exchange resin was from Bio-Rad (Richmond, CA); Bn, gastrin-releasing peptide (GRP), neuromedin B (NMB), and [Tyr⁴]Bn were from Bachem, (Torrance, CA); and bovine serum albumin was from ICN Pharmaceutical Inc. (Aurora, OH).

Cell Culture. Balb 3T3 cells stably expressing human BRS-3 receptor (hBRS-3), human NMB receptor (hNMBR), or human GRP receptor (hGRPR) were made as described previously (Benya et al., 1995; Mantey et al., 1997; Ryan et al., 1998a) and grown in Dulbecco's modified Eagle's cell medium supplemented with 300 mg/l G418 sulfate. The cells were mycoplasma-free and were used when they were in exponential growth phase after incubation at 37°C in 5% CO₂, 95% air.

Strategies Used and Preparation of Peptides. In this study, we attempted to develop or identify selective ligands for human Bn receptors, primarily concentrating on orphan receptor human BRS-3. We have reported previously that the Bn analog [D-Phe⁶,-Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 1, Peptide 2), has high affinity for all known Bn receptors (Mantey et al., 1997, 2001; Pradhan et al., 1998; Ryan et al., 1998a; Katsuno et al., 1999). Recently, analogs of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) have been described (Darker et al., 2001; Mantey et al., 2001, 2004; Weber et al., 2003, 2002) that are reported to have selectivity for hBRS-3 or the other Bn receptor subtypes. In three studies (Weber et al., 2002, 2003; Boyle et al., 2005), this selectivity was based on assessment of changes in [Ca²⁺]_i using a FLIPR assay with no direct assessment of receptor affinities. In the present study, we have used a number of strategies known to produce receptor subtype-selective analogs with other peptides to synthesize possible new analogs selective for human Bn receptor subtypes. Furthermore, we have synthesized these recently described Bn receptor subtype-selective analogs, as well as other related analogs, to fully characterize their selectivity for the human Bn receptors and attempted to identify analogs with specificity for hBRS-3 or the other Bn receptors. The peptides were synthesized using standard solid-phase methods as described previously (Mantey et al., 2001). In brief, solid-phase syntheses of peptide amides were carried out using Boc chemistry on methylbenzhydrylamine resin (Advanced ChemTech, Louisville, KY) followed by hydrogen fluoride-cleavage of free peptide amides. The crude peptides were purified by preparative high-pressure liquid chromatography on columns (2.5 x 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 the 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.

Preparation of ¹²⁵I-[D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14). This radioligand, with specific activity of 2200 Ci/mmol, was prepared as described previously (Mantey et al., 1997; Ryan et al., 1998b). In brief, 0.8 µg of 1,2,4,6-tetrachloro-3,6-diphenylglycouril 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 for 6 min at room temperature. The incubation was stopped with 300 µl of water. The radiolabeled peptide was separated using a Sep-Pak (Waters Associates, Milford, MA) and further purified by reverse-phase high-performance liquid chromatography on a C18 column. 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.

Binding of ¹²⁵I-Labeled BN-Related Peptides to Various Cells. Binding was performed as described previously (Mantey et al., 1993, 1997; Ryan et al., 1998b). The standard binding buffer contained 24.5 mM HEPES (pH 7.4), 98 mM NaCl, 6 mM KCl, 5 mM MgCl₂, 2.5 mM NaH₂PO₄, 5 mM sodium pyruvate, 5 mM sodium fumarate, 0.01% (w/v) soybean trypsin inhibitor, 1% amino acid mixture, 0.2% (w/v) bovine serum albumin, and 0.05% (w/v) bacitracin. BALB 3T3 cells stably expressing hGRPR (0.3 x 10⁶), hNMBR (0.03 x 10⁶), or hBRS-3 (0.3 x 10⁶) 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 10,000g for 1 min using a Beckman Microcentrifuge 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 of the experiments. Receptor affinities were determined using a least-square curve-fitting program (LIGAND) and the Cheng-Prusoff equation.

Measurement of [³H]IP. Changes in total [³H]inositol phosphates ([³H]IP) was measured as described previously (Benya et al., 1992, 1994, 1995; Ryan et al., 1998a). In brief, hBRS-3-, hGRPR-, or hNMBR-transfected Balb 3T3 cells were subcultured into 24-well plates (5 x 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 Ci/ml myo-[2-³H]inositol in growth media supplemented with 2% fetal bovine serum for an additional 24 h. Before the assay, the 24-well plates were washed by incubating for 30 min at 37°C with 1 ml/well 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, and 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 (Benya et al., 1992, 1994; Ryan et al., 1998a). In brief, samples were loaded onto Dowex AG1-X8 anion-exchange resin columns, washed with 5 ml of distilled water to remove free [³H]inositol, and then washed with 2 ml of 5 mM disodium tetraborate/60 mM sodium formate solution to remove [³H]glycerophosphorylinositol. Two milliliters of 1 mM ammonium formate/100 mM formic acid solution were added to the columns to elute total [³H]IP. Each eluate was mixed with scintillation cocktail and measured for radioactivity in a scintillation counter.

Measurement [Ca²⁺]_i Using ⁴⁵Ca Efflux and Fura-2. Changes in [Ca²⁺]_i using ⁴⁵Ca efflux and Fura-2 were measured as described previously (Benya et al., 1992). In a previous study, both methods demonstrate similar dose-response curves for Bn-related peptides (Benya et al., 1992). In brief, for [Ca²⁺]_i measurements using Fura-2, hBRS-3-, hGRPR-, or hNMBR-transfected Balb 3T3 cells were resuspended in binding buffer without bacitracin containing 4 x 10⁶ cells and 2 µM fura-2 for 45 min at 37°C. Fura-2-loaded cells were washed three times in binding buffer, and 2-ml samples were placed in quartz cuvettes in a Delta PTI Scan I spectrophotometer (PTI Instruments, Gaithersburg, MD). [Ca²⁺]_i was measured as described previously (Benya et al., 1992) after detecting fluorescence at 500 nm following excitation at 340 and 380 nm. For measuring changes of [Ca²⁺]_i using ⁴⁵Ca efflux, hBRS-3-, hGRPR-, or hNMBR-transfected Balb 3T3 cells were subcultured into 24-well plates (5 x 10⁴ cells/well) in regular propagation media. After aspirating the media, 1 ml of phosphate-free binding buffer containing 5 µCi/ml ⁴⁵Ca was added to each well, and the cells incubated for 90 min at 37°C in a 5% CO₂ atmosphere. Immediately before the assay, the cells were rapidly washed once in phosphate-free buffer and then incubated at 22°C in buffer containing the appropriated concentration of peptide. After 5 min, the supernatant was removed and discarded; the cells were lysed in 1% HCl in methanol, and the cell-associated radioactivity was determined in a liquid scintillation counter.

	Results

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Because with a number of other receptors the insertion of N-methyl groups in ligands results in subtype-selective analogs (Lin et al., 1990; Reissmann et al., 1996; Pradhan et al., 1998), a similar strategy was applied to the Bn analog [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 1, Peptide 1), which has high affinity for all human Bn receptor subtypes (Mantey et al., 1997, 2001, 2004; Pradhan et al., 1998; Ryan et al., 1998a) (Fig. 1; Table 2). N-Methyl groups were added to the Nle¹⁴, His¹², Ala¹¹, Val¹⁰, and Ala⁹ (Table 1, Peptides 3–7) of the nonselective peptide [D-Tyr⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6–14) or to Trp⁸ on the truncated analog [Ala¹¹,Phe¹³,Nle¹⁴]Bn(8–14) (Table 1, Peptide 8). The ability of the N-methyl peptides to inhibit binding of ¹²⁵I-[D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) and stimulate the generation of [³H]IP at the three human Bn receptor subtypes was investigated, because the activation of each of the three human Bn subtypes causes phospholipase C activation (Benya et al., 1995; Ryan et al., 1998a,b). [D-Tyr⁶,Ala¹¹,Phe¹³, Nle¹⁴]Bn(6–14) had greater potency for each of the three human Bn receptor subtypes than Bn, and Bn had a much greater affinity for the hGRPR and hNMBR than the hBRS-3 (Table 2, compare Bn with Peptide 1). With the exception of position 11 (Table 2, Peptide 5), the insertion of N-methyl groups at position 14, 12, 10, or 9 of [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) resulted in marked (>20-fold) decreases in affinity for each of the three human Bn receptors (Table 2; Fig. 1). In contrast, the insertion at the Ala¹¹ position of an N-methyl group (Table 2, Peptide 5) resulted in only a 2- to 16-fold decrease in affinity for the human Bn receptors. Neither the insertion of the N-methyl groups in various positions of [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) nor that in Trp⁸ of [D-Tyr⁸,Ala¹¹,Phe¹³,Nle¹⁴]Bn(8–14) (Table 2, Peptide 8) resulted in an increase in selectivity for hBRS-3 over hGRPR or hNMBR for any of the N-methyl-substituted analogs. The addition of N-methyl to Ala¹¹ (Table 2, Peptide 5) resulted in a small increase in selectivity for the hGRPR. Specifically, with this analog (Table 2, Peptide 5), the hGRPR had an 8-fold greater affinity over the hNMBR or hBRS-3 compared with [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14). Each of the N-methyl-substituted peptides (Peptides 3–8 in Table 2 and Fig. 1) was an agonist stimulating an increase in [³H]IP (Fig. 1, bottom panels). Their relative potencies for human Bn receptors were similar to the results for the affinities from binding studies, and no analog showed greater selective potency for activating hBRS-3.

View larger version (31K): [in this window] [in a new window]   Fig. 1. The ability of Bn and [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) (Peptide 1) with N-methyl-substituted analogs to inhibit binding and stimulate increase in [3H]IP formation at the hGRPR, hNMBR, and hBRS-3. In binding (top) Balb 3T3 cells stably transfected with hGRPR (0.3 x 106 cell/ml), hNMBR (0.03 x 106 cells/ml) or hBRS-3 (0.5 x 106 cells/ml) cells were incubated for 60 min at 22°C with 50 pM I125-[D-Tyr6,-Ala11,Phe13,Nle14]Bn(6–14), with or without the indicated concentrations of the various peptides added. Results are expressed as the percentage of saturable binding without unlabeled peptide added (percent control). Bottom, BALB 3T3 cells transfected with hGRPR, hNMBR, or hBRS-3 were subcultured and preincubated for 24 h at 37°C with 3 mCi/ml myo-[2-3H]inositol. The cells were then incubated with the ligands at the concentrations indicated for 60 min at 37°C. Values expressed are a percentage of total [3H]IP release stimulated by 1 µM [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14). Control and 1 µM [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14)-stimulated values were 1510 ± 98 and 8200 ± 210 dpm, respectively, for hGRPR; 1950 ± 82 and 22820 ± 540 dpm, respectively, for hNMBR; and 2510 ± 120 and 12600 ± 2305 dpm, respectively, for hBRS-3. Results are the mean ± S.E.M. from at least three experiments, and in each experiment, the data points were determined in duplicate. Numbers refer to the peptide number in Table 1.

View this table: [in this window] [in a new window]   TABLE 2 The ability of human Bn receptors to interact with and be activated by bombesin, [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) and various analogs with N-methyl substitutions Structures of peptides are listed in Table 1. Balb3T3 cells stably transfected with hGRPR (0.3 x 106 cells/ml), hNMBR (0.05 x 106 cells/ml), or hBRS-3 (0.5 x 106 cells/ml) were incubated with 50 pM iodinated [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14), with or without increasing concentrations of unlabeled ligand for 60 min at 22°C, as described in legend to Fig. 1. The affinities of [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) and Bn were calculated by a least-squares curve-fitting program (LIGAND). The remaining affinities were calculated using KaleidaGraph and the Cheng-Prusoff equation. All values are means ± S.E.M. from at least three experiments. Balb 3T3 cells stably transfected with hBRS-3, hGRPR, or hNMBR were incubated with [3H]inositol, and total [3H]IP was determined as described under Materials and Methods. For each peptide, a dose-response curve was performed with concentrations from 0.01 nM to 1 µM. Results are expressed as the concentration causing one-half the maximal increase EC50 seen with 1 µM peptide. Results were calculated from the dose-response curves shown in Fig. 1 for each peptide using KaleidaGraph. Each value is a mean ± S.E.M. from at least three experiments. For hBRS-3/BALB 3T3 cells, the control and 1 µM [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) values were 10,624 ± 571 and 37,660 ± 4106 dpm, respectively. For hGRPR/BALB 3T3 cells, the control and 1 µM [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) values were 9231 ± 2260 and 43,060 ± 9137 dpm, respectively. With hNMBR/BALB 3T3 cells, the control and 1 µM [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14) values were 2020 ± 166 and 40,667 ± 2371 dpm, respectively.

Recently, analogs of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Darker et al., 2001) have been reported to function as highly selective GRPR agonists in a [Ca²⁺]_i FLIPR assay. We synthesized three of the most potent analogs [Peptide 9 (compound 6 in Darker et al., 2001), Peptide 10 (compound 11 in Darker et al., 2001), and Peptide 12 (compound 13 in Darker et al., 2001) in Table 3] as well as six new related analogs, primarily with different position 12 substitutions (Table 6, Peptides 12–17), and assessed their abilities to interact with and activate each of the three human Bn receptors (Figs. 2 and 3; Table 3). In binding studies, we found [pGlu⁷,Ala¹¹,Phe¹³,Nle¹⁴]Bn(7–14) (Table 3, Peptide 9) to have a 300- to 1600-fold reduced affinity for each of the three human Bn receptors compared with [DPhe⁶, Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) and found no hGRPR selectivity for any human Bn receptor subtype (Fig. 2, top; Table 3). Furthermore, the Phe⁶ truncation made the peptide equipotent for activating and stimulating phospholipase C activity at the hGRPR and the hNMBR and 5-fold less potent for activating the hBRS-3 (Peptide 9 in Fig. 2 and Table 3). In binding studies, we found that Peptides 10 and 11 had a 100- to 8000-fold lower affinity for hGRPR than [D-Phe⁶, Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 3; Fig. 2). Furthermore, neither Peptide 10 nor 11 shows selectivity in our binding or [³H]IP assays for the hGRPR (Fig. 2; Table 3). In the calcium FLIPR assay in other cells (Darker et al., 2001), Peptides 9 and 10 (Table 3) were reported to have a 170- and >5000-fold higher affinity for hGRPR than hNMBR and 1000- and 10,000-fold affinity for hGRPR over BRS-3. To be certain that our results did not differ from the previous study (Darker et al., 2001) because of the different assays used in the two studies, we assessed the ability of Peptides 2, 9, and 11 (Table 1) to cause mobilization of cellular calcium by assessing changes in ⁴⁵Ca outflux (Fig. 2) or cause increases in [Ca²⁺]_i using Fura-2. In both assays, the EC₅₀s for altering cellular calcium at the hGRPR and hNMBR for Peptide 9 (0.5–1 nM), 11 (1–3 nM), or 2 (0.003 nM) did not differ, which resembled our results with stimulating an increase in [³H]IP (Table 3). Furthermore, similar to our results with [³H]IP, their relative affinities for stimulating hBRS-3 were Peptide 2 (EC₅₀ 0.3 nM) > Peptides 9 and 11 (Fig. 2).

View this table: [in this window] [in a new window]   TABLE 3 The affinities and potencies of human GRP, NMB, and BRS-3 receptors for bombesin, [D-Tyr6,Ala11,Phe13,Nle14]Bn(6–14), and various synthetic peptides reported to be selective for BRS-3 The binding affinity (Ki) and the EC50 for each peptide were determined as described in the legend for Table 1. Structures of peptides are listed in Table 1. Balb 3T3 cells stably expressing hGRPR, hNMBR, and hBRS-3 were incubated with 50 pM 125I ligand, with or without increasing concentrations of unlabeled ligand for 60 min at 22°C as described in Fig. 1 legend. The affinities were calculated using the Cheng-Prusoff equation. To assess phospholipase C activation, the cells were incubated with [3H]inositol, and total [3H]IP was determined as stated under Materials and Methods. A dose-response curve was determined for each ligand with concentrations of 0.01 nM to 0.1 µM. For each ligand, a concentration causing a half-maximal increase EC50 was calculated using KaleidaGraph. Each value is a mean ± 1 S.E.M from at least three experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 2. The ability to interact with human Bn receptors of various analogs of [D-Phe6,Ala11,Phe13,Nle14]Bn(6–14) reported to have selectivity for hGRPR, hNMBR, and hBRS-3. The experimental conditions were similar to those outlined in the legend to Fig. 1 or as described under Materials and Methods. The results in binding experiments (top) are expressed as the percentage of saturable binding without unlabeled peptide added (percent control). The results for [3H]IP (middle) and 45Ca efflux (bottom) stimulation are expressed as percentages of the response stimulated by a maximally effective concentration of [D-Phe6,Ala11,Phe13,Nle14]Bn(6–14). Results are the mean ± S.E.M. from at least three experiments, and in each experiment, the data points were determined in duplicate. Numbers refer to the peptide number in Table 1.

View larger version (31K): [in this window] [in a new window]   Fig. 3. The affinities and potencies of various position 12 and truncated analogs of [D-Phe6,Ala11,Phe13,Nle14]Bn(6–14) for human GRP, NMB, and BRS-3 receptors. The experimental conditions were similar to those outlined in the Fig. 1 legend, and the results are means ± S.E.M. from at least three experiments, with each data point determined in duplicate. Numbers refer to peptide numbers in Table 1.

Because the substitution of alanine in the 12th position of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) was reported to result in hGRPR-selective analogs with high affinity for hGRPR (Darker et al., 2001), we synthesized six related peptides to explore the importance of substitution in this position for determining Bn receptor selectivity (Peptides 12–17 in Table 3 and Fig. 3). Four analogs in this group of peptides (Table 3, Peptides 12, 13, 14, and 16) had a substitution of Tyr¹² or Phe¹², instead of Ala¹² in Peptide 11 (Table 1), in place of the His¹² in [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14). The substitution of a Tyr¹² or Phe¹² moiety (Table 3, Peptides 12 and 14) resulted in two peptides that, when compared with [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 1, Peptide 1), had a 32- and 208-fold decrease, respectively, in affinity for the hGRPR, and neither substitution increased its selectivity for hGRPR (Table 3; Fig. 3). In contrast to the effect on hGRPR, the substitution of Tyr¹² resulted in an analog (Peptide 12 in Table 3 and Fig. 3) that showed some selectivity for hBRS-3 (10- and 60-fold selective for hBRS-3 over hGRPR and hNMBR, respectively) in binding studies. hGRPR, hNMBR, and hBRS-3 demonstrate different degrees of receptor spareness for stimulating changes in [³H]IP (i.e., Peptide 2 demonstrates 3-, 14-, and 0-fold spareness, respectively) (Table 2). Because of the different receptor spareness that occurs with different human Bn receptors (Benya et al., 1995; Ryan et al., 1998a,b, 1999), there was no selectivity in activating hBRS-3 over the other Bn receptors (Table 3; Fig. 3).

In a previous study (Mantey et al., 1997), the presence of Ala¹¹ was reported to be important for high-affinity interaction with hBRS-3. To examine this point further, Peptide 14 with a Ala in position 11 and Peptide 13 without a Ala¹¹ were compared. Both peptides had a marked decrease in affinity and potency for activating each human Bn receptor subtype compared with [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 3, Peptide 1). Each of the two analogs demonstrated similar decreases in affinity for each receptor subtype demonstrating that, for low affinity Bn analogs, the presence of the Ala¹¹ did not have a greater effect on hBRS-3 affinity than the other human Bn receptors. Because N-terminal truncation of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) is reported to result in analogs that retain high affinity and show preference for GRPR (Darker et al., 2001), we synthesized two N-terminal truncated analogs, [D-Phe⁷,Ala¹¹,Phe¹³, Nle¹⁴]Bn(7–14) (Peptides 15 in Table 3 and Fig. 3) and [pGlu⁸,Ala¹¹,Phe¹³,Nle¹⁴]Bn(8–14) (Peptides 17 in Table 3 and Fig. 3). Each of these two analogs (Peptides 15 and 17 in Table 3 and Fig. 3) demonstrated >1000-fold decrease in affinity for each of the three human Bn receptors and had either no or minimal (<3-fold) selectivity for the hGRPR. In general, the ability of each of the new analogs in this series (Peptides 9–17 in Table 3 and Figs. 2 and 3) to activate the three human Bn receptors correlated with their binding affinities if differences in receptor spareness were considered, and no antagonists or partial agonists were found (Table 2; Figs. 2 and 3).

Recently, three studies (Weber et al., 2002, 2003; Boyle et al., 2005), which performed structure/activity studies on [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) using a calcium or FLIPR calcium assay, have described a number of shortened analogs (Table 4, Peptides 18, 19, 20, and 23) with selectivity for hBRS-3. We synthesized these four peptides and compared their selectivity for the different human Bn receptors to two other [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) analogs, [D-Tyr⁶,(R)Apa¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 3, Peptide 21) and [D-Tyr⁶,(R)Apa¹¹-4Cl,Phe¹³,Nle¹⁴]Bn(6–14) (Table 4, Peptide 22) (Mantey et al., 2001, 2004), that we had previously reported were selective for the hBRS-3 (Table 3). As seen in previous studies (Mantey et al., 2001, 2004), [D-Tyr⁶,(R)Apa¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 4, Peptide 21) and its chloro-substituted analog [D-Tyr⁶,(R)Apa¹¹, 4ClPhe¹³,Nle¹⁴]Bn(6–14) (Table 4, Peptide 22), when compared with the nonselective peptide [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14), had lower affinity for all of the human Bn receptor subtypes but showed less of a decrease in affinity for hBRS-3, resulting in a 50- to 229-fold selectivity for the hBRS-3 over the other human Bn receptors. In our assays, we found that two of the recently described hBRS-3-selective peptide-shortened analogs of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]-Bn(6–14) (Peptides 18 and 20 in Table 4 and Fig. 4) had very low affinities for each of the human Bn receptors (>5 µM), whether analyzed by binding studies or by their abilities to activate phospholipase C and increased [³H]IP generation. We found that the reported hBRS-3-selective short peptide (Weber et al., 2003), Peptide 19, had both very low affinity for hBRS-3 and the other two human Bn receptors (i.e., >10 µM) and very low potency (>5 µM) for activating any of the three human Bn receptors and stimulating phospholipase C (Table 4; Fig. 4). In a third study using a FLIPR calcium assay (Boyle et al., 2005), Ac-Phe,Trp,Ala,His(Bzl),Nip,Gly,Arg-NH₂, (Table 4, Peptide 23) was reported to have equal high affinity to [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) for hBRS-3 and to have greater than 1800-fold selectivity for hBRS-3 over hGRPR or hNMBR. In our binding study, Peptide 23 had a 185-fold lower affinity than [D-Phe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) for hBRS-3 (Table 4; Fig. 4). However, Peptide 23 had some selectivity for hBRS-3 because it had a 14-fold higher affinity for hBRS-3 than hNMBR and >20-fold higher affinity for hBRS-3 than hGRPR (Table 4; Fig. 4). Its potency for activating hBRS-3 was 30-fold greater than that for activating hNMBR and 74-fold greater than that for activating hGRPR. Compared with the most selective hBRS-3 agonists as described previously (Mantey et al., 2001, 2004) [(Table 4, analog 21) and [D-Tyr⁶,(R)Apa¹¹-4Cl,Phe¹³, Nle¹⁴]Bn(6–14) (Table 4, analog 22)], Peptide 23 was also an agonist at each of the human Bn receptors stimulating activation of phospholipase C in each (Fig. 4; Table 4). In terms of relative selectivity for activating each human Bn receptor subtype, Peptide 22 had the greatest selectivity for hBRS-3 over hGRPR (i.e., 98-fold) followed by Peptides 23 (i.e., 74-fold) and 21 (9-fold) (Table 4; Fig. 4). For selectivity of hBRS-3 over hNMBR, Peptide 23 had greatest selectivity (30-fold) compared with 20-fold for Peptide 22 and 6-fold for Peptide 21 (Fig. 4; Table 4).

View this table: [in this window] [in a new window]   TABLE 4 The affinities and potencies for human GRP, NMB, and BRS-3 receptors of [D-Tyr6, Ala11,Phe13,Nle14]Bn(6–14), and various short synthetic bombesin The binding affinity (Ki) and the EC50 for each peptide were determined as described in the legend for Table 2 and are from the data in Fig. 4. Structures of peptides are listed in Table 1.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Comparison of the ability of various short analogs and conformationally restricted analogs of [D-Phe6,Ala11,Phe13,Nle14]Bn(6–14) reported to have hBRS-3 selectivity to interact with human Bn receptors. The experimental conditions were similar to those outlined in the Fig. 1 legend, and the results are means ± S.E.M. from at least three experiments, with each data point determined in duplicate. Numbers refer to peptide numbers in Table 1.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we synthesized a series of Bn-related analogs using different strategies to attempt to identify selective ligands for Bn receptor subtypes (GRPR, NMBR, and BRS-3), as well as fully characterized ligands recently reported to have selectivity for hGRPR or hBRS-3. First, we synthesized conformationally constrained analogs by inserting N-methyl substitutions into the Bn COOH terminus, the biologically active portion (Jensen and Coy, 1991; Lin et al., 1996). Second, we synthesized analogs with either N-terminal truncations or amino acid substitutions in the position equivalent to His¹² of Bn. The former strategy was used because previous studies concluded that the COOH terminus of GRP/Bn exists in a folded conformation with an antiparallel -pleated sheet structure and a -bend centered on glycine 11 (Kull, et al., 1992; Erne and Schwyzer, 1987; Coy et al., 1988). It was proposed that this structure is maintained by hydrogen bonding between the Trp⁸ carbonyl (C = O) and Val¹⁰ NH, the Val¹⁰ C = O and NH of Leu¹³, as well as the NH of Val¹⁰ and Leu¹³ C = O (Coy et al., 1988). N-Methyl substitutions should introduce conformational restriction, disrupt hydrogen bonding, and thus have pronounced effects on the conformation of the COOH terminus (Lin et al., 1995, 1996). Substitution of N-methyl groups to produce conformationally restricted analogs has been widely used with analogs of somatostatin (Rajeswaran et al., 2001), bradykinin (Reissmann et al., 1996), cholecystokinin (Pradhan et al., 1998), endothelin (Cody et al., 1997), tachykinins (Wormser et al., 1986), glucose-dependent insulinotropic polypeptide (Hinke et al., 2003), enkephalins (Penkler et al., 1993), angiotensin (Khosla et al., 1976), insulin (Ogawa et al., 1987), and galanin (Rivera et al., 1994). N-Methyl substitution can result in analogs with enhanced selectivity, potency, or enhanced stability (Wormser et al., 1986; Lin et al., 1990; Schmidt et al., 1995; Cody et al., 1997; Pradhan et al., 1998). Previously, the results of N-methyl substitutions in the COOH terminus of Bn(7–14) was reported (Horwell et al., 1996). However, Bn(7–14) only has high affinity equal to the native ligand GRP for the GRPR, whereas it has a 20-fold lower affinity for the NMBR than NMB and has a very low affinity, similar to Bn, for the hBRS-3 receptor (Horwell et al., 1996; Lin et al., 1996). In the present study, to investigate the effect of N-methyl substitutions on affinity/selectivity for the three human Bn receptors, we made the substitutions in the analogs of [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14), which has high affinity for all human Bn receptors, as well as the frog bombesin receptor subtype 4, the GRPR, and the NMBR from a number of species (Mantey et al., 1997; Pradhan et al., 1998; Ryan et al., 1998a,b). For the hGRPR, N-methyl substitution in Ala⁹ or Val¹⁰ had the greatest effect in decreasing affinity (>1500-fold), whereas insertion into His¹² or Nle¹⁴ caused a 100- to 220-fold decrease in affinity and insertion into Ala¹¹ had almost no effect (i.e., 2-fold decrease). These results have both similarities and differences from the previously reported N-methyl scan of Bn(7–14) (Horwell et al., 1996). Our results are similar in that substitution on Ala⁹, Val¹⁰, Nle¹⁴, or Met¹⁴ caused a marked decrease in hGRPR affinity and substitutions on Trp⁸ resulted in a <100-fold affinity decrease. However, our results differ in that we found no effect from insertion of N-methyl into Ala¹¹ and a 112-fold decrease in affinity with His ¹², whereas in the previous study (Horwell et al., 1996), the reverse was found, with no change in affinity for insertion of N-methyl on His¹² but a 50-fold decrease for Gly¹¹. A number of our results are consistent with the previously proposed -pleated sheet model that envisages a bend at Gly¹¹ for the Bn COOH terminus upon binding with the GRPR. The insertion of N-methyl into the 11th position should lead to the stabilization of this conformation, which is consistent with our finding that this analog retains high affinity. In this model, the insertion of N-methyl in Val¹⁰ would be expected to disrupt hydrogen bonding and markedly decrease affinity, as was found. Unfortunately, the pharmacology of N-methyl-substituted analogs for hBRS-3 and hNMBR generally mirrored the changes in affinity with each substitution seen with the hGRPR. These results would support the conclusion that the active conformation of [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) for interacting with each of the three human Bn receptors is generally similar; thus, this approach was unsuccessful at yielding potent subtype-selective ligands.

The second strategy used to identify possible selective Bn receptor subtype ligands was to synthesize N-terminal truncated and/or His¹²-substituted analogs of [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14), because a recent study (Darker et al., 2001) reported that this approach yields potent GRPR-selective agonists. Six such analogs were synthesized. Three had His¹² replaced by Tyr¹², Phe¹², and Ala¹², and another three were made with various N-terminal truncations of [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14). This strategy did not yield any GRPR-selective agonists, although two analogs, [D-Tyr⁶, Ala¹¹,Tyr¹²,Phe¹³,Nle¹⁴]Bn(6–14) (Table 2, Peptide 12) and [D-Phe⁶,Ala¹¹,Phe¹²,Phe¹³,Nle¹⁴]Bn(6–14) (Table 2, Peptide 16), had moderate selectivity for hBRS-3 over hGRPR (11–25-fold) and hNMBR (59–2361-fold). These latter analogs may prove useful as templates in discovering more selective hBRS-3 ligands, but this approach does not seem useful for making GRPR-selective ligands.

The final strategy used to identify useful selective ligands was to fully characterize pharmacologically compounds that have been recently described as having selectivity for a Bn receptor subtype. This pharmacological characterization was performed because in the studies describing these selective compounds (Darker et al., 2001; Weber et al., 2003; Boyle et al., 2005), receptor affinity for each human receptor subtype was not determined from binding studies and also characterization of receptor potency by receptor subtype activation was often incomplete. This occurred because potencies were derived from calcium FLIPR assays, and detailed dose-response curves as well as determining whether agonists were full or partial agonists were usually lacking or unclear. In the present study, we performed complete dose-response curves with binding studies using a universal ligand, ¹²⁵I-[D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14), that had high affinity for all human Bn receptor subtypes (Mantey et al., 1997; Pradhan et al., 1998; Ryan et al., 1998b), determined their potencies to stimulate phospholipase C, and for three selected peptides, determined their abilities to alter cellular cytosolic calcium.

One study (Darker et al., 2001) reported that three truncated and His¹²-substituted analogs of [D-Phe⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) had hGRPR selectivity in the calcium FLIPR assay. [pGlu⁷,Ala¹¹,Phe¹³,Nle¹⁴]Bn(7–14) (Table 2, Peptide 9), [D-Phe⁶,Ala⁸,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) (Table 2, Peptide 10), and [D-Phe⁶,Ala¹¹,Ala¹², Phe¹³,Nle¹⁴]Bn(6–14) (Table 2, Peptide 11) were reported to have affinities of 0.01 to 2 nM for the hGRPR and to have a 5570-, 4-, and 170-fold greater affinity for hGRPR than hNMBR, respectively, and a 160- to 100,000-fold greater affinity for hGRPR than hBRS-3 (Darker et al., 2001). In our binding studies, each of these three analogs had relatively low affinity for hGRPR (63–5000 nM), and none was hGRPR-preferring. Each functioned as an agonist at each of the Bn receptor subtype; however, they had either no hGRPR selectivity (Table 2, Peptides 9 and 11) or less than a 1-fold selectivity (Peptide 10). This difference from the previous study (Darker et al., 2001) was not due to differences in the assays used, because when we assessed the abilities of these peptides to cause changes in cellular calcium in our cells, no Bn receptor subtype selectivity was seen. These results suggest that some difference in the Bn receptor-transfected cells used in these two studies (receptor number, G proteins, coupling, etc.) probably is the reason for these differences. In previous studies (Benya et al., 1992, 1994, 1995; Ryan et al., 1998a,b), we have demonstrated that Bn receptors stably expressed in BALB 3T3 cells, as used in the present study, behaved with similar pharmacology and cell activation to wild-type nontransfected Bn receptors.

The second group of possible selective Bn receptor ligands investigated was recently reported shortened, selective hBRS-3 ligands (Weber et al., 2002, 2003; Boyle et al., 2005). Each of these ligands had been identified from structure-function studies on [D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14), which we and others have previously shown has a high affinity for hBRS-3, but is not selective for hBRS-3, because it also has high affinity for hGRPR and hNMBR (Mantey et al., 1997, 2001; Pradhan et al., 1998; Ryan et al., 1998b; Reubi et al., 2002). In the present study, four of these shortened [D-Tyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6–14) analogs were synthesized [[H-D-Phe-Gln-D-Trp-NH(CH₂)₂C₆H₅ (Table 3, Peptide 18) (Weber et al., 2002); 3-phenyl-propionyl-Ala-D-Trp-NH(CH₂)₂C₆H₅ (Table 3, Peptide 19) (Weber et al., 2003); H-D-Phe-Gl-D-Trp-Phe-NH₂ (Peptide 20) (Weber et al., 2002); and Ac-Phe,Trp,Ala,His(Bzl),Nip,Gly,Arg-NH₂ (Table 3, Peptide 23) (Boyle et al., 2005)] and fully characterize pharmacologically at each Bn receptor subtype. In the original studies (Weber et al., 2002, 2003; Boyle et al., 2005) of these shortened analogs, the reported hBRS-3 selectivity was based primarily on results from calcium or calcium FLIPR studies. In these studies, the above shortened analogs were reported to have selectivity for hBRS-3 over hGRPR or hNMBR of 1941- (Peptide 19) (Weber et al., 2003), 151- (Peptide 23) (Boyle et al., 2005), >50- (Peptide 18) (Weber et al., 2002), and >3-fold (Peptide 20) (Weber et al., 2002). Furthermore, Peptides 23 (Boyle et al., 2005) and Peptide 19 (Weber et al., 2003) were reported to retain similar high affinity (i.e., nanomolar range) to [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) for the hBRS-3. In the present study, we found that three of these shortened [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) analogs, Peptide 18 (Weber et al., 2002), Peptide 19 (Weber et al., 2003), and Peptide 20 (Weber et al., 2002), not only had very low affinities (i.e., 5 µM for hBRS-3) but also hGRPR and hNMBR and, therefore, were not only not selective for hBRS-3 but had too low affinities to be useful. In contrast, Peptide 23 was found to have moderate affinity for hBRS-3 (i.e., 259 nM) and also have 15-fold higher selectivity for hBRS-3 than hNMBR and 19-fold higher selectivity for hBRS-3 than hGRPR. Furthermore, its potency for stimulating the hBRS-3 was >30-fold higher than hNMBR or hGRPR. We have previously described two conformationally restrained analogs of [D-Tyr⁶,Ala¹¹, Phe¹³,Nle¹⁴]Bn(6–14) in which Ala¹¹ was replaced by either aminopropionic acid (Peptide 21) (Mantey et al., 2001) or 4-chloro-aminopropionic acid (Peptide 22) (Mantey et al., 2004), which are hBRS-3-selective. We compared the affinities and selectivities of these two full-length analogs (Peptide 21 and 22) with those of the recently described shortened analog (Peptide 23), which also demonstrated hBRS-3 selectivity in our assays. The full-length analogs (Peptides 21 and 22) had 123- and 32-fold greater affinities for hBRS-3 than the shortened analog Peptide 23, and they were 4- and 20-fold, respectively, more selective than Peptide 23 for the hBRS-3. Therefore, the full-length analogs with Apa¹¹ substitution (Peptides 21 and 22) remain the most selective agonists for hBRS-3.

In conclusion, although this study does not identify more selective Bn receptor ligands, it provides important information and leads to future studies. It demonstrates that selective incorporation of N-methyl moieties in Bn analogs does not yield selective analogs and that a number of truncated and position 12-substituted Bn analogs recently reported to be selective ligands or a series of new position 12-substituted Bn analogs were not high affinity-selective Bn receptor ligands. However, the study demonstrates that the shortened analog, Peptide 23, represents an important lead compound with a novel structure that could be used as a template to develop more selective hBRS-3 ligand in the future.

	Footnotes

 
This research is supported by the Intramural Research Program of the NIDDK, National Institutes of Health and Tulane University Peptide Research Fund.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.107011.

ABBREVIATIONS: Bn, bombesin; GRPR, gastrin-releasing peptide receptor; NMBR, neuromedin B receptor; BRS-3, bombesin receptor subtype 3; [³H]IP, [³H]inositol phosphate(s); CNS, central nervous system; Ala, -alanine; Nle, norleucine; NMe, N-methyl; His(Bzl), histidine(benzl); Bzl, benzyl; Apa, 3-amino, propionic acid; FLIPR, fluorometric imaging plate reader; [Ca²⁺]_i, intracellular calcium; Nip, piperidine-3 carboxylic acid; Ac, acetyl.

Address correspondence to: Dr. Robert T. Jensen, DHHS/NIH, NIDDK, DDB, Bldg. 10, Rm. 9C103, 31 Center Drive, Bethesda, MD 20892. E-mail: robertj{at}bdg10.niddk.nih.gov

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