Characterization of putative GRP- and NMB-receptor antagonist's interaction with human receptors.

The mammalian bombesin (Bn) peptides neuromedin B (NMB) and gastrin-releasing peptide (GRP) actions are mediated by two receptors (NMB-receptor, GRP-receptor) which are widely distributed in the GI tract and CNS. From primarily animal studies NMB/GRP-receptor activation has physiological/pathophysiological effects in the CNS and GI tract including stimulating of growth of cancers and normal tissues. Whereas these Bn-receptors' effects have been extensively studied in nonhuman cells and animals, little is known of the physiological/pathological role(s) in humans, largely due to lack of potent antagonists. To address this issue we compared NMB/GRP-receptor affinity/potency of 10 chemical classes of putative antagonists (35 compounds) for human Bn-receptors by performing binding studies or assessing abilities to activate hGRP/hNMB-receptor [assessing phospholipase C activation] in four different cells containing native Bn-receptors or transfected receptors. From binding studies 23 were GRP-receptor-preferring, 4 were NMB-receptor, and 8 nonselective. For the hGRP-receptor-preferring analogues none showed hGRP-receptor agonist activity, but 13 were full or partial hNMB-receptor agonists at hNMB-receptors. For hNMB-receptor-preferring analogues none were agonists. Analogue #24 ([(3-Ph-Pr(6)), His(7), d-Ala(11), d-Pro(13), Psi(13-14), Phe(14)]Bn(6-14)NH2) and analogue #7 [d-Phe(6), Leu(13), Psi(CH(2)NH), Cpa(14)]Bn(6-14) were the most potent (0.2-1.4nM) and selective (>10,000-fold) for the hGRP-receptor with analogue #7.5 [d-Tpi(6), Leu(13), Psi(CH2NH), Leu(14)]Bn(6-14)[RC-3095] (0.2-1.4nM) slightly less selective. Analogue #34 (PD168368) had the highest affinity for hNMB-receptor (1.32-1.58nM) and the greatest selectivity (2298-6952-fold) for the hNMB-receptor. These results demonstrate numerous putative hGRP/hNMB-receptor antagonists identified in nonhuman cells and/or animals have agonist activity at the hNMB-receptor, limiting their potential usefulness. However, a number were identified which were potent/selective for human Bn-receptors and should be useful for investigating their roles in human physiological/pathophysiological conditions.

Secretin-receptor and secretin-receptor-variant expression in gastrinomas: correlation with clinical and tumoral features and secretin and calcium provocative test results.

CONTEXT/OBJECTIVES: The diagnosis of Zollinger-Ellison syndrome requires secretin testing in 60% of patients. Even with secretin, the diagnosis may be difficult because variable responses occur, and 6-30% have negative testing. The basis for variability or negative responses is unclear. It is unknown whether the tumor density of secretin receptors or the presence of a secretin-receptor-variant, which can act as a dominant negative, is important. The aim of this study was to investigate these possibilities. PATIENTS/METHODS: Secretin-receptor and variant mRNA expression was determined in gastrinomas using real-time PCR from 54 Zollinger-Ellison syndrome patients. Results were correlated with Western blotting, secretin-receptor immunohistochemistry, with gastrin-provocative test results and tumoral/clinical/laboratory features. RESULTS: Secretin-receptor mRNA was detectible in all gastrinomas but varied 132-fold with a mean of 0.89 +/- 0.12 molecules per beta-actin. Secretin-receptor PCR results correlated closely with Western blotting (r = 0.95; P < 0.0001) and receptor immunohistochemistry (P = 0.0015; r = 0.71). The variant was detected in all gastrinomas, but levels varied 102-fold and were 72-fold lower than the total. Secretin-receptor levels correlated with variant levels, Deltasecretin, but not Deltacalcium and with tumor location, but not growth, extent, or clinical responses. Variant levels did not correlate with the Deltasecretin. Detailed analysis provides no evidence that variant expression modified the secretin-receptor response or accounted for negative tests. CONCLUSIONS: Secretin-receptor and secretin-receptor-variant expressions occur in all gastrinomas. Because the expression of the total, but not variant, correlated with the secretin results and no evidence for dominant negative activity of the variant was found, our results suggest that the total secretin-receptor density is an important determinant of the secretin test response.

Identification of bombesin receptor subtype-specific ligands: effect of N-methyl scanning, truncation, substitution, and evaluation of putative reported selective ligands.

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-Tyr(6),betaAla(11),Phe(13),Nle(14)]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 [Ca(2+)](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-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn(6-14) reported to be potent selective BRS-3 ligands on [Ca(2+)](i) assays, only AcPhe,Trp,Ala,His(tauBzl),Nip,Gly,Arg-NH(2) had moderate selectivity for hBRS-3; however, it was less selective than previously reported Apa(11) 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.

Development of simplified vasoactive intestinal peptide analogs with receptor selectivity and stability for human vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptors.

Vasoactive intestinal peptide (VIP) is a widespread neurotransmitter whose physiological and pathophysiological actions are mediated by two receptor classes, VIP/pituitary adenylate cyclase-activating polypeptide (VPAC) 1 and VPAC2. VIP is a 28-amino acid peptide that is rapidly degraded and simplified; metabolically stable analogs are needed. In this study, we use information from studies of the VIP pharmacophore for VPAC1/VPAC2 to design nine simplified VIP analogs that could have high affinity and selectivity for each VPAC or that retained high affinity for both VPACs and were metabolically stable. From binding studies of their abilities to directly interact with hVPAC1 (T47D cells, hVPAC1-transfected cells) and hVPAC2 (Sup T1- and VPAC2-transfected cells) and to stimulate adenylate cyclase in each, two analogs [(Ala(2,8,9,11,19,22,24,25,27,28))VIP and (Ala(2,8,9,11,19,24-28))VIP] were found to have >2000- and >600-fold selectivity for hVPAC1. None of the nine analogs had hVPAC2 selectivity. However, two simplified analogs [(Ala(2,8,9,16,19,24))VIP and (Ala(2,8,9,16,19,24,25))VIP] retained high affinity and potency for both hVPACs. 125I-[Ala(2,8,9,16,19,24,25)]VIP was much more metabolically stable than 125I-VIP. The availability of these simplified analogs of VIP, which are metabolically stable and have either hVPAC1 selectivity or retain high affinity for both hVPACs, should be useful for exploring the role of VPAC subtypes in mediating VIPs' actions as well as being useful therapeutically and for exploring the usefulness of VIP receptor imaging of tumors and VIP receptor-mediated tumor cytotoxicity.

Development of high affinity camptothecin-bombesin conjugates that have targeted cytotoxicity for bombesin receptor-containing tumor cells.

Mammalian bombesin (BN) receptors are among those most frequently overexpressed by a number of common tumors including prostate, breast, lung, and colon cancers. The aim of this study was to develop a camptothecin-bombesin (CPT-BN) conjugate that interacts with all classes of BN receptors and possibly functions as a prodrug via a labile linker with site-specific cytotoxicity for cancer cells bearing these receptors. CPT was coupled to analogs of [D-Tyr6,beta-Ala11,Phe13,Nle14]BN-(6-14) (BA0) using carbamate linkers (L1 and L2) with built-in nucleophile-assisted releasing groups for intracellular cleavage of free cytotoxic agents. One conjugate, CPT-L2-BA3, bound to all three BN receptor classes with high affinity and functioned as a full agonist at each. 125I-CPT-L2-BA3 was rapidly internalized by cells expressing each BN receptor class and, using fluorescent imaging, was found to co-localize with BN receptors initially and later to be internalized in cytoplasmic compartments. HPLC analysis of internalized ligand showed that 40% was intact, 25% was metabolized by releasing free CPT, and 35% was metabolized to other breakdown products. CPT-L2-BA3 inhibited the growth of NCI-H1299 non-small cell lung cancer cells in 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) and clonal growth assays. CPT-L2-BA3 was cytotoxic in an MTT assay for cells transfected with each class of BN receptor; however, it had significantly less effect in cells lacking BN receptors. These results indicate that CTP-L2-BA3 is a potent agonist that is cytotoxic for cells overexpressing any of the three BN receptor classes and functions as a prodrug for receptor-mediated cytoxicity. It therefore should be a useful prototype to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism.

Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors.

Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.

Elucidation of vasoactive intestinal peptide pharmacophore for VPAC(1) receptors in human, rat, and guinea pig.

Vasoactive intestinal peptide (VIP) is a neurotransmitter involved in a number of pathological and physiological processes. VIP is rapidly degraded and simplified stable analogs are needed. VIP's action was extensively studied in rat and guinea pig. However, it is largely unknown whether its pharmacophore in these species resembles human. To address this issue we investigated the VIP pharmacophore for VPAC(1) (the predominant receptor subtype in cancers and widely distributed in normal tissues) by using alanine and D-amino acid scanning. Interaction with rat, guinea pig, and human VPAC(1) was assessed using transfected Chinese hamster ovary (CHO) and PANC1 cells and cells possessing native VPAC(1). Important species differences existed in the VIP pharmacophore. The human VPAC(1) expressed in CHO cells, which were used almost exclusively in previous studies, differed markedly from the native VPAC(1) in T47D cells. The most important amino acids for determining affinity are His(1), Asp(3), Phe(6), Arg(12), Arg(14), and Leu(23). Ser(2), Asp(8), Asn(9), Thr(11), Val(19), Asn(24), Ser(25), Leu(27), and Asn(28) are not essential for high-affinity interaction/activation. [Ala(2,8,9,11,19,24,25,27,28)]VIP, which contained 11 alanines, was synthesized and it was equipotent to VIP at VPAC(1) receptors in all species and was metabolically stable. Our results show in any design of simplified VIP analogs for VPAC(1) it will be important to consider species differences and it is essential to use transfected systems that reflect the native receptor's pharmacophore. Last, with our results a simplified, metabolically stable VIP analog was identified that should be useful as a prototype for design of selective agonists/antagonists that could be useful therapeutically.