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The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Hannan FM, Walls GV, Babinsky VN, Nesbit MA, Kallay E, Hough TA, Fraser WD, Cox RD, Hu J, Spiegel AM, Thakker RV - Endocrinology (2015)

Bottom Line: Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1.Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry.Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit (F.M.H., G.V.W., V.N.B., M.A.N., E.K., R.V.T.), Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX3 7LJ, United Kingdom; Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre (T.A.H., R.D.C.), MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, OX11 0RD, United Kingdom; Department of Medicine (W.D.F.), Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, United Kingdom; Laboratory of Bioorganic Chemistry (J.H.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892; and Albert Einstein College of Medicine (A.M.S.), Bronx, New York 10461.

ABSTRACT
Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

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Effect of NPS 2143 on the concentration-response curve of the mutant Gln723 CaSR in transfected HEK293 cells. HEK293 cells were transiently transfected with wild-type or the mutant CaSR-EGFP construct. Single, live cells were loaded with indo-1-acetoxymethylester, which emits fluorescence at 525 nm. NPS 2143 was added at 0nM, 20nM, 40nM, and 80nM concentrations to HEK293 cells transfected with the mutant Gln723 CaSR-EGFP. Cells transfected with the CaSR were selected by fluorescence-activated cell sorting, and the Ca2+o-evoked increases in Ca2+i concentrations were measured. The concentration-response curves of the untreated (dashed line) and NPS 2143-treated (dotted lines) mutant Gln723 receptor were compared with the untreated wild-type (WT) Leu723 CaSR-EGFP (solid line). The increments in Ca2+o concentrations from 0mM to 10mM are shown on the x-axis, and the Ca2+i response, which was measured as a percentage of the maximum normalized response, is shown on the y axis (mean ± SEM of 4 estimations).
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Figure 2: Effect of NPS 2143 on the concentration-response curve of the mutant Gln723 CaSR in transfected HEK293 cells. HEK293 cells were transiently transfected with wild-type or the mutant CaSR-EGFP construct. Single, live cells were loaded with indo-1-acetoxymethylester, which emits fluorescence at 525 nm. NPS 2143 was added at 0nM, 20nM, 40nM, and 80nM concentrations to HEK293 cells transfected with the mutant Gln723 CaSR-EGFP. Cells transfected with the CaSR were selected by fluorescence-activated cell sorting, and the Ca2+o-evoked increases in Ca2+i concentrations were measured. The concentration-response curves of the untreated (dashed line) and NPS 2143-treated (dotted lines) mutant Gln723 receptor were compared with the untreated wild-type (WT) Leu723 CaSR-EGFP (solid line). The increments in Ca2+o concentrations from 0mM to 10mM are shown on the x-axis, and the Ca2+i response, which was measured as a percentage of the maximum normalized response, is shown on the y axis (mean ± SEM of 4 estimations).

Mentions: The responses of wild-type and mutant CaSRs to alterations in Ca2+o concentrations were assessed, after transient transfection of HEK293 cells, by measurements of Ca2+i concentrations (Figure 2). In agreement with previous findings (26), the mutant Gln723 CaSR showed a significant leftward shift in its concentration-response curve when compared with the wild-type Leu723 CaSR, thereby demonstrating that the mutant CaSR is activated by a lower [Ca2+]o than the wild type, consistent with this leading to a gain of CaSR function. Indeed, the mutant Gln723 CaSR had a significantly (P < .01) reduced EC50 (1.94 ± 0.07mM) when compared with the EC50 of the wild type (2.53 ± 0.14mM) (Figure 2 and Table 1). A dose titration of the calcilytic agent, NPS 2143, in HEK293 cells expressing the mutant Gln723 CaSR revealed that NPS 2143 at a concentration of 20nM led to a rightward shift of the mutant receptor concentration-response curve (EC50 of 2.79 ± 0.19, P = .33 compared with wild type) (Table 1), so that this was indistinguishable to that of the wild-type Leu723 CaSR (Figure 2), and thus the function of the mutant receptor was normalized. The addition of higher doses of NPS 2143 (40nM and 80nM) led to a marked rightward shift of the concentration-response curve, so that the mutant Gln723 CaSR displayed a loss-of-function with significantly raised EC50 values of more than or equal to 4.0mM (P < .01) (Figure 2 and Table 1).


The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Hannan FM, Walls GV, Babinsky VN, Nesbit MA, Kallay E, Hough TA, Fraser WD, Cox RD, Hu J, Spiegel AM, Thakker RV - Endocrinology (2015)

Effect of NPS 2143 on the concentration-response curve of the mutant Gln723 CaSR in transfected HEK293 cells. HEK293 cells were transiently transfected with wild-type or the mutant CaSR-EGFP construct. Single, live cells were loaded with indo-1-acetoxymethylester, which emits fluorescence at 525 nm. NPS 2143 was added at 0nM, 20nM, 40nM, and 80nM concentrations to HEK293 cells transfected with the mutant Gln723 CaSR-EGFP. Cells transfected with the CaSR were selected by fluorescence-activated cell sorting, and the Ca2+o-evoked increases in Ca2+i concentrations were measured. The concentration-response curves of the untreated (dashed line) and NPS 2143-treated (dotted lines) mutant Gln723 receptor were compared with the untreated wild-type (WT) Leu723 CaSR-EGFP (solid line). The increments in Ca2+o concentrations from 0mM to 10mM are shown on the x-axis, and the Ca2+i response, which was measured as a percentage of the maximum normalized response, is shown on the y axis (mean ± SEM of 4 estimations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541614&req=5

Figure 2: Effect of NPS 2143 on the concentration-response curve of the mutant Gln723 CaSR in transfected HEK293 cells. HEK293 cells were transiently transfected with wild-type or the mutant CaSR-EGFP construct. Single, live cells were loaded with indo-1-acetoxymethylester, which emits fluorescence at 525 nm. NPS 2143 was added at 0nM, 20nM, 40nM, and 80nM concentrations to HEK293 cells transfected with the mutant Gln723 CaSR-EGFP. Cells transfected with the CaSR were selected by fluorescence-activated cell sorting, and the Ca2+o-evoked increases in Ca2+i concentrations were measured. The concentration-response curves of the untreated (dashed line) and NPS 2143-treated (dotted lines) mutant Gln723 receptor were compared with the untreated wild-type (WT) Leu723 CaSR-EGFP (solid line). The increments in Ca2+o concentrations from 0mM to 10mM are shown on the x-axis, and the Ca2+i response, which was measured as a percentage of the maximum normalized response, is shown on the y axis (mean ± SEM of 4 estimations).
Mentions: The responses of wild-type and mutant CaSRs to alterations in Ca2+o concentrations were assessed, after transient transfection of HEK293 cells, by measurements of Ca2+i concentrations (Figure 2). In agreement with previous findings (26), the mutant Gln723 CaSR showed a significant leftward shift in its concentration-response curve when compared with the wild-type Leu723 CaSR, thereby demonstrating that the mutant CaSR is activated by a lower [Ca2+]o than the wild type, consistent with this leading to a gain of CaSR function. Indeed, the mutant Gln723 CaSR had a significantly (P < .01) reduced EC50 (1.94 ± 0.07mM) when compared with the EC50 of the wild type (2.53 ± 0.14mM) (Figure 2 and Table 1). A dose titration of the calcilytic agent, NPS 2143, in HEK293 cells expressing the mutant Gln723 CaSR revealed that NPS 2143 at a concentration of 20nM led to a rightward shift of the mutant receptor concentration-response curve (EC50 of 2.79 ± 0.19, P = .33 compared with wild type) (Table 1), so that this was indistinguishable to that of the wild-type Leu723 CaSR (Figure 2), and thus the function of the mutant receptor was normalized. The addition of higher doses of NPS 2143 (40nM and 80nM) led to a marked rightward shift of the concentration-response curve, so that the mutant Gln723 CaSR displayed a loss-of-function with significantly raised EC50 values of more than or equal to 4.0mM (P < .01) (Figure 2 and Table 1).

Bottom Line: Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1.Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry.Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit (F.M.H., G.V.W., V.N.B., M.A.N., E.K., R.V.T.), Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX3 7LJ, United Kingdom; Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre (T.A.H., R.D.C.), MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, OX11 0RD, United Kingdom; Department of Medicine (W.D.F.), Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, United Kingdom; Laboratory of Bioorganic Chemistry (J.H.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892; and Albert Einstein College of Medicine (A.M.S.), Bronx, New York 10461.

ABSTRACT
Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

Show MeSH
Related in: MedlinePlus