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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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Schematic representation of the mechanism of action of NPS 2143. NPS 2143 binds to the TMD of plasma membrane-expressed CaSRs and decreases Ca2+o-mediated signaling responses such as Ca2+i mobilization. Prolonged exposure of CaSR-expressing cells to NPS 2143 may lead to the internalization of this calcilytic compound, which is considered to bind to newly formed CaSRs within the endoplasmic reticulum (ER), and destabilize their active conformation, thus leading to protein misfolding and proteasomal degradation (8), which would in turn diminish the pool of receptors available for trafficking to the plasma membrane. EC, extracellular.
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Figure 1: Schematic representation of the mechanism of action of NPS 2143. NPS 2143 binds to the TMD of plasma membrane-expressed CaSRs and decreases Ca2+o-mediated signaling responses such as Ca2+i mobilization. Prolonged exposure of CaSR-expressing cells to NPS 2143 may lead to the internalization of this calcilytic compound, which is considered to bind to newly formed CaSRs within the endoplasmic reticulum (ER), and destabilize their active conformation, thus leading to protein misfolding and proteasomal degradation (8), which would in turn diminish the pool of receptors available for trafficking to the plasma membrane. EC, extracellular.

Mentions: Autosomal dominant hypocalcemia type 1 (ADH1) and ADH2 (Online Mendelian Inheritance in Man [OMIM] numbers 601198 and 615361) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and G protein subunit α-11 (1–4), respectively, which play a pivotal role in the parathyroid and renal regulation of extracellular calcium (Ca2+o) concentrations. Gain-of-function CaSR mutations have been demonstrated to induce biased signaling responses that involve the preferential activation of phospholipase C-mediated intracellular calcium (Ca2+i) mobilizations (Figure 1) (5), which lead to decreased PTH secretion and increased urinary calcium excretion (2, 4, 6). ADH1-associated mutations may also enhance CaSR biosynthesis by stabilizing newly formed CaSRs in an active conformation that protects against proteasomal degradation (7, 8).


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)

Schematic representation of the mechanism of action of NPS 2143. NPS 2143 binds to the TMD of plasma membrane-expressed CaSRs and decreases Ca2+o-mediated signaling responses such as Ca2+i mobilization. Prolonged exposure of CaSR-expressing cells to NPS 2143 may lead to the internalization of this calcilytic compound, which is considered to bind to newly formed CaSRs within the endoplasmic reticulum (ER), and destabilize their active conformation, thus leading to protein misfolding and proteasomal degradation (8), which would in turn diminish the pool of receptors available for trafficking to the plasma membrane. EC, extracellular.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of the mechanism of action of NPS 2143. NPS 2143 binds to the TMD of plasma membrane-expressed CaSRs and decreases Ca2+o-mediated signaling responses such as Ca2+i mobilization. Prolonged exposure of CaSR-expressing cells to NPS 2143 may lead to the internalization of this calcilytic compound, which is considered to bind to newly formed CaSRs within the endoplasmic reticulum (ER), and destabilize their active conformation, thus leading to protein misfolding and proteasomal degradation (8), which would in turn diminish the pool of receptors available for trafficking to the plasma membrane. EC, extracellular.
Mentions: Autosomal dominant hypocalcemia type 1 (ADH1) and ADH2 (Online Mendelian Inheritance in Man [OMIM] numbers 601198 and 615361) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and G protein subunit α-11 (1–4), respectively, which play a pivotal role in the parathyroid and renal regulation of extracellular calcium (Ca2+o) concentrations. Gain-of-function CaSR mutations have been demonstrated to induce biased signaling responses that involve the preferential activation of phospholipase C-mediated intracellular calcium (Ca2+i) mobilizations (Figure 1) (5), which lead to decreased PTH secretion and increased urinary calcium excretion (2, 4, 6). ADH1-associated mutations may also enhance CaSR biosynthesis by stabilizing newly formed CaSRs in an active conformation that protects against proteasomal degradation (7, 8).

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