Design Of Selective Antagonists Research Articles

Abstract 5787: Discovery and Characterisation of Linear and Cyclic Peptide Agonists of Apelins

The recently discovered apelin family of peptides mediate their actions by a single G-protein coupled receptor, APJ. We have previously shown apelins have three major actions in the human cardiovascular system: endothelium-dependent vasodilatation; direct vasoconstriction by interacting with smooth muscle APJ receptor and increased cardiac contractility by action on cardiac myocytes. [Pyr 1 ]apelin-13 was identified as the predominant isoform in human heart. Our aim was to discover shorter sequences of [Pyr 1 ]apelin-13 retaining agonist activity and use a computational ligand based strategy to design cyclic peptides. Over 30 compounds were synthesised and tested in a competition binding assay against [Glp65,Nle75,Tyr77] [ 125 I]Apelin-13. Functional assays measured the vaso-constrictor action in endothelium denuded human saphenous veins in oxygenated Krebs’ solution (37°C) in organ baths for isometric tension recordings. Cumulative concentration response curves were constructed to the endogenous and synthetic analogues. Responses were expressed as percentage of the terminal KCl (0.1M) constriction. Data are expressed as mean ± S.E. Mean and n-values are the number of patients from whom tissues were obtained. One micromolar concentrations of the predicted cleavage product of the angiotensin converting enzyme 2 (ACE-2), apelin-13(1–12), inhibiting radiolabelled apelin binding by 60.0±7.0% (n=3). The most potent cyclic analogue identified, MM07 inhibited binding by 42.2±4.8% (n=3) compared with 76.0±4.5% inhibition by (Pyr 1 )apelin-13. Both compounds potently constricted endothelium denuded saphenous vein (Apelin-13(1–12): pD 2 9.07±0.40, E max 29.30±9.43% KCl, n=5; MM07: pD 2 10.53±0.24, E max 21.80±5.72% KCl, n=3) with a comparable potency and efficacy to (Pyr 1 )apelin-13 (pD2 (pD 2 = 8.8±0.3, E MAX = 26±4%, n=15). These results show unexpectedly that the cleavage product of ACE-2 retains full agonist activity. The novel cyclic peptide retains potency and can be used as a pharmacological tool to characterise the apelin system and in the design of selective antagonists.