Pharmacological characterization of a selective agonist for bombesin receptor subtype-3

Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor in the bombesin receptor family that still awaits identification of its natural ligand. BRS-3 deficient mice develop a mild late-onset obesity with metabolic defects, implicating BRS-3 plays a role in feeding and metabolism. We describe here the pharmacological characterization of a synthetic compound, 16a, which serves as a potent agonist for BRS-3. This compound is selective for BRS-3 as it does not activate neuromedin B or gastrin-releasing peptide receptors, two most closely related bombesin receptors, as well as a series of other GPCRs. We assessed the receptor trafficking of BRS-3 and found that compound 16a promoted β-arrestin translocation to the cell membrane. Neither central nor peripheral administration of compound 16a affects locomotor activity in mice. Therefore compound 16a is a potential tool to study the function of the BRS-3 system in vitro and possibly in vivo.     

Development and function of bombesin-like peptides and their receptors

Amphibian bombesin and its related peptides consist a family of neuropeptides in many vertebrate species. Bombesin and two major bombesin-like peptide in mammals, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to elicit various physiological effects. These include inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. Receptors for GRP and NMB (GRP-R and NMB-R), as well as third subtype of bombesin-like peptide receptor (BRS-3) have been cloned. These receptors are G-protein-coupled receptors and are expressed in various brain regions and in the digestive tract. In this paper, we will summarize studies on these peptides and their receptors, with special reference to research using gene-knockout mice. These studies clearly demonstrated the role of three receptors in vivo and in vitro. We will also discuss the phylogeny of these receptors.     

Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene

Bombesin (BN)-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5 kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human (Xq25) and mouse (XA7.1–7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression.     

Bombesin receptor subtype-3 agonists stimulate the growth of lung cancer cells and increase EGF receptor tyrosine phosphorylation

The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr⁶, (Ala¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr¹⁰⁶⁸ phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr⁶, R-Apa¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA2) and (DTyr⁶, R-Apa¹¹, 4-Cl,Phe¹³, Nle¹⁴) bombesin^6-14 (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB₂R antagonist) or PD168368 (BB₁R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH₂ (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific ¹²⁵I-BA1 binding to NCI-H1299-BRS-3 cells with an IC₅₀ values of 1.1, 21, 15 and 750 nM, respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.     

Novel chiral-diazepines function as specific,selective receptor agonists with variable coupling and species variability in human,mouse and rat BRS-3 receptor cells

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor which is classified in the bombesin receptor (BnR) family with which it shares high homology. It is present widely in the central nervous system and peripheral tissues and primarily receptor-knockout studies suggest it is involved in metabolic-glucose-insulin homeostasis, feeding and other CNS behaviors, gastrointestinal motility and cancer growth. However, the role of BRS-3 physiologically or in pathologic disorders has been not well defined because the natural ligand is unknown. Until recently, no selective agonists/antagonists were available; however, recently synthetic high-affinity agonists, chiral-diazepines nonpeptide-analogs (3F, 9D, 9F, 9G) with low CNS penetrance, were described, but are not well-categorized pharmacologically or in different labarotory species. The present study characterizes the affinities, potencies, selectivities of the chiral-diazepine BRS-3 agonists in human and rodents (mice,rat). In human BRS-3 receptors, the relative affinities of the chiral-diazepines was 9G > 9D > 9F > 3F; each was selective for BRS-3. For stimulating PLC activity, in h-BRS-3 each of the four chiral diazepine analogs was fully efficacious and their relative potencies were: 9G (EC₅₀: 9 nM) > 9D (EC₅₀: 9.4 nM) > 9F (EC₅₀: 39 nM) > 3F (EC₅₀: 48 nM). None of the four chiral diazepine analogs activated r,m,h-GRPR/NMBR. The nonpeptide agonists showed marked differences from each other and a peptide agonist in receptor-coupling-stiochiometry and in affinities/potencies in different species. These results demonstrate that chiral diazepine analogs (9G, 9D, 9F, 3F) have high/affinity/potency for the BRS-3 receptor in human and rodent cells, but different coupling-relationships and species differences from a peptide agonist.     

Discovery of novel chiral diazepines as bombesin receptor subtype-3 (BRS-3) agonists with low brain penetration

The discovery and optimization of a novel series of BRS-3 agonists are described. We explored a potent BRS-3 agonist with low brain penetration to avoid an adverse effect derived from central nervous system exposure. Through the derivatization process, chiral diazepines 9f and 9g were identified as possessing low brain penetration as well as potent in vitro activity against human and mouse BRS-3s.     

Systematic optimization of a lead-structure identities for a selective short peptide agonist for the human orphan receptor BRS-3.

The orphan receptor, human bombesin receptor subtype 3 (BRS-3) was assigned to the G-protein coupled bombesin receptor family because of its high sequence homology with the neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R). Since its pharmacology is stiIl unknown, new highly potent and selective tool-substances are needed, that may be able to elucidate its possible role in obesity and cancer. We have performed structure activity relationship studies on the high affinity peptide agonists [D-Phe6,beta-Ala11,Phe13,Nle14]Bn(6-14) and [D-Phe6,Phe13]Bn(6-13)propylamide, using their ability to mobilize intracellular calcium in BRS-3 transfected CHOGa-16 cells combined with receptor binding studies. It was demonstrated that for [D-Phe,beta-Ala11,Phe13,Nle14]Bn(6-14) the side chains of the residues Trp8 and Phe13, and to a smaller extent beta-Ala11, are the important amino acid side chains for receptor activation and binding, however for [D-Phe6,Phe13]Bn(6-13) propylamide His12 seems to be more important than Phe13. C-and N-terminal deletions and amino acid substitutions allowed further understanding. It was demonstrated that substitution of His 12 by Tyr leads to a high selectivity towards GRP-R. Using the acquired information, a small tetrapeptide library was designed with compounds presenting Trp and Phe at varying stereochemistry and distances, which led to the discovery of the lead-structure H-D-Phe-Gln-D-Trp-Phe-NH2. Systematic SAR revealed the important structural features of this peptide, C-terminal optimization resulted in the highly active and selective BRS-3 agonist H-D-Phe-Gln-D-Trp-1-(2-phenylethyl)amide. In summary, the size of the peptide was reduced from 8 or 9 amino acids to a tripeptide for BRS-3.     

Rational design of a peptide agonist that interacts selectively with the orphan receptor, bombesin receptor subtype 3

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-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]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-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14) or its d-Phe(6) 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 ([(3)H]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 [(3)H]inositol phosphate. Two peptides with an (R)- or (S)-amino-3-phenylpropionic acid substitution for beta-Ala(11) 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.     

Molecular basis of the pharmacological difference between rat and human bombesin receptor subtype-3 (BRS-3)

We cloned the gene and cDNA for rat bombesin receptor subtype-3 (BRS-3) and characterized its mRNA expression pattern and pharmacological properties. Despite the high degree of sequence similarity (80% identical), rat and human BRS-3 differ markedly in their pharmacological properties. Although the natural ligand for BRS-3 is still unknown, a synthetic peptide, dY-Q-W-A-V-(beta-A)-H-F-Nle-amide (dY-bombesin), activates human BRS-3 with an EC(50) of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC(50) = 2 microM). To understand the molecular basis of this pharmacological difference, we constructed chimeric receptors in which individual extracellular loops of rat BRS-3 were replaced with the corresponding human sequences. Switching the N-terminal region or the second extracellular loop did not significantly change receptor properties. However, switching the third extracellular loop (E3) in the rat BRS-3 resulted in a chimeric receptor (RB3-E3) that behaved almost identically to human BRS-3. RB3-E3 bound dY-bombesin with high affinity (K(i) = 1.2 +/- 0.7 nM), and was activated by dY-bombesin with high potency (EC(50) = 1.8 +/- 0.5 nM). Within the E3 loop, mutation of Y(298)E(299)S(300) to S(298)Q(299)T(300) (RB3-SQT) or of D(306)V(307)P(308) to A(306)M(307)H(308) (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A(302)E(303) to V(302)D(303) or of V(310)V(311) to I(310)F(311) had little effect on the dY-bombesin potency. These results indicate that the sequence variation in the E3 loop is responsible for the species difference between rat and human BRS-3, and multiple residues in the E3 loop are involved in interactions with the agonist dY-bombesin.     

Cloning, expression, and mapping of a gene that is upregulated in adipose tissue of mice deficient in bombesin receptor subtype-3.

To identify novel obesity-related genes in adipose tissue, differential display was performed using bombesin receptor subtype-3 (BRS-3)-deficient mice. These mice exhibit mild late-onset obesity. We report that a gene, Urb, is upregulated in these mice. Full-length Urb cDNA is approximately 3 kb long and comprises an open reading frame of 949 amino acid residues. Interestingly, Urb mRNA expression in brown adipose tissue of BRS-3-deficient mice is fourfold higher than that in wild-type controls. Enhanced Urb mRNA expression was also observed in brain, digestive tissues, kidney, and lung. Within the brain, Urb mRNA is detected in the dorsal endopiriform nucleus and choroid plexus. A T31 radiation hybrid mapping panel revealed that the Urb gene maps to mouse chromosome 16. Collectively, these findings suggest that Urb may have a unique function in the regulation of body weight and energy metabolism.     

Bombesin receptor subtype-3 modulates plasma insulin concentration

Mice lacking a functional bombesin receptor subtype-3 (BRS-3) develop mild obesity. However, the origin of obesity in BRS-3 knockout (KO) mice remains unclear. We used a strain-crossing strategy to investigate the physiological role of the BRS-3 pathway. We crossed female heterozygous BRS-3 KO mice (X-/X) and male KK-Ay mice (Ay/+) to obtain BRS-3 KO/KK-Ay hybrid animals. In X-/Y:Ay/+ mice, plasma insulin concentrations were significantly higher, and on the oral glucose tolerance test, the additional secretion of insulin was impaired compared to other genotypes. Our results indicate that the BRS-3 pathway contributes to the regulation of plasma insulin concentrations.     

Bombesin-like peptide and receptors in lung injury models: diverse gene expression, similar function

We previously demonstrated that bombesin-like peptide (BLP) mediates lung injury in premature infants with bronchopulmonary dysplasia (BPD). We now investigate gene expression and function of BLP (gastrin-releasing peptide, GRP) and BLP-receptors (GRP-R and BRS-3) in lung from two baboon BPD models. In the "interrupted gestation model," only GRP mRNA was up-regulated. In the "hyperoxic model," GRP-R mRNA was up-regulated. In lung explants from O2-treated animals, all BPD animals responded to 1nM bombesin, whereas non-BPD animals did not; the opposite effect was observed with a BLP blocking antibody. Cumulatively, these observations suggest that novel BLPs and/or BLP receptors are likely to be implicated in the pathogenesis of BPD.     

Mutation in bombesin receptor subtype-3 gene is not a major cause of obesity in the Japanese.

Bombesin receptor subtype-3 (BRS-3) is one of the candidate genes of obesity. The mice lacking BRS-3 have been shown to develop mild obesity. These mice also showed hypertension and impaired glucose metabolism, supporting these mice as a good model for human obesity. We screened 104 Japanese obese men (BMI > 26.4, 26.5-44.1) to investigate whether there is any genetic defect in BRS-3 gene. The DNA fragments containing each exon of BRS-3 gene were amplified by polymerase chain reaction (PCR) and were directly sequenced. No mutation, nor polymorphism was found in the coding region of BRS-3, suggesting that mutation of this gene is not a major cause of obesity in humans.     

Discovery of substituted biphenyl imidazoles as potent,bioavailable bombesin receptor subtype-3 agonists

We report SAR studies on a novel non-peptidic bombesin receptor subtype-3 (BRS-3) agonist lead series derived from high-throughput screening hit RY-337. This effort led to the discovery of compound 22e with significantly improved potency at both rodent and human BRS-3.     

The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.     

Characterization of the bombesin-like peptide receptor family in primates

In mammals, bombesin-like peptides mediate a broad range of physiological functions through binding to three highly conserved G-protein-coupled receptors: the neuromedin B-preferring, the gastrin-releasing peptide-preferring, and the bombesin-receptor subtype 3. Selective modulation of these receptors presents opportunities for the development of novel therapeutics. To ascertain if rhesus monkey could serve as a surrogate animal model for the development of modulators of bombesin-like receptor function, we undertook a search for additional receptor family members and studied the expression profiles of the three known bombesin-related receptors. We found no evidence for additional receptor family members in mammals, suggesting that the expression of the previously described bombesin-receptor subtype 4 is limited to amphibians. We studied the distribution of the three receptors in a broad array of human and rhesus monkey tissues. Based on the similarity between the human and the rhesus expression profiles, we conclude that the rhesus monkey may be a suitable animal model to evaluate the clinical efficacy and potential side effects of bombesin-like peptide ligands.     

Bombesin-like peptide receptor gene expression,regulation, and function in fetal murine lung

Bombesin-peptide (BLP) immunoreactivity occurs at high levels in fetal lung. Previous studies showed that bombesin promotes fetal lung development. To test the hypothesis that such effects are mediated by known mammalian bombesin receptors [gastrin-releasing peptide (GRP)/bombesin-preferring receptor (GRPR), neuromedin B (NMB) receptor (NMBR), and the orphan bombesin receptor subtype-3 (BRS-3)], we analyzed the ontogeny of GRPR, NMBR, and BRS-3 gene expression in mouse lung. We examined the regulation of these three genes by dexamethasone and bombesin, which modulate lung development. Using incorporation of [3H]thymidine and [3H]choline, we then assessed whether GRP, NMB, and Leu8-phyllolitorin modulate lung growth and maturation in fetal lung explants. GRPR gene expression was detected predominantly in utero, whereas NMBR and BRS-3 genes were expressed from embryonic days 13-16 and on multiple postnatal days. All three mRNAs are present in airway epithelium and mesenchymal cells but occur in different relative patterns. These genes were regulated differently. Dexamethasone and bombesin increased GRPR mRNA, bombesin downregulated NMBR, and neither agent affected BRS-3. GRP increased incorporation of [3H]thymidine and [3H]choline in explants, whereas NMB induced cell proliferation and Leu8-phyllolitorin yielded variable results. Cumulative data suggest the involvement of multiple BLP receptors, including novel molecules, and argue against simple functional redundancy within this gene family during lung development.     

Wound repair and proliferation of bronchial epithelial cells enhanced by bombesin receptor subtype 3 activation

The present study was designed to investigate the role of bombesin receptor subtype 3 (BRS-3) in airway wound repair. The results showed that: (1) There was few expression of BRS-3 mRNA in the control group. In contrast, the expression of BRS-3 mRNA was gradually increased in the early 2 days, and peaked on the fourth day, and then decreased in the ozone-stressed AHR animal. BRS-3 mRNA was distributed in the ciliated columnar epithelium, monolayer columnar epithelium cells, scattered mesenchymal cells and Type II alveolar cells; (2) The wound repair and proliferation of bronchial epithelial cells (BECs) were accelerated in a concentration-dependent manner by BRS-3 activation with P3513, which could be inhibited by PKA inhibitor H89. The study demostrated that activation of BRS-3 may play an important role in wound repair of AHR.