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

Y Masuda, T Sugo, T Kikuchi, A Kawata, M Satoh, Y Fujisawa, Y Itoh, M Wakimasu and T Ohtaki

Discovery Research Laboratories I, Takeda Chemical Industries, Ltd.

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.

Volume 279, Issue 2, pp. 675-685, 11/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

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