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Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus

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Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus BASIC RESEARCH www.jasn.org Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus Fre´de´ ric Jean-Alphonse,* Sanja Perkovska,* Marie-Ce´line Frantz,† Thierry Durroux,* Catherine Me´jean,* Denis Morin,* Ste´phanie Loison,† Dominique Bonnet,† Marcel Hibert,† Bernard Mouillac,* and Christiane Mendre* *CNRS UMR 5203, Institut de Ge´nomique fonctionnelle, INSERM U661, and Universite´ Montpellier I and II, Montpellier, France; and †CNRS UMR 7175, Institut Gilbert Laustriat, and Universite´ Louis Pasteur, Illkirch, France ABSTRACT X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV2R). Most of these mutations lead to intracellular retention of the hV2R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV2Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV2R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV2R, being unable to recruit arrestin, trigger receptor internaliza- tion, or stimulate mitogen-activated protein kinases. These characteristics make these hV2R agonist phar- macochaperones promising therapeutic candidates for cNDI. J Am Soc Nephrol 20: 2190–2203, 2009. doi: 10.1681/ASN.2008121289 The antidiuretic hormone arginine-vasopressin congenital nephrogenic diabetes insipidus (cNDI), (AVP) is crucial for osmoregulation, cardiovascular a rare disease characterized by the kidney’s inability control, and water homeostasis. The human AVP to concentrate urine despite normal or elevated 4 V2 receptor (hV2R), localized in the principal cells plasma concentrations of AVP. More than 200 dif- of the kidney collecting duct, mediates AVP antidi- ferent mutations have been described and are re- uretic effect and therefore helps in maintaining sponsible for polyuria, a main consequence of the physiologic plasma osmolality, blood volume, and disease. Most of the mutant receptors (cNDI- arterial pressure. Binding of AVP to hV2R first trig- hV2Rs), trapped in the endoplasmic reticulum gers a cAMP signal through activation of the G pro- ␣ tein s (Gs) subunit and adenylyl cyclase (AC). Received December 22, 2008. Accepted June 30, 2009. Then, the cAMP-activated protein kinase A phos- Published online ahead of print. Publication date available at phorylates aquaporin 2 water channels, resulting in www.jasn.org. their insertion into the luminal membrane of prin- F.J.-A., S.P., and M.-C.F. contributed equally to this work. cipal cells and finally to water reabsorption.1 AVP Correspondence: Dr. Christiane Mendre or Bernard Mouillac, IGF, binding to hV2R also induces arrestin recruitment, 141 rue de la Cardonille, 34094 Montpellier Cedex, France. Phone: receptor internalization,2 and mitogen-activated ϩ33-467-142-984/467-142-922; Fax: ϩ33-467-542-432; E-mail: protein kinase (MAPK) activation.3 [email protected] or [email protected] ᮊ Mutations in the hV2R gene lead to the X-linked Copyright 2009 by the American Society of Nephrology 2190 ISSN : 1046-6673/2010-2190 J Am Soc Nephrol 20: 2190–2203, 2009 www.jasn.org BASIC RESEARCH A (ER), cannot reach the cell surface and interact with AVP.5 cNDI is thus referred as a conformational or protein-misfold- ing disease.6 Various chaperones,7 either chemical (cellular osmolytes such as glycerol or DMSO) or pharmacologic (specific li- gands),8,9 are promising therapeutic agents for future clinical treatment of protein-misfolding disorders. Because a majority of cNDI-hV2Rs are misfolded and many ligands are available for hV2R, the pharmacochaperone-based strategy is of partic- B ular interest for cNDI. Considering an efficient therapy for this disease, the ideal drug should combine pharmacochaperone properties together with hV2R agonist and noninternalizing activities, for stimulating AC and maintaining a long-lasting cAMP signal. This would classify such a molecule as a biased agonist or functionally selective compound.10 Small nonpeptide AVP antagonists (commonly named vaptans)—such as the hV2R-selective antagonists SR121463 (satavaptan), VPA985 (lixivaptan),11 OPC41061 (tolpavtan), Figure 1. Structure of the three MCF nonpeptide compounds and OPC31260 (mozavaptan)12; the V receptor (V R) an- and snake-like plot of the hV R. (A) Chemical structures of 1a 1a 2 tagonist SR49059 (relcovaptan); and the nonselective V R/ MCF14, MCF18, and MCF57. (B) L44P, L59P, Y128S, A294P, and 1a 13 R337X cNDI mutants of the hV R used in the study. V2R antagonist YM087 (conivaptan) —were demonstrated 2 to promote adequate maturation and cell surface rescue of cNDI-hV2Rs, with restoration of their capacity to initiate a cell A B CD Figure 2. Binding profiles of AVP and MCF compounds for human AVP/OT receptor subtypes expressed in tsA201 cells. Competition experiments using [3H]AVP as radioligand on tsA201 transfected cells were done as described in the Concise Methods section and in 3 f F the legend to Table 1. (A through D) [ H]AVP competition experiments to each human c-myc–tagged AVP/OT receptors ( ,V2; ,V1a; ‚ E ,V1b; , OTR) were performed using increasing concentrations of unlabeled ligand: AVP (A), MCF14 (B), MCF18 (C), and MCF57 (D). Specific binding was expressed as percentage of the specific binding measured in the presence of vehicle only. Results illustrated correspond to an experiment representative of three independent experiments, each performed in duplicate. J Am Soc Nephrol 20: 2190–2203, 2009 V2R Biased Agonist Pharmacochaperones 2191 BASIC RESEARCH www.jasn.org Table 1. Affinity of the MCF nonpeptide compounds for the human AVP/OT receptor subtypes Receptors Compound c-myc-hV2R c-myc-hV1aR c-myc-hV1bR c-myc-hOTR Ki nM V2-SI Ki nM V2-SI Ki nM V2-SI Ki nM V2-SI AVP 5.3 Ϯ 0.5 1.0 1.1 Ϯ 0.2 0.2 1.4 Ϯ 0.3 0.3 6.3 Ϯ 0.7 1.2 MCF14 6.2 Ϯ 0.8 1.0 19.0 Ϯ 3.0 3.1 Ͼ1000 Ͼ100 37.0 Ϯ 8.0 6.0 MCF18 20.0 Ϯ 3.0 1.0 106.0 Ϯ 10.0 5.3 Ͼ1000 Ͼ100 31.0 Ϯ 7.0 1.5 MCF57 18.0 Ϯ 3.0 1.0 122.0 Ϯ 34.0 6.8 Ͼ1000 Ͼ100 214.0 Ϯ 55.0 12.0 Binding assays were performed as described in the Concise Methods section and are illustrated in Figure 2. ͓3H͔AVP binding was measured on whole tsA201 cells transfected with c-myc-hV2R, c-myc-hV1aR, c-myc-hV1bR, or c-myc-hOTR subtypes and displaced using increasing concentrations of AVP, MCF14, MCF18, ͓3 ͔ or MCF57. Inhibition constants (Ki in nM) were determined from the competition experiments performed using H AVP concentrations around Kd for the Ϯ ϭ various receptors (i.e., 1.5 nM). Data are means SEM of three distinct experiments each done in duplicate. V2-SI (normalized selectivity index) Ki MCF or ͓3 ͔ Ϯ Ϯ AVP for VxR/Ki MCF or AVP for V2R. The Kd for H AVP, calculated from saturation binding assays done on whole tsA201 cells were 2.20 0.40, 0.76 0.08, Ϯ Ϯ 0.85 0.04, and 3.10 0.30 nM for c-myc-hV2R, c-myc-hV1aR, c-myc-hV1bR, and c-myc-hOTR, respectively. Table 2. Agonist properties of the nonpeptide compounds on c-myc-hV2R–stimulated AC activity cAMP Accumulation Compound EC50 (nM) Emax (%) AVP 0.05 Ϯ 0.01 100 MCF14 4.00 Ϯ 2.00 96 Ϯ 5 MCF18 7.00 Ϯ 1.00 95 Ϯ 3 MCF57 6.00 Ϯ 2.00 103 Ϯ 7 Accumulation of cAMP was performed as described in the Concise Methods section and supplemental information, using an homogeneous time-resolved fluorescence-fluorescence resonance energy transfer technology developed by CisBio Int. TsA201 cells transfected with the myc-hV2R were stimulated for 30 min with increasing concentrations of AVP, MCF14, MCF18, or MCF57 in Figure 3. Determination of agonist properties of the nonpeptide the presence of 0.1 mM of the phosphodiesterase inhibitor RO 201724. The cAMP accumulation was expressed as the mean Ϯ SEM of cAMP compounds for the c-myc–tagged hV2R using cAMP accumula- concentration (nM) and reported as percentage of the maximal AVP response Ϯ tion as a readout. The tsA201 cells transfected with the c-myc- (Emax). EC50s in the table are the mean SEM from three distinct Њ hV2R were incubated for 30 min at 37 C in a cAMP buffer con- experiments performed in triplicate and described in the legend to Figure 3. taining 0.1 mM RO 201724 with or without increasing amounts of AVP (F), MCF57 (E), MCF14 (‚), or MCF18 (f). As described in RESULTS the Concise Methods section, cAMP accumulation was evaluated using the Cisbio International Dynamic 2 kit based on fluores- Nonpeptide Compounds Have a High Affinity for hV2R cence resonance energy transfer measurements. Basal cAMP ac- and Induce Receptor-Dependent cAMP Production cumulation values were subtracted from values obtained with To develop ligands exhibiting both hV R agonist properties AVP or MCFs. Data are means Ϯ SEM of cAMP concentration 2 and pharmacochaperone effects on cNDI-hV Rs, we used the (nM). The figure illustrates an experiment representative of three 2 distinct experiments, each performed in triplicate. Otsuka OPC23h and the Wyeth-Ayerst WAY-VNA-932 non- peptide hV2R agonist molecules as lead compounds. New mol- ecules derived from these antidiuretics were synthesized for response upon AVP binding.
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