AL-8810: A Novel Prostaglandin F2a Analog with Selective Antagonist Effects at the Prostaglandin F2a (FP) Receptor

A novel analog of prostaglandin F2a [AL-8810; (5Z, 13E)(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17, 18,19,20-pentanor-5,13-prostadienoic acid] has been discovered with uniquely low efficacy (Emax) at the endogenous prostaglandin F2a receptors (FP receptors) of A7r5 rat thoracic aorta smooth muscle cells and Swiss mouse 3T3 fibroblasts, as assayed by stimulation of phospholipase C activity. AL-8810 has weak agonist potency (EC50) of 261 6 44 nM (n 5 3) and Emax 5 19% (relative to the full FP receptor agonist cloprostenol) in A7r5 cells and EC50 of 186 6 63 nM (n 5 3) and Emax 5 23% in 3T3 fibroblasts. AL-8810 exhibited properties of an apparent competitive antagonist, i.e., produced parallel dextral shifts of the agonist concentration-response curves and no significant suppression of the maximal agonist-induced response, when the potent, selective FP receptor agonist fluprostenol was used. The inhibition parameters of AL-8810 were: pA2 5 6.68 6 0.23 and 6.34 6 0.09 (n 5 3–4) for A7r5 cells and 3T3 cells, respectively, with Schild slopes ranging from 0.80 to 0.92. AL-8810 concentrationdependently antagonized the response to 100 nM fluprostenol (Ki 5 426 6 63 nM; n 5 5) in A7r5 cells. However, even at 10 mM concentration, AL-8810 did not significantly inhibit functional responses of TP, DP, EP2, EP4, receptor subtypes in various cell lines. AL-8810 also did not antagonize the phospholipase Ccoupled V1-vasopressin receptor in A7r5 cells. These results suggest that AL-8810 is a unique, selective antagonist at the FP receptor, a heretofore unavailable pharmacological tool that should be valuable for studying FP receptor-mediated functional responses in complex biological systems. The classification of prostanoid [prostaglandin (PG)] receptors according to the binding affinities, potencies, and selectivities of the five known classes of endogenous PGs (PGD2, PGE2, PGF2a, PGI2, and thromboxane A2), as first proposed by Coleman et al. (1990), has been validated in numerous studies by using pharmacological methods and molecular biological techniques (Coleman et al., 1994; Narumiya, 1994). All PG receptors identified to date are seven-transmembrane proteins that couple to specific G proteins mediating the formation of cAMP or inositol trisphosphate/diacylglycerol second messengers (Coleman et al., 1994). Isoforms of some PG receptors, e.g., the EP, TP, and prostaglandin PGF2a (FP) receptors, have been positively identified, although full characterization of the pharmacological properties of all such isoforms generally has not yet been accomplished (Coleman et al., 1994; Narumiya, 1994; Sugimoto et al., 1994; Pierce et al., 1997). Potent, selective, synthetic agonists of some PG receptors have defined the specific functional responses coupled to activation of those receptors by use of cell lines expressing the respective cloned or endogenous PG receptors and also isolated tissues (Coleman et al., 1994). However, conclusive identification of the specific receptor(s) involved in PG-mediated functional responses of complex biological systems in vitro or in vivo requires potent, selective receptor antagonists. To date, of the eight known major PG receptor subtypes, experimentally useful antagonists, e.g., possessing both the requisite potency and selectivity, have been described only for the DP receptor (BW A868C) (Giles et al., 1989) and TP receptor (SQ-29,548) (Ogletree et al., 1985), whereas less robust antagonists with modest to poor selectivity have been characterized for the EP1 receptor (SC51089 and AH6809, which is also a weak antagonist at the EP2 and DP receptors) and EP4 receptor (AH23848) (Coleman et al., 1994). FP receptor agonists are potent, highly efficacious agents that reduce elevated intraocular pressure in humans and certain animals (Wang et al., 1990; Bito, 1997) and that have other pharmacological effects in the mammalian body (Coleman et al., 1994). The major FP receptor isoform has high-sequence homology among many animal species (Abramovitz et al., 1994, 1995; Lake et al., 1994; Sakamoto et al., 1994); a second, minor FP receptor isoform with a trunReceived for publication March 4, 1999. ABBREVIATIONS: PG, prostaglandin; FP receptor, prostaglandin PGF2a receptor; DMEM, Dulbecco’s modified Eagle’s medium; IP, inositol phosphate; AVP, Arg-vasopressin; PI, phosphoinositide; PLC, phospholipase C; Emax, maximal response (%) relative to the maximal response of a standard full agonist; RIA, radioimmunoassay; CHO, Chinese hamster ovary; EbTr, embryonic bovine tracheal; NPE, nonpigmented ciliary epithelial. 0022-3565/99/2903-1278$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:1278–1284, 1999 1278 at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from cated carboxyl terminus has been detected thus far only in a highly specialized cell type (large cell of mid-cycle corpus luteum) of sheep (Pierce et al., 1997). The structure, signaling properties, and pharmacological profile of the single FP receptor in humans and several species have been investigated. Agonist binding to the FP receptor activates phospholipase C (PLC), producing elevated diacylglycerol and inositol trisphosphate and a rapid increase in intracellular Ca as early-signaling events (Davis et al., 1987; Woodward et al., 1990; Sakamoto et al., 1994; Pierce et al., 1997; Griffin et al., 1997, 1998). Numerous such studies have demonstrated considerable similarity of FP receptor function across species, corroborating predictions based on the high interspecies homology of the FP receptor gene. However, the tissue distribution of the FP receptor in ocular as well as nonocular tissues varies greatly among species (Bhattacherjee and Paterson, 1994; Coleman et al., 1994; Lake et al., 1994; Narumiya, 1994; Abramovitz et al., 1995; Ocklind et al., 1997; Mukhopadhyay et al., 1999; Davis and Sharif, 1999), raising important questions about its fundamental physiologic functions. Despite substantial efforts in this area, there is still no well characterized FP receptor antagonist sufficiently potent and selective to address these issues. A few structural analogs of PGF2a, such as PGF2a dimethylamine and PGF2a dimethylamide (Stinger et al., 1982) and some nonprostanoid structures, i.e., phloretin (Kitanaka et al., 1993) and glibenclamide (Delaey and Van de Voorde, 1995), have been reported to be FP receptor antagonists. However, the potencies and selectivities of these compounds at the FP receptor relative to other prostanoid receptors are not well