Elsevier

Peptides

Volume 32, Issue 8, August 2011, Pages 1685-1699
Peptides

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs

https://doi.org/10.1016/j.peptides.2011.06.017Get rights and content

Abstract

The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r = 0.92–99, p < 0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤1 nM) for hGRP receptor with 15 selective for it (greatest = GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing β-alanine11 had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r = 0.131, p = 0.54), but hNMB receptor results correlated with rat NMB receptor (r = 0.71, p < 0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.

Highlights

• Pharmacology of 24 natural occurring/synthetic bombesin related agonists is compared for rat and human receptor bearing tissues. • Binding affinities/potencies and efficacy of cell activation are determined for all peptides. • 12 peptides had high affinity/potency for hGRP receptors, 8 for hNMB receptors, 1 for hBRS-3 receptor. • There was no correlation between affinities for hGRP and rat GRP receptors, but there was with h- and rat NMB receptors. • Results show the human and rat peptide agonist pharmacophore for human and rat GRP receptors differ markedly. • Results also report a number of high affinity/potency agonists that may be useful for human studies.

Introduction

The mammalian bombesin (Bn) receptor family consists of three closely related G protein-coupled receptors: the gastrin-releasing peptide receptor (GRP receptor) whose native ligand is the 27 amino acid peptide, GRP; the neuromedin B receptor (NMB receptor) mediating the action of the decapeptide, NMB, and the orphan receptor, BRS-3 which shares 47–51% protein homology with the GRP/NMB receptors, but whose native ligand is still unknown [7], [12], [19], [29], [41], [95]. Each of these receptors as well as their ligands are widely distributed in both the central nervous system [CNS] and peripheral tissues [7], [29], [34], [36], [56], [78].

Studies in animals suggest these receptors are involved in a broad range of physiological and pathophysiological processes [19], [29], [36], [41], [95]. The possible physiological effects include roles in regulation in the CNS/peripheral nervous system [thermoregulation, behavior, circadian rhythm, satiety, sensory nerve transmission], in the gastrointestinal tract [secretion, motility, growth], endocrine [energy homeostasis, secretion of numerous hormones/neurotransmitters, thyrotropin release], immunological [effects on leukocytes, lymphocytes, macrophages, dendritic cells], as well as effects in the respiratory system and urogenital system [19], [29], [36], [41], [95]. Important possible pathophysiological roles include proposed roles in lung injury/diseases, tumoral growth, thyroid disorders, human feeding disorders and disorders of energy homeostasis, pruritic responses, and various human CNS disorders [19], [29], [41], [54], [70], [95]. Of these latter disorders, the one that has received the most attention is the prominent role that GRP and NMB have on growth and/or differentiation of various human tumors [lung, squamous cell tumors of head/neck, prostate, colon, CNS, neuroendocrine tumors, pancreatic, gynecologic tumors], which include in some cases functioning as an autocrine growth factor [19], [29], [36], [41], [54], [95]. Furthermore, the GRP/NMB receptors are two of the G protein-coupled receptors that are most frequently overexpressed by various human neoplasms including cancers of the lung [small cell and non-small cell], pancreas, prostate, CNS (gliomas), head/neck (squamous cell tumors), breast, colon and various neuroendocrine tumors (bronchial, intestinal, thymic carcinoids) [19], [29], [31], [54], [67], [95].

Recently there is an increased interest in the use of possible Bn receptor agonists and antagonists to further define the role of Bn receptors in both human pharmacology and pathophysiology [18], [19], [25], [29], [41], [70]. This is particularly true for the marked interest in the possible use of Bn receptor agonists which could be preferentially internalized by Bn receptor-overexpressing human neoplasms, for use in localizing these neoplasms as well as for their treatment, by performing Bn receptor-mediated imaging or Bn receptor-mediated cytotoxicity [5], [29], [55], [67]. This has received considerable interest because of the marked clinical success of using radiolabeled somatostatin analogs to image neuroendocrine tumors either by nuclear medicine methods or by positron emission tomographic scanning and more recently the promising results with 111In-, 90Y-, or 177Lu-labeled somatostatin analogs for treatment of advanced malignant neuroendocrine tumors by peptide radioreceptor therapy [5], [19], [41], [66]. This increased interest in Bn receptors has occurred, because the successful results with human tumors containing overexpression of somatostatin receptors raise the possibility that a similar approach may be successful with more common neoplasms, which do not overexpress somatostatin receptors, but do overexpress human Bn receptors [5], [19], [41], [66].

Unfortunately, at present it can be difficult to select the appropriate Bn receptor agonist to use for these human Bn receptor-imaging/cytotoxicity studies as well as for other Bn receptor studies in humans. This has occurred because in contrast to Bn receptor antagonists [9], [18], [24], [29], [91], [92], there have been few systematic studies of human Bn receptor pharmacology or receptor activation, especially using cellular systems containing receptor expression densities that usually occur with native Bn receptors. This has led to a reliance on results from animal studies, particularly from rodents for optimum pharmacological design. This approach can lead to inaccurate conclusions, because with a number of different G protein-coupled receptors, including Bn receptors, there are reports of important species differences, especially between human and rodent-receptors, on agonist receptor interaction/activation [23], [27], [29], [35], [40], [68], [88], [92], [93]. Similarly, high densities of receptors, as frequently occur with transfected cell systems, including with Bn receptors [87], can have marked effects on agonist receptor activation/interaction, leading to conclusions which may not be applicable to native cells with lower Bn receptor densities, as seen in vivo.

The present study was performed to attempt to address these issues by systematically studying the affinities and potency of cellular activation at human Bn receptors of 24 putative agonists (12 natural and 12 synthetic Bn analogs) that either themselves or their analogs are commonly used in studies of human Bn receptors. To insure the results were representative of native Bn receptor responses two different cell lines were used, including one containing either native receptors or cells with Bn receptors expressed at the density seen in native cells. Results using human Bn cells were compared to results with cells containing native rat Bn receptors under identical experimental conditions. There was a marked discordance between results with rat GRP receptors and human, showing that data from the rat could not be used for human ligand design for hGRP receptors. A number of high affinity agonist ligands for each Bn receptor subtype could be identified which could be useful for investigating the role of GRP/NMB receptors in human physiological/pathophysiological conditions.

Section snippets

Materials

The following cells and materials were obtained from the sources indicated: Balb-3T3 (mouse fibroblast) cells, HuTu-80 (human duodenal cancer cell line), AR42J cells (rat pancreatic acinar cells) and C6 rat glioblastoma cells from the American Type Culture Collection, (Rockville, MD); NCI-H1299 (non-small cell lung cancer cells) were a gift from Herb Oie (National Cancer Institute-Navy Medical Oncology Branch, Naval Medical Center, Bethesda, MD); GENETICIN selective antibiotic (G418 Sulfate)

Human Bn receptor affinities-natural occurring peptides

In this study, we have compared the ability of a number of naturally occurring peptides as well as synthetic Bn-related peptide ligands (Table 1) that are reported to function as Bn receptor agonists in either human studies or in other species, to interact with and activate human Bn receptors [hGRPR, hNMBR, hBRS-3] and compared their affinities to those for Bn receptors of rat, a commonly used laboratory animal in various Bn receptor studies (rGRPR, rNMBR) (Table 1, Table 2, Table 3) [3], [11],

Discussion

The purpose of this study was to assess the abilities of various naturally occurring Bn-related and synthetic Bn agonist analogs, which are increasingly used in human studies [19], [41], [59], [95], to interact with and activate human bombesin receptors [29], [47], [59], [69]. This study was performed because most of the pharmacology of Bn receptors has been performed on nonhuman cells, particularly rat tissues [6], [11], [22], [29], [33], [47], [69], [90], [97] and the results generally

Acknowledgment

This work was partial supported by intramural funds of the NIDDK, NIH.

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