Comparative Pharmacology of Bombesin Receptor Subtype-3, Nonpeptide Agonist MK-5046, a Universal Peptide Agonist, and Peptide Antagonist Bantag-1 for Human Bombesin Receptors

Abstract

Bombesin-receptor-subtype-3 (BRS-3) is an orphan G-proteincoupled receptor of the bombesin (Bn) family whose natural ligand is unknown and which does not bind any natural Bnpeptide with high affinity. It is present in the central nervous system, peripheral tissues, and tumors; however, its role in normal physiology/pathophysiology is largely unknown because of the lack of selective ligands. Recently, MK-5046and Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate], a nonpeptide agonist and a peptide antagonist, respectively, for BRS-3 have been described, but there have been limited studies on their pharmacology. We studied MK-5046 and Bantag-1 interactions with human Bn-receptors-human bombesin receptor subtype-3 (hBRS-3), gastrin-releasing peptide receptor (GRP-R), and neuromedin B receptor (NMB-R)-and compared them with the nonselective, peptide-agonist [D-Tyr6,bAla11,Phe13, Nle14]Bn-(6-14) (peptide #1). Receptor activation was detected by activation of phospholipase C (PLC), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), paxillin, and Akt. In hBRS-3 cells, the relative affinities were Bantag-1 (1.3 nM) . peptide #1 (2 nM) . MK-5046 (37-160 nM) . GRP, NMB (.10 mM), and the binding-dose-inhibition curves were broad (.4 logs), with Hill coefficients differing significantly from unity. Curve-fitting demonstrated high-affinity (MK-5046, K i 5 0.08 nM) and low-affinity (MK-5046, K i 5 11-29 nM) binding sites. For PLC activation in hBRS-3 cells, the relative potencies were MK-5046 (0.02 nM) . peptide #1 (6 nM) . GRP, NMB, Bantag-1 (.10 mM), and MK-5046 had a biphasic dose response, whereas peptide #1 was monophasic. Bantag-1 was a specific hBRS-3-antagonist. In hBRS-3 cells, MK-5046 was a full agonist for activation of MAPK, FAK, Akt, and paxillin; however, it was a partial agonist for phospholipase A 2 (PLA 2 ) activation. The kinetics of activation/ duration of action for PLC/MAPK activation of MK-5046 and peptide #1 differed, with peptide #1 causing more rapid stimulation; however, MK-5046 had more prolonged activity. Our study finds that MK-5046 and Bantag-1 have high affinity/selectivity for hBRS-3. The nonpeptide MK-5046 and peptide #1 agonists differ markedly in their receptor coupling, ability to activate different signaling cascades, and kinetics/duration of action. These results show that their hBRS-3 receptor activation is not always concordant and could lead to markedly different cellular responses.

Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor

“…125 I-[D-Tyr 6 , β-Ala 11 , Phe 13 , Nle 14 ]Bn-(6–14), with specific activity of 2,200 Ci/mmol, was prepared by a modification of methods described elsewhere [12,14]. In brief, 0.8 μg of IODO-GEN (in 0.02 mg/ml chloroform) was transferred to a vial, dried under a stream of nitrogen, and washed with 100 μl of 0.5 M KH 2 PO 4 , pH 7.4.…”

“…The incubation was stopped by the addition of 100 μl of distilled water. Radiolabeled peptide was separated using a Sep-Pak (Waters Associates, Milford, MA) and high-performance liquid chromatography as described previously elsewhere [12,14]. The radioligand was stored with 0.5% BSA at −20°C.…”

“…This has occurred because its native ligand is unknown, it has low affinity for all natural Bn peptides, and unlike BB 2 or BB 1 receptor, for which numerous selective agonists and antagonists are described [2,6–11], until recently no agonist or antagonist with sufficient selectivity to be useful for in vivo studies, existed for BB 3 receptor [2,3,12–17]. A high affinity BB 3 receptor agonist has been described, [D-Tyr 6 , β-Ala 11 , Phe 13 , Nle 14 ]Bn(6–14) (peptide #1), which allowed studies of BB 3 receptor’s signaling cascades, demonstrating it was coupled to phospholipase C, A2 and D activation as well as tyrosine kinase cascades [4,14,18–21]. However, peptide #1 was not useful for pharmacological/pathological studies because it was nonselective, having high affinity for BB 2 receptor / BB 1 receptor in all species [12,22–24], as well as human BB 3 receptor [25–27], but not rat/mouse BB 3 receptor [27,28].…”

“…Recently, the BB 3 receptor selective peptide antagonist Bantag-1 was described [14,15,35], however nothing is known of the molecular basis for its high affinity/selectivity for BB 3 receptor. With other Bn receptors [36], as with other GI hormone/neurotransmitter GPCR’s [36,37], there are only limited studies of the molecular basis of high affinity, selectivity of peptide antagonists [36–39].…”

Bombesin-like receptor 3 regulates blood pressure and heart rate via a central sympathetic mechanism

“…Mechanism-based adverse hemodynamic effects are unacceptable in pharmacological treatment for obesity, and it is unclear if attenuation could be used to mitigate this problem. MK-5046 has different activation properties than a BRS-3 peptide ligand (38), suggesting that partial or biased (52) BRS-3 agonists may exist. Discovery of a BRS-3 ligand with anti-obesity but not cardiovascular activities would be welcome.…”