title: Receptor binding and antagonist properties of a novel endothelin receptor antagonist, TAK-044 [cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl) carbonyl]-L-alanyl-L-alpha-aspartyl-D-2-(2-thienyl) glycyl-L-leucyl-D-tryptophyl]disodium salt], in human endothelinA and endothelinB receptors.
abstract: Receptor binding and antagonist properties of an endothelin (ET) receptor antagonist, TAK-044 ¿cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl) carbonyl]-L-alanyl-L-alpha-aspartylD-2-(2-thienyl) glycyl-L-leucyl-D-tryptophyl]disodium salt¿, were investigated using recombinant human ETA and ETB receptors expressed in Chinese hamster ovary cells. The membranous ETA receptor was shown to be heterogeneous in ET-3 binding affinity (Hill coefficient = 0.54, Kd1 = 390 pM and Kd2 = 8.1 nM). This heterogeneity disappeared upon the addition of guanosine-5'-O-3-thiotriphosphate (Hill coefficient = 0.95, Kd = 7.8 nM). The Kd (from a computer program LIGAND analysis) and Ki (from Dixon plot analysis) values of TAK-044 were 95 and 120 pM for the membranous ETA receptor and 41 and 60 nM for the ETB receptor, respectively. The Kd values of TAK-044 for the ETA receptor was comparable to that of ET-1. The Ki values of TAK-044 for the cellular ETA and ETB receptors were 130 pM and 130 nM at 5,000 cells/well and 1.3 and 590 nM at 50,000 cells/well, respectively. Dixon plot analysis indicated that TAK-044 is a competitive inhibitor of ET-1 binding. TAK-044 inhibited ET-1-induced phosphatidylinositol hydrolysis and arachidonic acid release at 50,000 cells/well in a competitive manner with respective pA2 values of 8.5 and 8.7 in the ETA-expressing cells and 7.4 and 6.6 in the ETB-expressing cells. TAK-044 suppressed ET-1-induced transient increase in intracellular Ca+2 concentration in the ETA- and ETB-expressing cells with respective IC50 values of 2.8 and 230 nM. TAK-044 is a potent and competitive ETA receptor antagonist which simultaneously exhibits definite antagonist activity at the ETB receptor.
authors.affiliation: Discovery Research Laboratories I, Takeda Chemical Industries, Ltd.
authors.forename: Y
authors.initials: Y
authors.lastname: Masuda
authors.forename: T
authors.initials: T
authors.lastname: Sugo
authors.forename: T
authors.initials: T
authors.lastname: Kikuchi
authors.forename: A
authors.initials: A
authors.lastname: Kawata
authors.forename: M
authors.initials: M
authors.lastname: Satoh
authors.forename: Y
authors.initials: Y
authors.lastname: Fujisawa
authors.forename: Y
authors.initials: Y
authors.lastname: Itoh
authors.forename: M
authors.initials: M
authors.lastname: Wakimasu
authors.forename: T
authors.initials: T
authors.lastname: Ohtaki
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D065128:Endothelin Receptor Antagonists; D006801:Humans; D010456:Peptides, Cyclic / Q000378:metabolism / Q000494:pharmacology*; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D065128:Endothelin Receptor Antagonists; D010456:Peptides, Cyclic; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011994:Recombinant Proteins; C088196:TAK 044; D016718:Arachidonic Acid; D002118:Calcium