Pyridinesulfonylureas and pyridinesulfonamides as selective bombesin receptor subtype-3 (BRS-3) agonists

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor belonging to the subfamily of bombesin-like receptors. BRS-3 is implicated in the development of obesity and diabetes. We report here small-molecule agonists that are based on a 4-(alkylamino)pyridine-3-sulfonamide core. We describe the discovery of 2a, which has mid-nanomolar potency, selectivity for human BRS-3 versus the other bombesin-like receptors, and good bioavailability.     

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.     

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.     

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.     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus

The gene encoding the human homologue of the guinea pig uterine bombesin receptor [(1992) Eur. J. Biochem. 208,405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26–q28 on chromosome X.     

Characterization of the bombesin receptor on mouse pancreatic acini by chemical cross-linking

Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18–27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of ¹²⁵I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used ¹²⁵I-GRP and chemical cross-linking techniques to characterized the mouse pancreatic BN receptor. After binding of ¹²⁵I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18–27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of ¹²⁵I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure.     

Anti-analgesia of a selective NPFF2 agonist depends on opioid activity

NPFF agonists designed to be selective NPFF(2) receptor probes were synthesized. D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH(2) (dNPA) displays a very high affinity (0.027nM) for NPFF(2) receptors transfected in CHO cells, and a very high selectivity with a discrimination ratio greater than 100 versus NPFF(1) receptors. dNPA acts as a potent and selective agonist in [(35)S]GTPgammaS binding experiments and inhibits intracellular cAMP production with the same efficacy as NPA-NPFF. In SH-SY5Y cells expressing NPFF(2) receptors dNPA, in the presence of carbachol, stimulates Ca(2+) release from the intracellular stores. In vivo, after intracerebroventricular injection dNPA increases body temperature in mice and reverses the morphine-induced analgesia. Also, dNPA displays anti-opioid activity after systemic administration. So far, dNPA exhibits the highest affinity and selectivity for NPFF(2) receptors and reveals that its behavioral anti-opioid activity depends on the degree of opioid-induced analgesia.     

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.     

SR 57227A is a partial agonist/partial antagonist of 5-HT3 receptor and inhibits subsequent 5-HT- or SR 57227A-induced 5-HT3 receptor current

The serotonin (5-hydroxytryptamine) type 3 (5-HT₃) receptors are transmembrane ligand-gated ion channels. Although several 5-HT₃ receptor agonists have been used as preclinical tools, SR 57227A is the most commonly used 5-HT₃ receptor agonist with the ability to cross the blood brain barrier. However, the precise pharmacological profile of SR 57227A remains unclear. Therefore, we examined the pharmacological profile of SR 57227A at the 5-HT₃A and 5-HT₃AB receptors. We microinjected Xenopus laevis oocytes with human 5-HT₃A complementary RNA (cRNA) or a combination of human 5-HT₃A and human 5-HT₃AB cRNA and performed two electrode voltage clamp recordings of 5-HT₃A and 5-HT₃AB receptor current in the presence of SR 57227A. Results showed that SR 57227A acts as partial agonist/partial antagonist at the 5-HT₃ receptor. Interestingly, SR 57227A specifically reduced subsequent current amplitudes induced by 5-HT or SR 57227A. Based on its 5-HT₃ receptor partial agonist/partial antagonist properties, we predict that SR 57227A functions as a serotonin stabilizer.     

The design of a new potent and selective ligand for the orphan bombesin receptor subtype 3 (BRS3).

Extensive SAR studies on the unselective BRS3 agonist, [H-D-Phe6,beta-Ala11,Phe13,Nle14]-bombesin-(6-14)-nonapeptide amide, have highlighted structural features important for BRS3 activity and have provided guidance as to the design of selective agonists. A radically modified heptapeptide agonist, maintaining only the Trp-Ala moiety of the parent [H-D-Phe6,betaAla11,Phe13,Nle14]-peptide amide, and with a very different carboxyl terminal region, has been produced which was potent at BRS3 and essentially had no NMB or GRP receptor activity. Its structure is Ac-Phe-Trp-Ala-His(tauBzl)-Nip-Gly-Arg-NH2.     

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.     

Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6–14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6–14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6–14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.     

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.     

Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.     

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.