Design and synthesis of a specific endothelin 1 antagonist: Effects on pulmonary vasoconstriction

The 21-amino acid vasoconstrictor peptide endothelin (Et) contains two disulfide bonds. We investigated the importance of the outer disulfide bond in Et-1 by replacing it with an amide linkage. Bioactivity was assessed in an isolated guinea pig lung preparation (perfused at constant flow with Ringer's solution/0.5% albumin) in which pulmonary artery pressure was monitored. Et-1 produced concentration-dependent pulmonary vasoconstriction at concentrations of 1 × 10^-10 M and higher. [Dpr¹,Asp¹⁵]Et-1 (where Dpr is diaminopropionic acid), in which the outer disulfide was replaced by an amide bond and the inner disulfide was left intact, showed no agonist activity at 1 × 10^-6 M but 1 × 10^-7 M [Dpr,Asp¹⁵]Et-l inhibited Et-1-induced pulmonary vasoconstriction: effects of 1 × 10^-10 M, 2 × 10^-10 M, and 1 × 10^-9 M Et-1 were inhibited by 98%, 75%, and 65%, respectively. Furthermore, this analog did not alter pulmonary vasoconstriction induced by thrombin, norepinephrine, or, most significantly, Et-3. A monocyclic Et-1 analog with the same sequence but in which the amide bond was not formed showed weak pulmonary vasoconstrictor activity (300-500 times less potent than Et-1) but had no antagonist activity. In addition, both the monocyclic control peptide and [Dpr¹,Asp¹⁵]Et-l competed effectively with ¹²⁵I- labeled Et-1 for binding to cultured rat pulmonary artery smooth muscle cells. Thus, an Et-1 structural analog produced by replacement of the outer disulfide bond with an amide linkage displayed potent and specific Et-1 antagonism.