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Bombesin receptor antagonists are potential therapeutic agents due to their ability to act as inhibitors of cellular proliferation. On the basis of our hypothesis concerning the mechanism of action of gastrin associating an activating enzyme to the receptor and on the results reported in the literature, we have synthesized bombesin analogs which have been modified in the C-terminal part. Potent bombesin receptor antagonists were obtained by replacement of Leu-13 with a statyl residue or with a residue bearing an hydroxyl group in place of the carbonyl function of Leu-13. Several inhibitors were able to recognize the bombesin receptor on rat pancreatic acini and antagonized bombesin stimulated amylase secretion in the nanomolar range. These compounds were also able to recognize the bombesin receptor and to inhibit [3H] thymidine incorporation in 3T3 cells with the same potency.

]Bn(6 -14)), and a pseudopeptide analogue, JMV641 (D-Phe-Gln-Trp-Ala-Val-GlyHis-Leu(CHOH-CH 2 )-(CH 2 ) 2 -CH 3 ), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 Å of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr 297 , Phe 302 , and Ser 305 of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-interactions and hydrogen bonding are important for their high affinity interaction.The gastrin-releasing peptide (GRP) 1 receptor, which mediates the diverse actions of the mammalian bombesin (Bn)-related peptide (1, 2), GRP, has numerous high affinity peptide antagonists (3-5). This is in contrast to most other gastrointestinal (GI) hormone/neurotransmitter receptors for which no high affinity peptide antagonists exist (6). These GRP receptor antagonists are now widely used in both in vitro studies (5) and in vivo studies in animals (3, 7-11) and humans (12). Recent studies show that for many nonpeptide antagonists, differences in amino acids in the transmembrane domains between receptor subtypes are frequently particularly important for determining receptor subtype selectivity (13,14). A recent study (15) shows a similar result with the peptoid antagonist PD168368 for the neuromedin B receptor. However, with peptide antagonists of non-GI hormone/neurotransmitter receptors, interactions with transmembrane regions (16, 17) or extracellular domains (18) are important for high affinity interaction or receptor subtype selectivity. Which if any of these results apply to the different classes of GRPR peptide antagonists, at present, is unclear.GRP and neuromedin B (NMB), mammalian homologues of the amphibian tetradecapeptide bombesin, have structurally related carboxyl termini (19). These peptides mediate a spectrum of biological activi...

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