Systematic optimization of a lead-structure identities for a selective short peptide agonist for the human orphan...

The orphan receptor, human bombesin receptor subtype 3 (BRS-3) was assigned to the G-protein coupled bombesin receptor family because of its high sequence homology with the neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R). Since its pharmacology is stiIl unknown, new highly potent and selective tool-substances are needed, that may be able to elucidate its possible role in obesity and cancer. We have performed structure activity relationship studies on the high affinity peptide agonists [D-Phe6,beta-Ala11,Phe13,Nle14]Bn(6-14) and [D-Phe6,Phe13]Bn(6-13)propylamide, using their ability to mobilize intracellular calcium in BRS-3 transfected CHOGa-16 cells combined with receptor binding studies. It was demonstrated that for [D-Phe,beta-Ala11,Phe13,Nle14]Bn(6-14) the side chains of the residues Trp8 and Phe13, and to a smaller extent beta-Ala11, are the important amino acid side chains for receptor activation and binding, however for [D-Phe6,Phe13]Bn(6-13) propylamide His12 seems to be more important than Phe13. C-and N-terminal deletions and amino acid substitutions allowed further understanding. It was demonstrated that substitution of His 12 by Tyr leads to a high selectivity towards GRP-R. Using the acquired information, a small tetrapeptide library was designed with compounds presenting Trp and Phe at varying stereochemistry and distances, which led to the discovery of the lead-structure H-D-Phe-Gln-D-Trp-Phe-NH2. Systematic SAR revealed the important structural features of this peptide, C-terminal optimization resulted in the highly active and selective BRS-3 agonist H-D-Phe-Gln-D-Trp-1-(2-phenylethyl)amide. In summary, the size of the peptide was reduced from 8 or 9 amino acids to a tripeptide for BRS-3.