Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

Abstract

IntroductionActivating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.MethodsAll 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914.ResultsAll BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.ConclusionThe calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.

GENETICS IN ENDOCRINOLOGY: Gain and loss of function mutations of the calcium-sensing receptor and associated proteins: current treatment concepts

“…Two ADH and two BS type 5 CASR mutants were independently tested by different research groups. The results were consistent except for the CASR mutant A843E where NPS-2143 was reported to decrease cell surface expression (107) but not signaling activity (108,109,110,111). Very recently NPS-2143 has been demonstrated to mitigate the gain of function of activating GNA11mutations R181Q and F341L from ADH type 2 patients (93).…”

“…NPS-2143 and its close relative ronacaleret are amino-alcohols, whereas ATF936 and AXT914 are quinazolinones. These different classes of calcilytics have partly different bindings sites (48) and data from four publications show that some but not all ADH CASR mutants were sensitive to NPS-2143 (108,111,112,113). In contrast all five ADH and all four BS5 mutants tested so far were sensitive to ATF936 and AXT914 (111).…”

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels

“…This structural similarity is highlighted by the fact that NPS 2143, Calhex-231, NPS R-568, Cinacalcet, and Calindol all target a common allosteric site within the seventh transmembrane domain of the CaSR. Calcilytics based on an unrelated quinazolinone structure have also been developed, namely ATF936 and AXT914 (John et al, 2014, Letz et al, 2014). In the future, it will be interesting to investigate if these agents also have CaSR-dependent and -independent actions in the vasculature, and whether they inhibit VGCC channels.…”

Calcimimetic and calcilytic therapies for inherited disorders of the calcium‐sensing receptor signalling pathway

“…ATF 936 and AXT 914 rectify the gain‐of‐function caused by constitutively active CaS receptor mutants (Letz et al ., )…”