Rational design of a peptide agonist that interacts selectively with the orphan receptor, bombesin receptor subtype 3

The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin -releasing peptide receptor (GRP-R)). This receptor is widely distributed i n the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiolo gical and pathological processes remain unknown due to lack of selective li gands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Saint, E., Akeson, M., Ryan, R. R. Pradhan, T . K., Searles, R. P., Spindel, E. R., Battey, J. F., Coy, D. H., and Jensen , R. T. (1997) J. Biol. Chem. 272, 26062-26071) that [D-Tyr(6),beta -Ala(11 ),Phe(13),Nle(14)]Bn-(6-14) has high affinity for BRS-3 and using this liga nd showed BRS-3 has a unique pharmacology with high affinity for no known n atural Bn peptides. However, use of this ligand is limited because it has h igh affinity for all known Bn receptors. In the present study we have attem pted to identify BRS-3 selective ligands using a strategy of rational pepti de design with the substitution of conformationally restricted amino acids into the prototype ligand [D-Tyr(6),beta -Ala(11),Phe(13),Nle(14)]Bn-(6-14) or its D-Phe(6) analogue. Each of the 22 peptides synthesized had binding affinities determined for hBRS-3, hGRPR, and hNMBR, and hBRS-3 selective li gands were tested for their ability to activate phospholipase C and increas e inositol phosphates ([H-3]inositol phosphate). Using this approach we hav e identified a number of BRS-3 selective ligands. These ligands functioned as receptor agonists and their binding affinities were reflected in their p otencies for altering [H-3]inositol phosphate. Two peptides with an (R)- or (S)-amino-3-phenylpropionic acid substitution for beta -Ala(11) in the pro totype ligand had the highest selectivity for the hBRS-3 over the mammalian Bn receptors and did not interact with receptors for other gastrointestina l hormone s/neurotransmitters. Molecular modeling demonstrated these two se lective BRS-3 ligands had a unique conformation of the position 11 beta -am ino acid. This selectivity was of sufficient magnitude that these should be useful in explaining the role of hBRS-3 activation in obesity, glucose hom eostasis, hypertension, and other physiological or pathological processes.