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

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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 internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV2R agonist pharmacochaperones 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

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A (ER), cannot reach the cell surface and interact with AVP.5 cNDI is thus referred as a conformational or protein-misfolding disease.6 Various chaperones,7 either chemical (cellular osmolytes such as glycerol or DMSO) or pharmacologic (specific ligands),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

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.

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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 nonpeptide hV2R agonist molecules as lead compounds. New molecules derived from these antidiuretics were synthesized for response upon AVP binding. Although the vaptans display the optimizing preferential affinity and activity toward hV2R. The expected pharmacochaperone beneficial effects, their antago- pharmacologic properties of one of these hits, MCF57 (Figure nistic activity limits their use as a result of their inability to 1A), were compared with those of OPC23h (termed MCF14 stimulate membrane-targeted cNDI-hV2Rs directly. Compar- here) and WAY-VNA-932 (named MCF18) in terms of hV2R atively, agonist pharmacochaperones would combine crucial affinity and Gs/AC activation. The three ligands fully displaced advantages for treating cNDI. [3H]AVP binding on tsA201 cells expressing the c-myc–tagged

Here, we identified the first functionally selective hV2Rag- AVP hV2R and hV1aR receptors, and the human oxytocin (OT) onist pharmacochaperones. The Wyeth-Ayerst WAY-VNA- receptor (hTOR) (Figure 2, A through D) but were unable to 3 932 and the Otsuka OPC23h nonpeptide hV2R antidiuret- compete with [ H]AVP binding to the hV1bR. They all dis- 14,15 ics, as well as a novel compound MCF57, were tested for played a preferential affinity (6 to 20 nM) for the c-myc-hV2R their capacity to recruit intracellularly trapped cNDI-hV2Rs versus other receptor subtypes (Table 1). MCF57 possessed the 3 and to restore their functionality. In addition, we determined best hV2R selectivity index. [ H]AVP saturation binding ex- the capacity of the three ligands to act as hV2R biased agonists periments performed with various MCF57 concentrations on (i.e., their behavior in terms of Gs/AC agonism and arrestin c-myc-hV2R–expressing cells revealed that the nonpeptide in- antagonism). hibited AVP binding competitively (data not shown).

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A B

E F

125

receptor) 100 2

25 Cell surface expression surface Cell

(% of membrane Myc-hV 0 l o 3 4 8 7 tr 6 F1 F1 5 n 4 o 1 C CF C 2 MC M M SR1

Figure 4. Cell surface rescue of the different N-terminally c-myc–tagged cNDI-hV2Rs with the nonpeptide compounds. (A through F) Transiently transfected tsA201 cells with c-myc–tagged L44P (A), A294P (B), R337X (C), Y128S (D), L59P (E) or mV2 (F) receptors were incubated for 18 h at 37ЊC with nonpeptide compounds at 10 ␮M (SR121463, MCF14, MCF18, and MCF57) or with vehicle only (DMSO 0.1%, control). Each of these receptors f Ⅺ ( ) was compared with the wild-type hV2R( ). ELISA measurements with an anti–c-myc (9E10) antibody were performed on nonpermeabilized cells as described in the Concise Methods section. Results are expressed as percentage of the maximal hV2R expression under control conditions done in the same experiment and are the means Ϯ SEM of at least three independent experiments, each performed in triplicate. For each nonpeptide compound, a statistical analysis between control and treated cells was done: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001.

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very effective and their rescue properties equivalent to those of the reference pharmacochaperone SR121463. By contrast, whereas SR121463 was still efficient to rescue Y128S and L59P, MCFs were either less active or inactive, respectively (Figure 4, D through E). Interestingly, the agonists increased significantly

membrane expression of the mV2R, such as SR121463 (Figure 4F). The three MCFs exhibited similar pharmacochaperone behavior, being active on several receptors (L44P, A294P, and

R337X), less active on others (Y128S and mV2R), or inactive (L59P). To avoid potential escape from the cell quality control sys- tem as a result of mutant overexpression, we defined condi-

tions in which no cNDI-hV2Rs could be detected at the cell surface in control situation using varying plasmid quantities.

Even with low cNDI-hV2R plasmid quantities, membrane targeting of the intracellularly retained cNDI-hV2Rs using MCF57 as a pharmacochaperone reference was evidenced (Supplemental Figure 1).

We also checked membrane rescue of cNDI-hV2Rs by directly visualizing the effects of MCF57 using confocal microscopy. We performed immunofluorescence experiments with nonpermeabilized cells to label only cell surface receptors. L44P, A294P, and R337X were shown to respond efficiently to Figure 5. Confocal microscopy visualization of cell surface res- the pharmacochaperone treatment. Incubation of cells with cue of different cNDI-hV2Rs. The tsA201 cells were transfected with different N-terminally c-myc–tagged human V2 receptors, either wild-type or mutants (A294P, L44P, or R337X). Then, cells were treated for 18 h at 37ЊC with MCF57 at 10 ␮M or with vehicle only (DMSO 0.1%, control), fixed, and incubated with antibodies. Bar ϭ 50 ␮m.

We characterized the agonist properties of the three MCFs to the c-myc-hV2R through their capacity to induce a cAMP accumulation (which correlates to Gs/AC stimulation). The ligands exhibited efficiencies similar to AVP (Emax values were close to that for AVP maximal response) but lower potencies

(nanomolar EC50 values), as illustrated by a rightward shift of the dose-response curves (Figure 3; Table 2). The three MCFs, however, are full agonists of the hV2R regarding the Gs/AC pathway.

Nonpeptide Agonist Compounds Are

Pharmacochaperones for Several cNDI-hV2Rs We determined the capacity of MCFs to trigger plasma mem- 16 brane expression of several cNDI-hV2Rs: L44P, L59P, Y128S, Figure 6. Effect of the MCF14 pharmacochaperone treatment 11 A294P, and R337X (Figure 1B). These mutants are known to on the maturation of the cNDI-hV2Rs. The tsA201 cells were be sequestered mainly either in the ER or in the ER-Golgi in- transfected with different N-terminally c-myc–tagged human V2 termediate compartment (although Y128S was shown to be receptors, either wild-type or mutants (A294P, L44P, or R337X). Њ ␮ 11 Then, cells were treated for 18 h at 37 C with MCF14 (10 M), partially present at the cell surface ) and functionally rescued ␮ by the hV R antagonist SR121463. We also tested the murine with SR121463 (10 M), or with vehicle only (DMSO 0.1%, con- 2 trol). Cell lysates from various conditions were immunoprecipi- V R(mVR), which is predominantly retained within the ER 2 2 tated with c-myc antibodies. Immunoprecipitates were treated or 17 as an immature protein but also membrane-targeted by the not with EndoH and analyzed by Western blotting with c-myc 18 vaptans. Treatment of the cells with each MCF led to a sig- antibodies as described in the Concise Methods section. Each nificant increase in the L44P, A294P, and R337X membrane result illustrated here corresponds to an experiment representa- expression levels, compared with control cells treated with tive of three independent experiments. Molecular weight markers 0.1% DMSO only (Figure 4, A through C). The MCFs were are indicated on the left of the panels.

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In control conditions, the three cNDI-hV Rs 2 were detected only as immature proteins (apparent molecular weight approximately 40 kDa, or 36 kDa for R337X) compared with

the complex-glycosylated hV2R (apparent molecular weight approximately 50 kDa; Fig- ure 6). Treatment of the receptors with En-

doH deglycosylated cNDI-hV2Rs immature forms without effect on the mature hV2R forms, indicating that the three cNDI-hV2Rs are probably high-mannose glycosylated proteins trapped in the ER. Interestingly, treatment of the cells with the pharmacochaperone MCF14 allowed efficient complex

glycosylation of the cNDI-hV2Rs, an effect comparable to that of SR121463. This confirmed the ability of MCF compounds to promote maturation and plasma membrane translocation of L44P, A294P, and R337X.

Membrane-Rescued cNDI-hV2Rs Are Functional We checked the functionality of mem-

brane-rescued cNDI-hV2Rs by measuring their ability to generate a cAMP response upon AVP stimulation. As in previous studies with antagonist pharmacochaper- 11,19,20 ones, we determined cNDI-hV2Rac- tivity after washing the cells (elimination of the pharmacochaperone) and subsequently stimulating them with AVP. First, optimal plasmid quantities were determined to

avoid cNDI-hV2R plasma membrane expression and a cAMP response in control conditions (Supplemental Figure 1). Then, using an 18-h preincubation with MCF57 Figure 7. Functional rescue of various cNDI-hV2Rs by the nonpeptide MCF57 com- ␮ pound. Transiently transfected tsA201 cells with N-terminally c-myc–tagged A294P (50 concentrations from 30 nM to 10 M, we ng of PRK5-c-myc-A294P), L44P (25 ng of PRK5-c-myc-L44P), or R337X (50 ng of were able to evidence further the cAMP re- PRK5-c-myc-R337X) were incubated for 18 h at 37ЊC in serum-free medium supple- sponses to AVP with different efficiencies mented with increasing concentrations of MCF57 (from 30 nM to 10 ␮M), with AVP (1 for L44P, A294P, and R337X (Figure 7). ␮M in DMSO 0.1%), or with vehicle only (DMSO 0.1%, control). After two PBS washes, The L44P and A294P mutants were sensi- 2 min each, cells were incubated again for 30 min at 37ЊC in serum-free medium tive to AVP after pretreatment with 30 nM containing 0.1 mM RO 201724 and BSA 0.5% with or without (basal) increasing MCF57. R337X was sensitive to AVP only concentrations of AVP (from 10 pM to 10 ␮M). The cAMP accumulation was measured when preincubated with 10 ␮M MCF57. Ϯ as described in the Concise Methods section. Data are means SEM of cAMP An 18-h AVP preincubation had no effect concentration (nM) and illustrated from a representative experiment performed at least on the cAMP response whatever the cNDI- three times. hV2R considered, as in control situation, confirming that AVP is not a pharmaco- MCF57 allowed detection of a clear surface labeling that was chaperone (Figure 7). These data show that rescued L44P, not seen in control cells treated with DMSO (Figure 5). A294P, and R337X are functional once targeted to the plasma membrane using low pharmacochaperone concentrations.

Agonist Pharmacochaperones Promote cNDI-hV2R The results obtained with MCF57 were reproducible with Maturation MCF14 and 18, although statistically less significant, these li- ␮ To evidence maturation of cNDI-hV2Rs under MCF treatment, gands used at 10 M being more difficult to eliminate, even we analyzed the glycosylation pattern of L44P, A294P, and R337X. though extensive washes were performed (data not shown).

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A B

C D

Figure 8. Agonist properties of the nonpeptide compounds toward the cNDI-hV2Rs. (A through D) Transiently transfected tsA201 cells Њ ␮ with N-terminally c-myc–tagged hV2R (A), A294P (B), L44P (C), or R337X (D) were incubated for 18 h at 37 C with 10 M MCF14, MCF18, MCF57, SR121463, MCF57 and SR121463 mixed together (10 ␮M each), or vehicle only (DMSO 0.1%, control condition). Then, the medium was supplemented with 0.1 mM RO 201724, and cells were incubated for 30 min at 37ЊC. The cAMP accumulation was measured, and the results are expressed as in Figure 7. A statistical analysis between control and treated cells was done: *P Ͻ 0.05 and **P Ͻ 0.01.

Nonpeptide Pharmacochaperones Directly Activate (Figure 8). Agonist properties of the MCFs were evaluated for cNDI-hV2Rs L44P, A294P, and R337X. Compared with the strong cAMP Because MCFs are able to membrane rescue cNDI-hV2Rs and response obtained for hV2R (Figure 8A), a lower but signifi- trigger an hV2R-dependent cAMP accumulation, we measured cant cAMP accumulation for A294P (Figure 8B) and L44P their agonist activity on mutant receptors. Cells expressing (Figure 8C) mutants was measured. The pharmacochaperone cNDI-hV2Rs were treated with various pharmacochaperones SR121463 was unable to stimulate a cAMP production, what- or DMSO 0.1% (control condition) and then directly incu- ever the receptor considered. The competitive effect of bated for 30 min with RO 201724 for accumulating cAMP SR121463 to MCF57-induced cAMP signal confirmed the

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␤ Figure 9. Inhibitory effects of the nonpeptide compounds on the AVP-induced -arrestin 1–YFP/hV2R-Rluc interactions. Real-time BRET experiments for arrestin recruitment were performed using tsA201 cells transiently co-transfected with hV2R-Rluc and ␤-arrestin 1–YFP and incubated at 37ЊC with coelenterazine-h either with the nonpeptide compounds alone or in combination with ␤ ࡗ AVP. (A and B) Mobilization of -arrestin 1–YFP was measured after treatment of the hV2R-expressing cells by 50 nM AVP ( ), 1 ␮M MCF14 (F)or10␮M MCF14 (E) alone (A) or by 50 nM AVP (ࡗ) in combination with either 1 ␮M(F)or10␮M MCF14 (E; B). ␤ ࡗ ␮ (C and D) Mobilization of -arrestin 1–YFP was measured after treatment of the hV2R-expressing cells by 50 nM AVP ( ), 1 M SR121463 (F), or 10 ␮M SR121463 (E) alone (C) or by 50 nM AVP (ࡗ) in combination with either 1 ␮M(F)or10␮M SR121463 (E; D). (E) Complete analysis of arrestin recruitment measured after a 600-s treatment in the absence (control) or presence of the nonpeptide compounds at 1 or 10 ␮M. In each condition, a 50 nM concentration of AVP was added (f) or not (basal; Ⅺ). Data are means Ϯ SEM obtained from at least three distinct experiments. A statistical analysis between control and treated cells was done: *P Ͻ 0.05 and ***P Ͻ 0.001.

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receptor-associated pathway. Indeed, hV2R internalization was investigated by ELISA using the N-terminal c-myc tag as a readout to measure disappearance of the receptor from the cell surface upon ligand binding. Neither a 30-min nor a long- lasting (18 h) application of the various compounds (MCF14, ␮ 18, or 57, 10 M) provoked hV2R internalization (Figure 10). The antagonist SR121463 displayed the same behavior. As a

positive control, AVP induced a significant hV2R internalization for a short or a long incubation period (31 Ϯ 4 and 62 Ϯ

5% of hV2R were internalized, respectively). The AVP-induced internalization could be inhibited by 10 ␮M of the various MCF compounds (data not shown). The MCFs are noninter- Figure 10. Inability of the various nonpeptide compounds to nalizing hV2R agonists. trigger hV2R internalization. Transiently transfected tsA201 cells Ⅺ with the c-myc–tagged hV2R were incubated for 30 min ( )or Nonpeptide Pharmacochaperones Are Antagonists of 18h(f)at37ЊC with vehicle (control), 100 nM AVP, 10 ␮M the MAPK Signaling Pathway SR121463, MCF14, MCF18, or MCF57. Cell surface expression was measured by ELISA on nonpermeabilized cells. Results are AVP activation of the hV2R leads to arrestin recruitment, which in turn behaves as a scaffold protein for MAPK stimu- expressed as percentage of the maximal hV2R expression under 22,23 control conditions. Data are means Ϯ SEM taken from at least lation. MAPK activity can be measured through modifica- three independent experiments, each performed in triplicate. A statistical analysis between control and treated cells was done for each incubation time, 30 min or 18 h: *P Ͻ 0.05 and ***P Ͻ 0.001.

specificity of interactions between cNDI-hV2Rs and the different pharmacochaperones; therefore, MCF ligands were unable to stimulate R337X (Figure 8D).

Nonpeptide Pharmacochaperones Are Antagonists of Arrestin Recruitment

The capacity of the MCF agonists to elicit hV2R-arrestin binding was investigated by bioluminescence resonance energy transfer measurements (BRET), using the receptor fused with Luciferase as a donor and ␤-arrestin 1 or 2 fused with the yellow fluorescence protein as an acceptor. Control cells were treated with DMSO 0.1% and stimulated by AVP. MCF14, representative of the three agonists, could not induce ␤-arrestin 1 recruitment (Figure 9A) but inhibited AVP-stimulated

hV2R-arrestin binding (Figure 9B). These effects were mim- icked by the inverse agonist SR121463 (Figure 9, C and D), contrasting with what has been published previously.21 Statis- tical results of the ␤-arrestin 1 mobilization with each MCF alone or combined with AVP are shown in Figure 9E. MCF18 was unable to mobilize arrestin but inhibited the AVP effect. Figure 11. Effects of the various nonpeptide compounds on the

MCF57 induced a slight increase in arrestin recruitment hV2R-mediated MAPK activation. Stably transfected HEK293 cells Ͻ Њ ( 10% of the AVP response) and inhibited AVP-dependent with the c-myc–tagged-hV2R were incubated for 3 min at 37 Cin arrestin mobilization. The effects of MCF14 and SR121463 on serum-free medium (control conditions) with either vehicle (Ⅺ)or arrestin recruitment were identical using ␤-arrestin 2 (Supple- 100 nM AVP (f). Other cells were treated with 10 ␮M of MCF14, Ⅺ mental Figure 2). Contrary to AVP, the MCFs exhibited antag- MCF18, MCF57, or SR121463 alone (basal; ) or in combination with 100 nM AVP (f). Then, p-ERK and total ERK from cell lysates onistic effects on hV2R-dependent arrestin recruitment. They consequently display functional selectivity properties. were analyzed by Western blot. Molecular weight markers are indicated on the left of the top panel. Results are expressed as percentage of the AVP maximal p-ERK/total ERK ratio under Nonpeptide Pharmacochaperones Are Unable to control conditions and are the means Ϯ SEM taken from at least Induce hV2R Internalization three independent experiments, each performed in duplicate. A Because arrestins are involved in hV2R internalization, we statistical analysis between control and treated cells was done: evaluated the capacity of the MCFs potentially to induce this *P Ͻ 0.05.

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Figure 12. Various transduction pathways induced or inhibited by the biased agonist pharmacochaperones. The nonpeptide pharmacochaperone compounds (MCFs) are able to cross the various cellular membranes and interact with intracellularly ER-retained cNDI-hV2Rs (step 1), enabling their maturation and translocation to the plasma membrane through the ER-Golgi intermediate compartment (Ergic) and the Golgi systems (step 2). Once the cNDI-hV2Rs are at the cell surface, the agonist pharmacochaperones activate the cAMP pathway through interaction between receptors with the Gs protein and stimulation of AC (step 3). Contrary to the hormone AVP, the MCF ligands do not recruit arrestins and are unable to internalize hV2Rs and to activate the MAPK pathway (step 4). tion of the level of extracellular signal–regulated kinase 1 and 2 nadotropic hypogonadism were rescued by the inverse agonist (ERK1/2) phosphorylation (p-ERK). We evaluated the effect 9-cis-retinal and an antagonist, respectively.26,27 The ␦ opioid of the MCFs used alone or in combination with AVP on the receptor, displaying low intrinsic maturation efficiency, was 28 hV2R-dependent MAPK activity (Figure 11). Contrary to AVP, membrane-targeted with selective opioid ligands. Consider- MCF14, 18, and 57 were not able to induce ERK phosphoryla- ing AVP receptors, the nonpeptide antagonists SR121463 and tion, their effect being not significantly different from the con- VPA985 were first shown to rescue cell surface expression of 11 trol (cells treated with DMSO). The hV2R inverse agonist several cNDI-hV2Rs. This effect was reproduced using the SR121463 was not able to induce MAPK activation on its AVP antagonists SR49059,29 YM087,13 OPC41061, and 21 12 own. The MCF compounds were able to inhibit significantly OPC31260. Because most of the cNDI-hV2Rs are functional the AVP-induced p-ERK. These results confirmed that once targeted to the plasma membrane, a pharmacochaper- MCF14, 18, and 57 are biased agonists. one-based therapy represents a potential general treatment of the disease. Using antagonists as pharmacochaperones, functional res- DISCUSSION cue is a subtle balance between the ability of the ligand to target

cell surface expression of the cNDI-hV2Rs and its possibility to Chemical chaperones, pharmacochaperones, or cell-penetrat- be displaced by AVP for receptor activation.12 In this regard, ing peptides have been developed to correct plasma membran- considering the antagonist affinity as an important feature for e–targeting deficiency of misfolded G protein-coupled recep- this challenge, either low concentrations of antagonists with tors (GPCRs).24,25 Pharmacochaperones are the most high affinity (SR121463)11,12 or higher concentrations of non- promising candidates because of their target specificity. For specific hV2R antagonists with low affinity (SR49059) may be instance, the P23H rhodopsin mutant responsible for autoso- used.13 Although SR49059 behaved as an efficient pharmaco- mal dominant retinitis pigmentosa and mutants of the gona- chaperone in patients with cNDI,13 it revealed hepatic toxicity dotropin-releasing hormone receptor responsible for hypogo- during phase II of its clinical development. An important al-

J Am Soc Nephrol 20: 2190–2203, 2009 V2R Biased Agonist Pharmacochaperones 2199 BASIC RESEARCH www.jasn.org

ternative to the antagonists would be the use of agonists able erone MCFs after binding to cNDI-hV2Rs is shown in Figure both to restore membrane trafficking of cNDI-hV2Rs and to 12. Functional selectivity or biased agonism describes the ca- promote their activation directly. These ligands would behave pacity of ligands to induce selectively one or several pathways as more beneficial cNDI drugs. among all those usually activated by the endogenous related 10 We characterized here some hV2R nonpeptide agonists dis- agonist. It is of major importance for future drug develop- playing such properties. OPC23h,15 WAY-VNA-932,14 and ment. Indeed, the ␮-opioid agonist herkinorin activates Gi

MCF57 have a nanomolar affinity for hV2R and behave as full protein coupling and MAPKs but not arrestin recruitment and 34 agonists for AC activation. They possess a lower affinity for hV1aR receptor internalization. Herkinorin represents a promising and hOTR. These molecules do not bind to hV1bR, in agreement opiate analgesic with limited adverse effects such as tolerance. with available data for OPC23h and WAY-VNA-932.14,15 They J-2156, a peptidomimetic analogue of somatostatin is a super- membrane-rescued the L44P, Y128S, A294P, and R337X mutants agonist of the somatostatin receptor 4 subtype but does not 35 and the mV2R but were inactive toward L59P. This suggests that cause desensitization. This makes it clinically useful for con- these agonists preferentially rescue some given misfolded cNDI- trolling cellular proliferation in tumorous tissues. The OT an- hV2Rs, whereas the antagonist pharmacochaperones (SR121463, tagonist atosiban, which is used clinically for preventing pre- VPA985, YM087, and SR49059) may have a larger spectrum of term birth, inhibits Gq and concomitantly stimulates Gi, 11,30 36 efficiency. Efficiency of cNDI-hV2R membrane translocation leading to MAPK activation and cell proliferation. The hV2R between the various pharmacochaperones could be directly re- agonist pharmacochaperones, devoid of arrestin recruitment lated to the affinity of each ligand for each mutant12; however, the and acting as noninternalizing ligands, seem to be particularly nonpeptide agonists having an hV2R relatively high affinity (6 to adapted for the treatment of the cNDI, potentially providing a 20 nM) are unable to rescue L59P, contrary to the low-affinity long-lasting cellular response during drug administration. SR49059 antagonist.13,29 Even though pharmacochaperones need to bind to mutants, affinity seems not to be the only critical pa- rameter. The mutation L59P itself may also change the whole CONCISE METHODS structure of the receptor and explain why the agonist cannot interact anymore with the binding site. Finally, different conformational changes induced by agonists rather than antagonists may Cell Culture and Transfection explain the differences observed in terms of rescuing.18 Alto- Human embryonic kidney HEK293 T cells and the SV40-transformed gether, a more exhaustive series of cNDI-hV2Rs should be inves- HEK293 cell line tsA201 were used and transfected as described in the tigated and several other agonist pharmacochaperones should be Supplemental Concise Methods. developed to rescue a larger panel of cNDI-hV2Rs. In terms of signaling, contrary to the antagonist pharmacochaperones, the MCF compounds directly activated mem- [3H]AVP Binding Assays 3 ϫ 6 brane-targeted L44P and A294P cNDI-hV2Rs but not the [ H]AVP binding to whole cells was performed as follows: 10 10 R337X, despite restoration of membrane trafficking. Poor tsA201 cells were electroporated (20 ␮g of total DNA) with the N-termi- functional recovery of R337X, potentially reflecting an impair- nally c-myc–tagged receptors hV2R, hV1aR, hV1bR, and hOTR (100, 250, ment of this mutant in its ligand affinity and/or its ability to 500, and 6000 ng, respectively) and seeded at a density of 3.3 ϫ 105 cells interact with Gs, has been previously described.11 For L44P and per well in 24-well plates pretreated with polyornithine. Forty-eight A294P mutants, a single molecule having both pharmacochap- hours after transfection, the plates were placed on ice and cells were erone and agonist properties seems highly beneficial and may washed three times with 250 ␮l of ice-cold buffer A (PBS [pH 7.4], 5 mM represent an alternative to antagonists for cNDI treatment. MgCl2, 1 mg/ml BSA, and 0.1 mM phenylalanine). For competition stud- A drawback of most GPCR agonists regarding long-term ies, cells were transfected with the receptors and incubated with buffer A efficacy is their propensity to trigger internalization of target containing approximately 1.5 nM [3H]AVP and increasing concentra- receptors, leading to desensitization and decrease in the inten- tions (10Ϫ10 to 10Ϫ5 M) of the various compounds (MCF14, MCF18, sity of cell response. Arrestins are key players regarding desen- and MCF57). An excess of unlabeled AVP (1 ␮M) was also added in the sitization/internalization of GPCRs or MAPK activation.31 medium to determine the nonspecific binding. All of the determinations These phenomena were shown to occur upon AVP stimulation were made in the presence of 0.1% DMSO. After four washings with 32,33 ␮ of the hV2R. In contrast to AVP, MCF compounds were ice-cold buffer A, the cells were lysed for 15 min at 37°C with 400 lof0.1 unable to induce arrestin mobilization (both ␤-arrestin 1 and M NaOH. Then 100 ␮l of 0.4 M acetic acid was added. The lysates were 2). Together with their Gs agonism, they thus displayed func- transferred into scintillation vials, and their radioactivity was determined. tional selectivity properties. Moreover, they could antagonize The specific binding was calculated as the difference between the total the strong and long-lasting interaction of hV2R with arrestins binding and the nonspecific binding and expressed as percentage of max- upon AVP binding. Accordingly, these molecules could not imum [3H]AVP binding in control conditions. The ligand-binding data promote hV2R internalization and MAPK stimulation, con- were analyzed by nonlinear least-squares regression using the software firming their cAMP selectivity. A schematic view of the trans- Ligand (Elsevier Biosoft, Cambridge, UK) and GraphPad Prism (Graph- duction pathways induced or inhibited by the pharmacochap- Pad Software, Inc., San Diego, CA).

2200 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 2190–2203, 2009 www.jasn.org BASIC RESEARCH cAMP Accumulation Assays transfection, the medium was removed and replaced by serum-free For measuring the cAMP accumulation triggered by each nonpeptide medium containing compounds at 10 ␮M in 0.1% DMSO. Control compound (Gs/AC agonist activity), tsA201 cells were electroporated cells were incubated at 37°C in medium containing only 0.1% DMSO. with 100 ng of the PRK5-c-myc-hV2R plasmid and seeded at a density Eighteen hours later, cells were first rinsed once with PBS at room of 75 ϫ 103 cells per well into 96-well plates pretreated with polyor- temperature. All subsequent steps were done at room temperature. nithine. Forty-eight hours after transfection, cells were treated for 30 The cells were fixed for 5 min in 4% paraformaldehyde-PBS and min at 37°C in the cAMP buffer with or without increasing com- rinsed once rapidly with PBS and twice again with PBS (10 min each pound concentrations (10Ϫ12 to 10Ϫ5 M) in the presence of the phos- time). Then, cells were washed twice with PBS-BSA 1% (blocking phodiesterase inhibitor RO 201724 0.1 mM (Sigma). The cAMP pro- buffer) for 10 min and incubated for 2 h with the rabbit anti–c-myc duction for each condition was expressed in nM and compared with antibody (A14) at 1 ␮g/ml. Then, the cells were washed twice with the maximal AVP response obtained under the same conditions. blocking buffer, and Cy3-labeled secondary anti-rabbit antibody For the evaluation of the functional activity of cNDI rescued re- (0.62 10Ϫ10 g/ml) was added for 1 h. The cells were rinsed twice with ceptors by nonpeptide pharmacochaperones, tsA201 cells were trans- PBS and H2O. Slides were mounted in Fluomount (Interchim), and fected with 50 ng of DNA plasmids and seeded at a density of 50 ϫ 103 images were collected on a Bio-Rad fluorescence confocal micro- cells per well into polyornithine-treated Costar 96-well plates. scope. Twenty-four hours after transfection, cells were treated with 10Ϫ5 M MCF compounds in serum-free DMEM (final DMSO concentration Immunoprecipitation and Analysis of Glycosylation of 0.1%). Eighteen hours later, cells were washed for 2 min three times Pattern of the Receptors with PBS 1ϫ at 37°C. Then, cells were stimulated for 30 min at 37°C After transfection of the cells with 250 ng of c-myc–tagged L44P, with AVP in the cAMP buffer (serum-free DMEM, 0.5% BSA) sup- A294P, and R337X or 100 ng of c-myc-hV2R, tsA201 cells were seeded plemented with 0.1 mM RO 201724. For evaluation of the direct ag- at a density of 15 ϫ 106 cells per 150-mm dish with complete medium. onist effect of nonpeptide compounds on cNDI receptors, tsA201 Twenty-four hours after transfection, the medium was removed and cells were treated with 10Ϫ5 M MCF compounds in serum-free replaced by serum-free medium without (control conditions contain- DMEM (final DMSO concentration of 0.1%). Eighteen hours later, ing only 0.1% DMSO) or with either MCF14 or SR121463 at 10 ␮Min the medium was supplemented with RO 201724 0.1 mM, the concen- 0.1% DMSO. Eighteen hours later, cells were rinsed twice with PBS at tration of the MCF being constant at 10Ϫ5 M. Then, cells were incu- 4°C and resuspended into lysis buffer (50 mM Tris-HCl [pH 7.4], 1 bated for 30 min at 37°C. mM EDTA, 200 mM NaCl, 1 mM Na3VO4, 1% Triton X-100, 10 For both types of experiments, the accumulated cAMP was quan- ␮g/ml leupeptin, and 10 ␮g/ml aprotinin) and mixed at 4°C for 30 tified using the cAMP Dynamic 2 Kit from Cisbio Int. (Bagnols-sur- min. Then the lysate was centrifuged at 21,000 ϫ g to eliminate insol- Ce`ze, France) based on a homogeneous time-resolved fluorescence- uble cell fragments, and the protein concentration was measured into fluorescence resonance energy transfer technology.37 The principle of the supernatant. C-myc antibodies (4 ␮g) were covalently linked to the kit is described in the Supplemental Concise Methods. agarose-protein G (50 ␮l) using 20 mM dimethyl-pimelimidate reagent (Sigma-Aldrich, St. Louis, MO). After clearing, protein lysate (1 ELISA Experiments mg/ml) was incubated overnight at 4°C with the c-myc–coupled aga- TsA201 cells were electroporated as described already with 100 and rose beads. Immune complexes were collected by centrifugation and

250 ng of DNA plasmids coding for N-terminally c-myc–tagged hV2R washed twice with washing buffer (50 mM Tris-HCl [pH 7.4], 1 mM 3 ␮ and cNDI receptors, respectively, and seeded at a density of 75 ϫ 10 EDTA, 0.1% Triton X-100, 1 mM Na3VO4,10 g/ml leupeptin, and per well in polyornithinilated Greiner 96-well plates. Twenty-four 10 ␮g/ml aprotinin). The beads were divided into two fractions, and hours after transfection, cells were treated with MCF compounds (10 100 ␮l of washing buffer containing or not 12.5 mU of EndoH was ␮M in 0.1% DMSO) in serum-free DMEM or with 0.1% DMSO at added to the beads and mixed for3hat37°C. Beads were then cen- 37°C. All subsequent steps were done at room temperature. Eighteen trifuged at 10,000 ϫ g, and 100 ␮lof2ϫ Laemmli was added before hours later, cells were fixed for 5 min with 4% formaldehyde diluted in Western blotting analysis. PBS 1ϫ and then washed with PBS 1ϫ. Cells were blocked for 30 min Protein samples (30 ␮l) separated using 12% SDS/PAGE were with PBS 1ϫ containing 1% FCS. Then, anti–c-myc antibody (9E10; blotted onto nitrocellulose membranes (Hybond-C; Amersham Bio- 1 ␮g/ml) was added for 30 min. After blocking buffer washes, anti- sciences, Buckinghamshire, UK). Nitrocellulose membranes were in- mouse horseradish peroxidase (HRP)-coupled secondary antibody cubated overnight at 4°C with 5 ␮g/ml c-myc antibodies. HRP-la- (0.55 ␮g/ml) was added for 30 min. After blocking buffer and PBS beled secondary anti-mouse (from sheep) antibodies (Amersham washes, the Super signal reagent was added and luminescence was Biosciences) at 0.22 ␮g/ml were then used for primary antibody de- recorded using the ANALYST apparatus. The cell surface receptor tection. Immunoreactivity was detected using an enhanced chemilu- expression was expressed as a percentage of c-myc-hV2R expression minescence method according to manufacturer’s instructions (ECL (maximal expression) in the same cells. detection reagent; Amersham Biosciences).

Immunocytochemistry BRET Measurements TsA201 cells were seeded on polyornithine-precoated glass coverslips TsA201 cells (5 ϫ 106) were electroporated with 5 ␮g of DNA: 150 ng ϫ 5 ␮ ␤ at a density of 1.5 10 cells per 18-mm dish. Twenty-four hours after of hV2R-Rluc and 1 gof -arrestin 1–YFP or 800 ng of hV2R-Rluc

J Am Soc Nephrol 20: 2190–2203, 2009 V2R Biased Agonist Pharmacochaperones 2201 BASIC RESEARCH www.jasn.org and 1.8 ␮gof␤-arrestin 2–YFP and empty vector. A total of 2.5 ϫ 106 luminescence and fluorescence measurements. We thank the Fonda- cells from the electroporation step were subsequently seeded per well tion pour la Recherche Me´dicale for providing financial support to of a six-well plate. Forty-eight hours after transfection, the cells were F.J.A. and the Ministe`re de l’Enseignement Supe´rieur et de la Recher- washed twice with KREBS buffer (146 mM NaCl, 4.2 mM KCl, 0.5 che for supporting M.-C.F. and S.P. mM MgCl2, 1 mM CaCl2, 10 mM HEPES [pH 7.4], and 1 mg/ml We thank Dr. Peter Deen (Radboud University Nijmegen Medical glucose) and resuspended in 1 ml of KREBS. BRET measurements Centre, Nijmegen, Netherlands) for providing the plasmid pEGF-N1- were performed into Greiner 96-well plates with a final volume of 50 L44P-hV2R. We are grateful to Nicole Lautredou-Audouy from the ␮l using 30 ␮l of cellular suspension corresponding to approximately MRI platform for confocal microscopy analysis. Thanks to Dr. Jean- 75,000 cells with 10 ␮l of the Rluc substrate Coelenterazine-h (final Philippe Pin for critically reading the manuscript, Dr. Mohammed concentration 5 ␮M) and 10 ␮l of KREBS buffer containing 5ϫ Akli Ayoub for introducing us to BRET measurements, and Muriel DMSO, AVP, MCF, or AVP and MCF mixed together (to determine Asari-Gien for iconography. We also thank Laure Boudier for her the antagonist effect on arrestin mobilization). The mix suspension participation in the characterization of the ligands. was incubated at 37°C, and BRET measurements were acquired on a BertholdTech Mithras LB940 using the MikroWin software. The BRET signal corresponded to the ratio between the light emitted at 530 nm (EYFP) and the light emitted at 480 nm (RLuc) from cells DISCLOSURES ␤ ␤ transfected with V2R-Rluc and -arrestin 1–YFP (or -arrestin None. 2–YFP) corrected for the background signal that corresponds to the same ratio from cells transfected with the receptor alone (V2R-Rluc). The BRET net signal was calculated from the difference between the corrected ratio 530 nm/480 nm of agonist-treated cells and vehicle- REFERENCES treated cells. All compounds were diluted in KREBS with a final DMSO concentration of 0.1% as in control conditions. 1. Birnbaumer M: Vasopressin receptors. Trends Endocrinol Metab 11: 406–410, 2000 2. Robben JH, Knoers NV, Deen PM: Regulation of the vasopressin V2 ERK Activation and Immunoblotting receptor by vasopressin in polarized renal collecting duct cells. 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