Characterization of a novel nonpeptide vasopressin V2‐agonist, OPC‐51803, in cells transfected human vasopressin receptor subtypes

We discovered the first nonpeptide arginine-vasopressin (AVP) V2-receptor agonist, OPC-51803. Pharmacological properties of OPC-51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V2, V1a and V1b) and compared with those of 1-desamino-8-D-arginine vasopressin (dDAVP), a peptide V2-receptor agonist. OPC-51803 and dDAVP displaced [3H]-AVP binding to human V2- and V1a-receptors with Ki values of 91.9±10.8 nM (n=6) and 3.12±0.38 nM (n=6) for V2-receptors, and 819±39 nM (n=6) and 41.5±9.9 nM (n=6) for V1a-receptors, indicating that OPC-51803 was about nine times more selective for V2-receptors, similar to the selectivity of dDAVP. OPC-51803 scarcely displaced [3H]-AVP binding to human V1b-receptors even at 10−4 M, while dDAVP showed potent affinity to human V1b-receptors with the Ki value of 13.7±3.2 nM (n=4). OPC-51803 concentration-dependently increased cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) production in HeLa cells expressing human V2-receptors with an EC50 value of 189±14 nM (n=6). The concentration-response curve for cyclic AMP production induced by OPC-51803 was shifted to the right in the presence of a V2-antagonist, OPC-31260. At 10−5 M, OPC-51803 did not increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells expressing human V1a-receptors. On the other hand, dDAVP increased [Ca2+]i in HeLa cells expressing human V1a- and V1b-receptors in a concentration-dependent fashion. From these results, OPC-51803 has been confirmed to be the first nonpeptide agonist for human AVP V2-receptors without agonistic activities for V1a- and V1b-receptors. OPC-51803 may be useful for the treatment of AVP-deficient pathophysiological states and as a tool for AVP researches. Keywords: OPC-51803, vasopressin, nonpeptide V2-agonist, receptor binding, cyclic AMP Introduction Vasopressin (AVP) is a neurohypophysial hormone which is synthesized in the hypothalamus and released from the pituitary. AVP plays an important role in the regulation of body fluid homeostasis. Physiological actions of AVP are exerted through seven transmembrane G-protein-coupled receptors (Laszlo et al., 1991; Liu & Wess, 1996). V1a-receptors are the most widespread and are coupled with Gq/11; they are responsible for vasoconstriction, platelet aggregation, glycogenolysis and so on, and V1b-receptors are also coupled with Gq/11 and are responsible for adrenocorticotropic hormone (ACTH) secretion, via stimulation of inositol phosphate turnover. V2-receptors are coupled to Gs, and responsible for promotion of water reabsorption in the collecting ducts of the kidney via stimulation of cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) production. In the 1990's, we reported orally effective nonpeptide antagonists of AVP receptors, V1a-selective OPC-21268 (Ogawa et al., 1993; Yamamura et al., 1991), V2-selective OPC-31260 (Ogawa et al., 1996; Yamamura et al., 1992) and OPC-41061 (Kondo et al., 1999; Yamamura et al., 1998). Then several nonpeptide antagonists of AVP receptors, such as V1a-selective SR-49059 (Serradeil-Le Gal et al., 1993), V2-selective SR-121463A (Serradeil-Le Gal et al., 1996), and VPA-985 (Albright et al., 1998), and nonselective YM-087 (Tahara et al., 1997) followed. These nonpeptide antagonists could overcome the problem of intrinsic agonistic activity associated with peptide vasopressin antagonists. Since peptide antagonists have an intrinsic agonistic activity, they acted as an agonist when tested in human. Though it has been difficult to develop antagonists without an agonistic activity for human AVP V2-receptors by peptide analogues, nonpeptide compounds succeeded in acting as an antagonist in human (Ohnishi et al., 1993; 1995). The approach for developing the nonpeptide ligands for peptide hormone receptors may enable us to produce functionally separate ligands, agonist and antagonist, and elucidate the mechanisms of receptor-ligand interactions. Recently, nonpeptide agonists have been targeted for the seven transmembrane G-protein-coupled receptors as well as antagonists. In fact, nonpeptide agonists have been reported in the angiotensin system (Kivlighn et al., 1995; Perlman et al., 1995) and the cholecystokinin system (Aquino et al., 1996; Willson et al., 1996). During our development of nonpeptide antagonists for AVP receptors, some compounds were also found to possess antidiuretic effects in rats. Since molecular cloning of human AVP receptor subtypes was identified (Birnbaumer et al., 1992; de Keyzer et al., 1994; Hirasawa et al., 1994; Lolait et al., 1992; Sugimoto et al., 1994; Thibonnier et al., 1994), we designed the stable expression of each human AVP receptor in HeLa cells (Yamamura et al., 1998) and attempted to develop functionally acting, human V2-receptor agonists. After the lead optimization of congeners of OPC-31260 and OPC-41061, OPC-51803 was selected as the first nonpeptide AVP V2-receptor agonist. In this study, we used cells stably expressing each human AVP receptor subtype (V2, V1a, and V1b) and tried to characterize the binding and functional properties of OPC-51803, (5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl) benzoyl) -2,3,4,5- tetrahydro-1H-1- benzazepin-5-yl] -N- isopropylacetamide (Figure 1) and compared it with dDAVP, a standard peptide AVP V2-receptor agonist. Figure 1 Structure of OPC-51803. Methods Materials OPC-51803 and OPC-31260 were synthesized by the Tokushima Second Factory, Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). AVP was purchased from Peptide Institute Inc. (Osaka, Japan), [3H]-AVP from NEN™ Lifescience Products, Inc. (Boston, MA, U.S.A), dDAVP, [deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin, penicillin, streptomycin, 3-isobutyl-1-methylxanthine (IBMX), ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), bovine serum albumin (BSA), MnCl2, and trypsin inhibitor from Sigma (St. Louis, MO, U.S.A), lipofectamine, foetal bovine serum (FBS), and trypsin-EDTA from GIBCO BRL (Rockville, MD, U.S.A), Dulbecco's minimum Eagle's medium (DMEM) from Nissui Pharmaceutical Co. (Tokyo, Japan), phosphate-buffered salts from Takara Shuzo Co., Ltd. (Kusatsu, Japan), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (HEPES), Fura-2 and Fura-2 acetoxymethyl ester (Fura-2-AM) from Dojindo Laboratories (Kumamoto, Japan), polyethylene glycol mono-p-isooctylphenyl ether (Triton X-100) from Nacalai Tesque (Kyoto, Japan), and geneticin disulphate (G418) and other chemicals from Wako Pure Chemicals (Osaka, Japan).