The rescue potentials of OPC-41061, OPC-31260 and OPC-21268 on mutant AVPR2s that cause nephrogenic diabetes insipidus

doi:10.21203/rs.3.rs-3146169/v1

Download PDF

Research Article

The rescue potentials of OPC-41061, OPC-31260 and OPC-21268 on mutant AVPR2s that cause nephrogenic diabetes insipidus

https://doi.org/10.21203/rs.3.rs-3146169/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose

Nephrogenic Diabetes insipidus (NDI) is a rare disease which is characterized by polyuria and polydipsia. AVPR2 mutations can cause NDI. Mutants may not function properly since the mutations can affect their folding process and make them be trapped in the Endoplasmic reticulum (ER). Recent studies showed that pharmacological chaperones (PCs) may rescue mutant AVPR2s and they can be functional again. OPC-41061, OPC-31260 and OPC-21268 are these kind of PCs and they can specifically bind to the mutant AVPR2s and rescue them from ER via helping their folding process. In this study, the effects of OPC-41061, OPC-31260 and OPC-21268 on AVPR2 mutants (R68W, ΔR67-G69/G107W, V162A and T273M) were analyzed.

Methods

We performed cell surface and total ELISA, and cAMP accumulation assay for mutant AVPR2s after the treatment with 3 different PCs.

Results

It was observed that OPC-41061 improved cell surface expressions of all mutants except V162A and ΔR67-G69/G107W, and cell surface expressions of R68W and ΔR67-G69 were increased with all three PCs. Then mutants were stimulated with their agonists to measure cAMP accumulation in the cell as a functional analysis. As a result, we found that R68W and ΔR67-G69 (except T273M) were rescued and could be functional after the treatment with OPC-41061 and OPC-31260.

Conclusion

Showing specific rescue potential of these kind of PCs has an importance on the development of new treatment strategies on NDI. Determining the responses of mutant proteins to various chaperones is extremely important for current treatment approaches and identifying the new PCs.

Nephrogenic Diabetes insipidus

AVPR2

Pharmacological chaperone

Arginine vasopressin receptor 2 (AVPR2) is a G protein-coupled receptor (GPCR) and it plays an important role in regulation of human body water homeostasis by kidney [1]. When body needs water, antidiuretic hormone arginine vasopressin (AVP) is released from pituitary gland and through the bloodstream it comes to the kidney where its specific receptor AVPR2 locates. After binding of AVP to AVPR2, cellular signaling process is activated and through aquaporin water channels, water is reabsorbed from urine to the bloodstream. When there is a mutation in AVPR2, urine cannot be concentrated since the water reabsorbtion mechanism has a problem. AVPR2 mutations can cause Nephrogenic Diabetes insipidus (NDI) which is a rare disease characterized by hypotonic polyuria and excessive thirstiness [2–4]. AVPR2 mutations can cause misfolding of the receptor protein and as a result, mutant AVPR2s can be trapped by the Endoplasmic reticulum (ER) which is the quality control mechanism of the cell [5, 6]. However, mutant AVPR2s can be still functional even if they have problems with folding state to their native conformations. Pharmacological chaperones (PCs) are mostly used to correct the folding state of misfolded protein and allow the mutant AVPR2s pass the quality control system of the cell and reach to the cell membrane [7, 8]. OPC-41061, OPC-31260 and OPC-21268 are these kind of PCs which are non-peptide and cell-permeable antagonists [9]. These molecules are called as vaptans and in addition to their usage for the treatment of hyponatremia, they have been used as PCs for the rescue studies of mutant AVPR2s [10–12]. This study aims to analyze rescue potantials of OPC-41061, OPC-31260 and OPC-21268 on R68W, ΔR67-G69/G107W, V162A and T273M mutant AVPR2s and to perfom functional analysis to search whether if these mutants can be functional again.

Chemicals and Reagents

OPC-41061, OPC-31260 and OPC-21268 (Sigma-Aldrich) were prepared as 1 mM stock in DMSO (Cell culture grade; AppliChem GmbH). COS-7 and COS-1 cell lines were purchased from Cell Line Service GmbH (Lot number: 605470-1012) in 2014 and AddexBio Technologies (T0014001, Lot number: 0013255) in 2017, respectively. They have been kept as stocks in liquid nitrogen. These cell lines are mostly used in functional analysis studies of mutant AVPR2s [13–15]. For the cell culture studies, fetal bovine serum (FBS) originated in South America and Dulbecco’s modified Eagle’s medimum (DMEM) High Glucose with stable Glutamine and sodium pyruvate (Biowest SAS France facility) were used. TurboFect™ Transfection Reagent (Thermo Fisher Scientific, IL, USA) was used for the transient transfection. Monoclonal HA-tag (6E2) Mouse mAb, HRP-conjugated (Cell Signaling Technology, Inc.) and monoclonal anti-FLAG M2 (Sigma-Aldrich, MO, USA) were used for ELISA experiments. For cAMP accumulation assay, AlphaScreen cAMP Detection Kit (PerkinElmer Inc.) and [Arg⁸]-Vasopressin acetate salt (Sigma-Aldrich Co. LLC.) were used.

Cell Culture, Transfection and Treatment with PCs

DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 10 µg/ml streptomycin was used as media and cells were incubated in 5% CO₂ in air at 37°C. Cells were seeded on 96-well plates as 15.000 cells/well (100 µl) for MTT assay and cAMP accumulation assay, 40.000 cells/well (250 µl) on 48-well plates for cell surface ELISA, and 400.000 cells/well (2 ml) on 6-well plates for total ELISA. COS-1 cell line was used for only the cAMP accumulation assay. Approximately 18 h after seeding the cells, they were transiently transfected with wild type and mutant (R68W, ΔR67-G69/G107W, V162A and T273M) pLV2R plasmids which were constructed by site-directed mutagenesis during our previous studies [14, 16]. pEGFP was used for the control of the efficiency of transient transfection [14, 16]. To analyze non-toxic concentration of OPC-41061, OPC-31260 and OPC-21268, MTT assay was performed. Then, 48 h after transfection, cells were treated with 10^− 5 M of OPC-41061, OPC-31260 and OPC-21268 for 16 h, separately.

Cell Surface and Total Expression of Mutants

After the treament with OPC-41061, OPC-31260 and OPC-21268, sandwich ELISA method was used to analyze total expression level of double-tagged AVPR2s (N-termianl HA-tag and C-terminal FLAG-tag) and direct cellular ELISA method was used for cell surface expression of N-terminal HA-tagged AVPR2 mutants. The EnSight™ Multimode plate reader (PerkinElmer) was used for the reads of ELISA experiments. All ELISA experiments were performed as described in earlier studies [14, 16]. All ELISA data were analyzed using GraphPad Prism 8 for macOS (Version 8.4.3., GraphPad Software, LLC.).

cAMP Accumulation Assay

AlphaScreen (PerkinElmer) technology was used for the measurements of cellular cAMP accumulation as described in earlier studies [14, 16]. For the stimulation of wild type and mutant AVPR2s, different concentrations of AVP (10 µM, 1 µM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM and without AVP) were used for 1 h at 37°C in 5% CO₂ in air. According to the manufacturer’s protocol, standards for measurements and acceptor and donor beads were prepared. After the incubation of cell lysates with beads, the emission was read on the EnSight™ Multimode plate reader (PerkinElmer). All data were analyzed using GraphPad Prism 8 for macOS (Version 8.4.3., GraphPad Software, LLC.).

Statistical Analysis

The differences between the PC-treated and untreated samples were evaulated with the Mann-Whitney U test and the significance level was taken as P < 0.05 in all instances. In all tables, the data were presented as mean ± S.D. and ± S.E.M. All statistical analysis was performed by using GraphPad Prism 8 for macOS (Version 8.4.3., GraphPad Software, LLC.).

Cell surface and total expressions of mutant AVPR2s

Total expression of the mutant AVPR2s in COS-7 cells were as same nearly with the wild type receptor (Table 1) and almost all three different PC-treatments did not change the total expression levels of mutants in the cell. For the cell surface expression levels, only the V162A and DR67-G69/G107W mutants were not affected from the treatment with PCs (Table 1 and Figure 1). DR67-G69 and R68W were rescued with all three PCs and OPC-41061 was the most successful PC in terms of rescuing mutants except V162A and DR67-G69/G107W (Table 1 and Figure 1). Also, cell surface expression levels of DR67-G69 mutant were significantly different after the treatment with OPC-41061, OPC-31260 and OPC-21268, separately (Table 3).

cAMP accumulation

After treatment with PCs, cAMP accumulation assay was performed for only R68W, DR67-G69 and T273M mutants because according to the cell surface ELISA results, these mutants were found to be rescued. After the separate treatments with OPC-41061 and OPC-31260, R68W and DR67-G69 mutants (except T273M) showed increased E_max values but none of them were statistically significant (Table 2 and 3). Among the mutants which had increased E_max values, mostly right-shifted concentration-response curves (EC₅₀; relative potency) were seen except the treatment of DR67-G69 mutant with OPC-31260 (Table 2 and Figure 2). Only the treatment of DR67-G69 mutant with OPC-41061 showed significant difference in EC₅₀ value (Table 3).

NDI is a rare disease and AVPR2 mutations are responsible for the 90% of the congenital cases [17]. AVPR2 is a GPCR, which has seven transmembrane domains, locates on the plasma membrane of the kidney [18]. Recent studies showed that GPCR mutations can cause misfolding and intracellular retention because they cannot pass through the ER quality control system of the cell [5, 6, 8, 16, 19]. Since they are trapped in the ER, their presence on the plasma membrane are reduced and consequently their agonist sensitivity were reduced. Some of the intracellularly trapped mutants can be rescued using with PCs and it is shown that AVPR2 mutants could be rescued by this strategy [14, 20–22]. With the help of PCs, which are cell-permeable small molecules, mutant receptors can fold properly and pass the ER quality control system. PC specifically binds to the mutant receptor and act as a chaperone in the folding process. By this way, rescued mutant receptors can reach to the cell membrane and can be functional because some mutations affect only the folding state of the receptor not the function of it.

PCs used in this study are OPC-41061 (tolvaptan), OPC-31260 (mozavaptan) and OPC-21268. OPC-41061 and OPC-31260 are non-peptide AVPR2 antagonists, OPC-21268 is a non-peptide AVPR1 antagonist [23]. OPC-41061 is an orally active molecule and used for the treatment of severe hyponatremia, autosomal dominant polycystic kidney disease (ADPKD) and syndrome of inappropriate antidiuretic hormone, etc [24–26]. Both OPC-41061 and OPC-31260 were found to be useful even at low concentrations to increase cell surface expression of mutant receptors [8, 11, 27, 28]. OPC-21268 is an orally effective [29, 30] and also can be used to rescue misfolded AVPR2s [12]. As it mentioned before OPC-41061 is approved for the ADPKD, but it was reported that the use of tolvaptan in patients should be carefully decided by evaluating benefits and risks due to its reversible acute liver injury effects [31, 32].

Because of the therapuetical potentials of PCs on treatment of NDI associated with misfolded AVPR2 mutants, we aimed to analyze the rescue potantials of OPC-41061, OPC-31260 and OPC-21268 on R68W, ΔR67-G69/G107W, V162A and T273M mutants. For the ΔR67-G69/G107W mutant, we also analyzed both mutations (ΔR67-G69 and G107W) separately. Our point is to identify the responses of different types of mutants to selected chaperones an by this help the selection of new potential therapeutic chaperones.

According to our previous study, all mutants showed reduced cell surface expressions except V162A but their total expression in the cell were found to be not affected by mutations [16]. The change of amino acid residue of the codon 162 is from valine to alanine do not change the nonpolarity of the codon because both of them are nonpolar amino acids and this codon is not conserved among the mammalian orthologs [16]. Therefore, we can say that there is no functional change in terms of cell surface expression and cAMP accumulation in the cell for the V162A mutant [16, 33]. On the other hand, R68W and T273M mutants showed the most decreased cell surface expression levels among others (Fig. 1 and Table 1). At the codon 68, change from basic amino acid arginine to a hydrophobic amino acid tryptophan could cause misfolding since the mutant receptor had a bulky residue at the intracellular loop 1 of the receptor (ICL1) [16]. Codon 273 is a highly conserved residue in the transmembrane domain 6 (TMD6) and it is close to the cytoplasm which can be important for the function of the receptor. The residue substitution from threonine to methionine changes the polarity of the codon which could be the cause of decreased cell surface expression level.

In this study, cell surface expression levels of mutants, except V162A and ΔR67-G69/G107W, were increased when OPC-41061 was used as a PC (Fig. 1 and Table 1) and these increases were statistically significant for ΔR67-G69, R68W and T273M mutants (Table 3). For the ΔR67-G69/G107W mutant, cell surface expressions of double mutant and separate mutants were decreased compared to the wild type receptor (Table 1) and usage of PCs could increase these levels. However, E_max and EC₅₀ values for this double mutant could not be measured because of the unsaturated concentration-response curve. Therefore, we only functionally analyzed the ΔR67-G69 mutant, which was one of the mutation that double mutant had, according to the results of ELISA experiments. ΔR67-G69 was the only mutant that its cell surface expression level was significantly increased after the treatment with all 3 PCs (Table 3). Starting from this point of view, we decided to functionally analyse R68W and ΔR67-G69 after the treatment with OPC-41061, OPC-31260 and OPC-21268, and also T273M after the treatment with only OPC-41061. By this way, we tried to understand whether they could be functional when they were rescued by these PCs. We can say that the treatment with OPC-41061 and OPC-31260 were succesful to make R68W and ΔR67-G69 mutants be functional again (Fig. 2a, 2b and Table 2). Their mutations seemed to affect only the folding processes not the functions of receptors since their E_max values were incerased compared to the untreated samples. However, except ΔR67-G69 mutant-treated with OPC-31260, other mutants showed very highly right-shifted concentration response curves which mean they need much more ligand concentrations to be half-stimulated compared to the wild type receptor (Fig. 2a, 2b and Table 2). The rescue potantial of OPC-21268 was not good for both R68W and ΔR67-G69 mutants because even if OPC-21268 increased cell surface expressions of these mutants, it was not enough to make them reach to the cell membrane which could be seen from the results of E_max values (Table 2). T273M mutant was only rescued by OPC-41061 to the cell membrane (Fig. 1 and Table 1) but according to the cAMP accumulation assay, its maximum response to ligand stimulation was very low compared to the untreated sample (Fig. 2 and Table 2). Even if T273M mutant was rescued by OPC-41061, it could not function properly because of the polarity change at the highly conserved residue and important location (TMD6). Therefore, OPC-41061 might not succesful since the mutation could effect the functionality of T273M even if cell surface expression of it was increased.

As a result, we can say that OPC-41061 was the most succesful PC among others which were used in this study (Fig. 1, 2A and 3). OPC-21268 was not succesful to rescue mutant AVPR2s even they did not show any functional loss. In addition, OPC-41061 and OPC-31260 showed their actions specifically to the mutation types because maximum responses of R68W and ΔR67-G69 were different from each other when they were treated with different PCs. For example, maximum response of the ΔR67-G69 mutant was higher when it was treated with OPC-41061 than OPC-31260. The situation was totally opposite for the R68W mutant (Table 2). The promising potentials of OPC-41061 and OPC-31260 in this study were coherent with other studies [27, 34].

Our results showed that R68W and ΔR67-G69 mutant AVPR2s could be pharmacologically rescued and translocated to the cell membrane after the treatment with OPC-41061 and OPC-31260. Also, they could be functional via initiating cAMP signaling when they were stimulated with the ligand. This study demonstrated that non-peptide AVPR2 antagonists such as OPC-41061 and OPC-31260 could act as PCs to functionally rescue misfolded R68W and ΔR67-G69 mutant AVPR2s. Using such PCs and showing their rescuing effects on several type of mutants has an importance on development of the new therapeutic strategies for NDI.

Funding

This work was supported by The Scientific and Technological Research Council of Turkey (Project number: 216S304) and Hacettepe University Scientific Research Project Coordination Unit (Project number: FBA-2017-14395).

Acknowledgments

The data of this study except cAMP accumulation assay were excerpted from the master thesis of the author. All authors have no conflicts of interest.

Author contributions

D.S., B.E.T. and E.S.O. performed the experiments. B.E.T. and E.S.O. wrote the main manuscript text and prepared all figures and tables. H.M. funded the experimental processes through the projects. All authors reviewed the manuscript.

N. Makita, K. Manaka, J. Sato, T. Iiri, (2020) V2 vasopressin receptor mutations. In: Vitam. Horm., 20190913th ed. pp 79–99
M. Christ-Crain, D.G. Bichet, W.K. Fenske, M.B. Goldman, S. Rittig, J.G. Verbalis, A.S. Verkman, Diabetes insipidus. Nat. Rev. Dis. Prim. 5, 54 (2019)
Q. Li, D. Tian, J. Cen, L. Duan, W. Xia, Novel AVPR2 mutations and clinical characteristics in 28 Chinese families with congenital nephrogenic diabetes insipidus. J. Endocrinol. Invest. 44, 2777–2783 (2021)
S. Selvaraj, D. Rodrigues, N. Krishnamoorthy, K.A. Fakhro, L.R. Saraiva, M.C. Lemos, Clinical, Genetic and Functional Characterization of a Novel AVPR2 Missense Mutation in a Woman with X-Linked Recessive Nephrogenic Diabetes Insipidus. J. Pers. Med. (2022). https://doi.org/10.3390/jpm12010118
A. Ulloa-Aguirre, A.C. Hanyaloglu, T. Zarinan, J.A. Janovick, (2020) Protein homeostasis and regulation of intracellular trafficking of G protein-coupled receptors. Protein Homeost. Dis. Mech. Nov. Ther. Academic Press, 247–277
S. Nademi, J.G. Dickhout, Protein misfolding in endoplasmic reticulum stress with applications to renal diseases. Adv. Protein Chem. Struct. Biol. 118, 217–247 (2019)
L. Liguori, M. Monticelli, M. Allocca, B.H. Mele, J. Lukas, M.V. Cubellis, G. Andreotti, Pharmacological Chaperones: A Therapeutic Approach for Diseases Caused by Destabilizing Missense Mutations. Int. J. Mol. Sci. (2020). https://doi.org/10.3390/ijms21020489
L. Szalai, A. Sziraki, L.S. Erdelyi et al., Functional Rescue of a Nephrogenic Diabetes Insipidus Causing Mutation in the V2 Vasopressin Receptor by Specific Antagonist and Agonist Pharmacochaperones. Front. Pharmacol. 13, 811836 (2022)
B. Mouillac, C. Mendre, Vasopressin receptors and pharmacological chaperones: From functional rescue to promising therapeutic strategies. Pharmacol. Res. 83, 74–78 (2014)
G.L. Robertson, Vaptans for the treatment of hyponatremia. Nat. Rev. Endocrinol. 7, 151–161 (2011)
C. Mendre, B. Mouillac, (2019) Misfolding of vasopressin receptors: biased agonist pharmacochaperones as potential therapeutics. In: Adv. Protein Chem. Struct. Biol. Elsevier, pp 249–272
F. Prosperi, Y. Suzumoto, P. Marzuillo et al., Characterization of five novel vasopressin V2 receptor mutants causing nephrogenic diabetes insipidus reveals a role of tolvaptan for M272R-V2R mutation. Sci. Rep. 10, 16383 (2020)
J.P. Morello, U.E. Petaja-Repo, D.G. Bichet, M. Bouvier, Pharmacological chaperones: a new twist on receptor folding. Trends Pharmacol. Sci. 21, 466–469 (2000)
B. Erdem Tuncdemir, H. Mergen, E. Saglar Ozer, Evaluation of pharmacochaperone-mediated rescue of mutant V2 receptor proteins. Eur. J. Pharmacol. 865, 172803 (2019)
I. Boselt, D. Tramma, S. Kalamitsou et al., Functional characterization of novel loss-of-function mutations in the vasopressin type 2 receptor gene causing nephrogenic diabetes insipidus. Nephrol. Dial Transplant. 27, 1521–1528 (2012)
B. Erdem, A. Schulz, E. Saglar, F. Deniz, T. Schöneberg, H. Mergen, Functional characterization of AVPR2 mutants found in Turkish patients with nephrogenic diabetes insipidus. Endocr. Connect. (2018). https://doi.org/10.1530/EC-17-0236
D.G. Bichet, D. Bockenhauer, Genetic Forms of Nephrogenic Diabetes Insipidus (NDI): Vasopressin Receptor Defect (X-Linked) and Aquaporin Defect (Autosomal Recessive and Dominant). Best Pr Res Clin Endocrinol Metab. 30, 263–276 (2016)
T.M. Fujiwara, D.G. Bichet, Molecular biology of hereditary diabetes insipidus. J. Am. Soc. Nephrol. 16, 2836–2846 (2005)
J.H. Robben, N.V. Knoers, P.M. Deen, Cell Biological Aspects of the Vasopressin Type-2 Receptor and Aquaporin 2 Water Channel in Nephrogenic Diabetes Insipidus. Am. J. Physiol. Ren. Physiol. 291, F257–F270 (2006)
Y.X. Tao, Molecular chaperones and G protein-coupled receptor maturation and pharmacology. Mol. Cell. Endocrinol. 511, 110862 (2020)
T. Schoneberg, I. Liebscher, Mutations in G Protein-Coupled Receptors: Mechanisms, Pathophysiology and Potential Therapeutic Approaches. Pharmacol. Rev. 73, 89–119 (2021)
J.A. Janovick, T.P. Spicer, T.D. Bannister, L. Scampavia, P.M. Conn, Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias. PLoS One. 12, e0181830 (2017)
G. Decaux, A. Soupart, G. Vassart, Non-peptide arginine-vasopressin antagonists: the vaptans. Lancet. 371, 1624–1632 (2008)
J.H. Yi, H.J. Shin, H.J. Kim, V2 receptor antagonist; tolvaptan. Electrolyte Blood Press. 9, 50–54 (2011)
B. Rangarajan, V. Binoy, S.S. Hingmire, V. Noronha, Tolvaptan South. Asian J. Cancer. 3, 182–184 (2014)
D.G. Bichet, What is the role of vaptans in routine clinical nephrology? Clin. J. Am. Soc. Nephrol. 7, 700–703 (2012)
J.H. Robben, M. Sze, N.V. Knoers, P.M. Deen, Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes insipidus. Am. J. Physiol. Ren. Physiol. 292, F253–F260 (2007)
D. Wesche, P.M.T. Deen, N.V. Knoers, Congenital nephrogenic diabetes insipidus: the current state of affairs. Pediatr. Nephrol. 27, 2183–2204 (2012)
M. Thibonnier, P. Coles, D.M. Conarty, C.L. Plesnicher, M. Shoham, A molecular model of agonist and nonpeptide antagonist binding to the human V(1) vascular vasopressin receptor. J. Pharmacol. Exp. Ther. 294, 195–203 (2000)
T. Imaizumi, S. Harada, Y. Hirooka, H. Masaki, M. Momohara, A. Takeshita, Effects of OPC-21268, an orally effective vasopressin V1 receptor antagonist in humans. Hypertension. 20, 54–58 (1992)
K. Takahashi, N. Makita, K. Manaka et al., V2 vasopressin receptor (V2R) mutations in partial nephrogenic diabetes insipidus highlight protean agonism of V2R antagonists. J. Biol. Chem. 287, 2099–2106 (2012)
H.A. Blair, Tolvaptan: A Review in Autosomal Dominant Polycystic Kidney Disease. Drugs. 79, 303–313 (2019)
I. Boselt, H. Rompler, T. Hermsdorf, D. Thor, W. Busch, A. Schulz, T. Schoneberg, Involvement of the V2 vasopressin receptor in adaptation to limited water supply. PLoS One. 4, e5573 (2009)
W. Wang, D.Y. Guo, Y.X. Tao, (2019) Therapeutic strategies for diseases caused by loss-of-function mutations in G protein-coupled receptors. In: Prog. Mol. Biol. Transl. Sci. Elsevier, pp 181–210

Table 1. Cell surface and sandwich ELISA results of mutant AVPR2s. The values of treatment with OPC-41061, OPC-31260 and OPC-21268, and untreated sample are seen. The data are given as mean ± S.E.M. of independent experiments. The WT was set to 100% as a reference for all experiments and the numbers in parentheses indicate independent experiments. WT: Wild type, a: Results are from our previous study [16].

		WT	R68W		DR67-G69		G107W		V162A		T273M		DR67-G69/G107W
		Mean	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Cell surface (WT%)	^aWithout tr. (7)	100	28.3	2.3	59.3	3.6	77.6	7.0	93.6	6.8	27.3	3.6	68.0	7.8
	OPC41061 (3)	100	79.3	3.2	87.0	4.1	85.9	1.2	87.4	5.2	74.4	4.8	48.5	5.4
	OPC31260 (3)	100	74.2	2.8	81.5	1.8	82.0	2.0	86.5	4.2	27.8	2.4	30.7	3.3
	OPC21268 (3)	100	55.8	4.6	77.7	3.9	73.1	8.5	76.4	10.6	11.9	3.3	29.7	1.2
Total (%)	^aWithout tr. (5)	100	85.6	11.4	82.0	10.4	90.4	4.9	88.4	9.2	91.6	7.3	94.0	7.7
	OPC41061 (3)	100	98.6	12.3	102.1	3.8	90.9	10.5	83.3	13.5	87.5	9.6	97.7	5.0
	OPC31260 (3)	100	112.9	7.0	104.7	3.2	95.5	4.7	90.6	7.1	94.1	9.8	90.8	3.2
	OPC21268 (3)	100	113.2	2.2	109.3	4.8	87.2	7.1	97.3	10.5	79.8	8.6	95.7	3.7

Table 2. cAMP accumulation assay results of R68W, DR67-G69 and T273M mutants. The values of treatment with OPC-41061, OPC-31260 and OPC-21268, and untreated sample are seen. For all experiments, except EC₅₀ values, the WT was set to 100% as a reference. The numbers in the parentheses indicate independent experiments and the data are given as mean ± S.E.M. of independent experiments. WT: Wild type, a: Results are from our previous study [16]. *cAMP accumulation assay was not performed because cell surface expressions of T273M after the treatment with OPC-31260 and OPC-21268 were not increased.

		WT			R68W		DR67-G69		T273M
		Mean			Mean	SEM	Mean	SEM	Mean	SEM
E_max (WT%)	^aUntreated (9)	100			47.8	7.9	81.7	10.8	44.9	8.1
	OPC41061 (3)	100			70.1	4.7	123.6	17.6	35.1	7.8
	OPC31260 (3)	100			82.0	20.1	118.5	16.2	*
	OPC21268 (3)	100			18.5	9.9	67.2	33.4	*
EC₅₀ (nM)	^aUntreated	0.4 (9)	0.29	0.01	0.28 (9)	0.05	0.39 (8)	0.08	31.0 (3)	18.0
	OPC41061 (3)	4000.0	5000.0	3000.0	477,0	81.0	1000.0	283.0	1000.0	373.0
	OPC31260 (3)	1.36	2.23	1.29	1.01	0.55	0.25	0.19	*
	OPC21268 (3)	0.02	0.03	0.02	0.09	0.06	0.04	0.03	*

Table 3. Statistical p values of Mann-Whitney U test. Significant p values are seen and The test was performed on GraphPad Prism 8 for macOS (Version 8.4.3., GraphPad Software, LLC.).

		Untreated sample vs Treated with PCs
		OPC-41061	OPC-31260	OPC-21268
DR67-G69	EC₅₀ value	0.0364	-	-
DR67-G69	Cell surface expression	0.0167	0.0167	0.0250
R68W	EC₅₀ value	0.0364	-	-
R68W	Cell surface expression	0.0250	-	-
T273M	Cell surface expression	0.0167	-	0.0333

Download PDF

Version 1

posted

You are reading this latest preprint version

The rescue potentials of OPC-41061, OPC-31260 and OPC-21268 on mutant AVPR2s that cause nephrogenic diabetes insipidus

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Methods

Chemicals and Reagents

Cell Culture, Transfection and Treatment with PCs

Cell Surface and Total Expression of Mutants

cAMP Accumulation Assay

Statistical Analysis

Results

Cell surface and total expressions of mutant AVPR2s

cAMP accumulation

Discussion

Declarations

References

Tables

Status:

Version 1