Systematic optimization of a lead‐structure identities for a selective short peptide agonist for the human orphan receptor BRS‐3

Abstract

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.

International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States

“…Molecular modeling demonstrated that these two selective BB 3 receptor ligands had a unique conformation of the position of the 11␤-amino acids, which probably accounted for their selectivity (Mantey et al, 2001). In a second study ]bombesin 6 -14 retained high affinity for the BB 3 receptor and was 227-fold more selective for the BB 3 receptor than for the human BB 2 receptor and 800-fold more selective than the human BB 1 receptor Weber et al (2002), and 3-phenylpropionyl-Ala,DTrp,NH(CH 2 ) 2 C 6 H 5 , compound 17d in Weber et al (2003)] were found (Mantey et al, 2006) in binding studies and studies of potency for activation of phospholipase C to have affinities Ͼ5 M for all three human bombesin receptor subtypes and therefore not to be useful. The novel compound Ac-Phe,Trp,Ala,His(tBzl), Nip,Gly,Arg-NH 2 [compound 34 in Boyle et al (2005)] had 14-and 20-fold higher affinities for the BB 3 receptor than for the BB 1 receptor BB 2 receptor, respectively (Mantey et al, 2006); however, its selectivity for the BB 3 receptor was less than that of [D-Tyr 6 ,Apa-4Cl 11 ,Phe 13 ,Nle 14 ]-bombesin 6 -14 (i.e., Ͼ100 fold selectivity) (Mantey et al, 2006) (Table 2).…”

Identification of Bombesin Receptor Subtype-Specific Ligands: Effect ofN-Methyl Scanning, Truncation, Substitution, and Evaluation of Putative Reported Selective Ligands

“…We have reported previously that the Bn analog [D-Phe 6 ,␤-Ala 11 ,Phe 13 ,Nle 14 ]Bn(6 -14) ( Table 1, Peptide 2), has high affinity for all known Bn receptors (Mantey et al, 1997(Mantey et al, , 2001Pradhan et al, 1998;Ryan et al, 1998a;Katsuno et al, 1999). Recently, analogs of [D-Phe 6 ,␤Ala 11 ,Phe 13 ,Nle 14 ]Bn(6 -14) have been described (Darker et al, 2001;Mantey et al, 2001Mantey et al, , 2004Weber et al, 2003Weber et al, , 2002 that are reported to have selectivity for hBRS-3 or the other Bn receptor subtypes. In three studies (Weber et al, 2002(Weber et al, , 2003Boyle et al, 2005), this selectivity was based on assessment of changes in [Ca 2ϩ ] i using a FLIPR assay with no direct assessment of receptor affinities.…”

“…Recently, analogs of [D-Phe 6 ,␤Ala 11 ,Phe 13 ,Nle 14 ]Bn(6 -14) have been described (Darker et al, 2001;Mantey et al, 2001Mantey et al, , 2004Weber et al, 2003Weber et al, , 2002 that are reported to have selectivity for hBRS-3 or the other Bn receptor subtypes. In three studies (Weber et al, 2002(Weber et al, , 2003Boyle et al, 2005), this selectivity was based on assessment of changes in [Ca 2ϩ ] i using a FLIPR assay with no direct assessment of receptor affinities. In the present study, we have used a number of strategies known to produce receptor subtype-selective analogs with other peptides to synthesize possible new analogs selective for human Bn receptor subtypes.…”

“…Recently, three studies (Weber et al, 2002(Weber et al, , 2003Boyle et al, 2005), which performed structure/activity studies on [D-Phe 6 ,␤Ala 11 ,Phe 13 ,Nle 14 ]Bn(6 -14) using a calcium or FLIPR calcium assay, have described a number of shortened analogs (Table 4 (Mantey et al, 2001, that we had previously reported were selective for the hBRS-3 (Table 3). As seen in previous studies (Mantey et al, 2001 Bn receptor subtypes but showed less of a decrease in affinity for hBRS-3, resulting in a 50-to 229-fold selectivity for the hBRS-3 over the other human Bn receptors.…”

“…There are relatively few selective synthetic Bn analogs that are metabolically stable for the various Bn receptor subtypes. This has been a particular problem with the BRS-3 receptor because its natural ligand is unknown and only a few synthetic ligands have been described that interact with this receptor with moderate to high affinity (Mantey et al, 1997;Pradhan et al, 1998;Ryan et al, 1998b;Weber et al, 2002Weber et al, , 2003Boyle et al, 2005). This is important because recent studies using BRS-3-deficient mice, produced by targeted disruption, develop hypertension, obesity, and diabetes (Ohki-Hamazaki et al, 1997); however, little is known about the role of BRS-3 in these processes because of the lack of selective ligands.…”