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

Abstract

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.

Figure 1

Comparison of affinities of various selected human GRP receptor-preferring putative antagonists for BALB 3T3 cells transfected with hGRP-receptor (left) or hNMB-receptor (right). The experimental conditions were similar to those outlined in the legend to Table 1 and as described under Materials and Methods. Values are mean ± S.E.M. from at least four experiments. >10,000 means the affinity was greater than 10,000 nM. Numbers refer to the peptide number in Table 1.

Figure 2

Comparison of affinities of various selected human NMB receptor-preferring putative antagonists for BALB 3T3 cells transfected with hGRP-receptor (left) or hNMB-receptor (right). The experimental conditions were similar to those outlined in the legend to Table 1 and as described under Materials and Methods. Values are mean ± S.E.M. from at least four experiments. >10,000 means the affinity was greater than 10,000 nM. Numbers refer to the peptide number in Table 1.

Figure 3

Affinities of various non-selective putative antagonists for BALB 3T3 cells transfected with hGRP-receptor (left) or hNMB-receptor (right). The experimental conditions were similar to those outlined in the legend to Table 1 and as described under Materials and Methods. Values are mean ± S.E.M. from at least four experiments. Numbers refer to the peptide number in Table 1.

Figure 4

Comparison of the ability of various selected putative human Bn receptor antagonists to stimulate [³H]IP generation in Balb 3T3 cells transfected with hGRP-receptor (left) or hNMB-receptor (right). hGRP-receptor-transfected BALB 3T3 and hNMB-receptor-transfected BALB 3T3 were incubated with each ligand at the indicated concentrations for 60 min. EC₅₀ is the concentration of the indicated peptide causing half-maximal stimulation seen with 1 μM GRP or NMB. The results are the means ± S.E.M. from at least three experiments performed in duplicate. The control and maximal stimulated [³H]IP values for the hGRP-R-transfected BALB 3T3 cells were 2,547 ± 210 and 25,720 ± 1,785 dpm, respectively. The control and maximal stimulated [³H]IP values for the hNMBR-transfected BALB 3T3 cells were 1,895 ± 230 and 71,466 ± 3,010 dpm, respectively. Numbers refer to the peptide number in Tables 3 and 4.

Figure 5

Comparison of the ability of various selected putative human GRP-preferring receptor antagonists without agonist activity, to inhibit the stimulation of [³H]IP generation by GRP in Balb 3T3 cells transfected with hGRP-receptor or NMB in cells transfected with hNMBR. hGRP-receptor-transfected BALB 3T3 and hNMB-receptor-transfected BALB 3T3 were incubated with 1 nM GRP (control: 4,210 ± 350 dpm; maximal: 25,020 ± 2,356 dpm) or 1 nM NMB (control: 2,280 ± 310 dpm; maximal: 33,288 ± 2,130 dpm) and each antagonist at indicated concentrations for 60 min. Results are expressed as the percentage of total [³H]IP released by 1 nM GRP or 1 nM NMB in the presence of various concentrations of each antagonist. The results are the means ± S.E.M. from at least three experiments performed in duplicate. Numbers refer to the peptide number in Tables 3 and 4.

Figure 6

Comparison of the ability of various selected putative human NMB receptor preferring antagonists without agonist activity, to inhibit the stimulation of [³H]IP generation by GRP in Balb 3T3 cells transfected with hGRP-receptor or NMB in cells transfected with hNMB-receptor. hGRP-receptor-transfected BALB 3T3 and hNMB-receptor-transfected BALB 3T3 were incubated with 1 nM GRP (control: 4,210 ± 350 dpm; maximal: 25,020 ± 2,356 dpm) or 1 nM NMB (control: 2,280 ± 310 dpm; maximal: 33,288 ± 2,130 dpm) and each compound at indicated concentrations for 60 min. Results are expressed as the percentage of total [³H]IP released by 1 nM GRP or 1 nM NMB in the presence of various concentrations of each ligand. The results are the means ± S.E.M. from at least three experiments performed in duplicate. Numbers refer to the peptide number in Tables 3 and 4.