Structure-Function Analyses of Prostaglandin E2 Receptor Subtypes 1 and 3

Abstract

Prostaglandin E2 (PGE2) is a lipid autacoid, which is an oxidative metabolite of arachidonic acid synthesized by cyclooxygenase and prostaglandin E synthases. PGE2 is proposed to mediate in part the pathophysiology of hypertensive and diabetic kidney diseases. To probe the structure and function of a cell surface receptor for PGE2, single amino acid substitutions of the mouse EP3 receptor were generated, each having a single cysteine-to-alanine missense mutation. These studies demonstrated a critical disulfide bond between the first and second extracellular loops of mouse EP3 at position C107 and C184. Receptors substituted at either of these cysteines had attenuated expression and were functionally inactive. To study EP1 and EP3 receptors in mouse models of chronic kidney disease, selective antagonists of EP1 and/or EP3 were synthesized and characterized. A 4-chlorophenylsulfonamide was a functional antagonist of EP1 and EP3, lacked the off-target activity of the lead antagonist, and was efficiently metabolized in vitro to a small subset of metabolites. Subcutaneous injection of this compound prolonged plasma exposure of this antagonist sufficiently to maintain receptor coverage with once daily injection. Subcutaneous injection of this antagonist significantly blunted the vasopressor response of EP1 and EP3 agonists in mice. Thus, this represents a novel, selective EP1 and EP3 antagonist that is bioavailable in mice and suitable for use in studies of chronic kidney disease.