Activating variants in the gene that encodes the calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia type-1 (ADH1), characterized by a decrease in the set-point for systemic calcium homeostasis with consequent hypercalciuria and hypocalcemia. Calcilytics (CaSR antagonists) that act as negative allosteric modulators decrease the sensitivity of the CaSR to extracellular Ca²⁺ concentration and thereby increase the set-point for systemic calcium homeostasis. As such, they have the potential to correct the functional genetic disorder of ADH1. In Phase 2 and 3 studies, the orally-active calcilytic, encaleret was found to normalize mineral metabolism in patients with ADH1. ADH1 patients with variants in the extracellular or intracellular domains of the CaSR required titration to a wide range of encaleret doses (4.5 mg to 162 mg). However, in a small number of patients with variants affecting TMD-6 or TMD-7, titration to higher doses of encaleret was required. We investigated the encaleret concentration-response relationship in vitro.

HEK-293 cells were transfected with pcDNA3.1 containing wild-type or variant CASR genes. After 48 h, cells were exposed to various extracellular Ca²⁺ concentrations in the absence or presence of encaleret (1 or 10 µM) and assays of IP₁ and pERK were used as signalling readouts. ADH1 variants studied included W818R, A835D, A835T, V836I, V836L, and A840V. 

Encaleret (1 µM) suppressed responses and increased EC₅₀ values for extracellular Ca²⁺ in A835T, V836I, and V836L variants. However, a higher concentration (10 µM) of encaleret was required to suppress responses in A835D and A840V variants while W818R was unaffected at either concentration.  Thus, signaling responses of CaSRs harbouring variants near the binding pocket of encaleret require higher concentrations of encaleret to be suppressed. Assessing CaSR variants in this manner may provide a tool for predicting patient responses in vivo.