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

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-alanine(11) 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.

Figure 1

Comparison of affinities for binding and potencies for activation of various natural Bn related peptide agonists for native human GRP receptors on the HuTu-80 cell line [left panels] and Balb 3T3 cells transfected with hGRPR [right panels]. The upper panels [Panels A and B] show the affinities from binding studies using 50 pM ¹²⁵I-[Tyr⁴] Bn as the ligand. The lower panels [panels C and D] show their potencies for activating phospholipase C and stimulating changes in total cellular [³H]inositol phosphates ([³H]IP). The experimental conditions were as outlined in the legend to Table 2 and as described under Materials and Methods. Values are mean ± S.E.M. from at least four experiments. Full names of peptide abbreviations are shown in Table 1.

Figure 2

Comparison of affinities for binding and potencies for activation of various natural Bn related peptide agonists for human NMB receptors overexpressed in low density on NCI-H1299 lung cancer cells [left panels] or in Balb 3T3 cells transfected with hNMBR [right panels] The upper panels [Panels A and B] show the affinities from binding studies using 50 pM ¹²⁵I-[D-Tyr⁰]NMB as the ligand. The lower panels [panels C and D] show their potencies for activating phospholipase C and stimulating changes in total cellular [³H]inositol phosphates ([³H]IP). The experimental conditions were as outlined in the legend to Table 2 and as described under Materials and Methods. Values are mean ± S.E.M. from at least four experiments. Full names of peptide abbreviations are shown in Table 1.

Figure 3

Comparison of affinities for binding and potencies for activation of various synthetic Bn related peptide agonists for native human GRP receptors on the HuTu-80 cell line [left panels] and Balb 3T3 cells transfected with hGRPR [right panels]. The upper panels [Panels A and B] show the affinities from binding studies using 50 pM ¹²⁵I-[Tyr⁴]Bn as the ligand. The lower panels [panels C and D] show their potencies for activating phospholipase C and stimulating changes in total cellular [³H]inositol phosphates ([³H]IP). The experimental conditions were as outlined in the legend to Table 2 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 affinities for binding and potencies for activation of various synthetic Bn related peptide agonists for human NMB receptors overexpressed in low density on NCI-H1299 lung cancer cells [left panels] or in Balb 3T3 cells transfected with hNMBR [right panels] The upper panels [Panels A and B] show the affinities from binding studies using 50 pM ¹²⁵I-[D-Tyr⁰]NMB as the ligand. The lower panels [panels C and D] show their potencies for activating phospholipase C and stimulating changes in total cellular [³H]inositol phosphates ([³H]IP). The experimental conditions were as outlined in the legend to Table 2 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 5

Correlations of the affinities and potencies of the various natural occurring Bn-related peptides and synthetic peptides for the different human Bn receptor cells and for both rat GRP receptors and rat NMB receptors on AR42J cells or rat C6 glioblastoma cells, respectively. Each point represents data from a single Bn analogue shown in Table 1–4. In all graphs there are 27 points, although in most graphs <27 points are seen because the majority are clustered near the origin and superimposed on each other. Correlation coefficients were calculated using a least-squares analysis from the data shown in Fig. 1–4, and Tables 2–4 as described in Materials and Methods.