Effect of the Calcimimetic R-568 [3-(2-Chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine] on Correcting Inactivating Mutations in the Human Calcium-Sensing Receptor

Abstract

Over 257 mutations in the human calcium-sensing receptor (hCaSR) gene have been reported. Heterozygous inactivating mutations can result in familial hypocalciuric hypercalcemia (FHH), whereas homozygous inactivating mutations can cause life-threatening neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the hCaSR can result in hypercalciuria and hypocalcemia. A recent publication on the successful treatment of a patient suffering from FHH with the hCaSR positive allosteric modulator cinacalcet prompted our interest in exploring the molecular pharmacology of calcimimetics to correct signaling defects associated with inactivating hCaSR mutations. We prepared 11 mutant hCaSRs, previously identified in patients suffering from NSHPT or FHH, and tested their ability to couple to inositol phosphate accumulation and intracellular calcium mobilization in transiently transfected human embryonic kidney 293 and Chines hamster ovary cells using the calcimimetic R-568 [3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine]. We found that extracellular Ca 2ϩ was significantly less potent on the mutated receptors compared with wild-type hCaSR. However, R-568 was able to enhance the potency of extracellular Ca 2ϩ toward the mutant hCaSRs. Furthermore, R-568 increased the maximal agonist response on several of the mutant CaSRs. We applied a novel operational model of allosteric modulation/agonism that provided a common mechanism to account for the behavior of the wild-type and mutant hCaSRs. The data provide evidence for the potential use of calcimimetics to treat diseases such as FHH and NSHPT where severe hypercalcemia can be lifethreatening.

Discovery of a Calcimimetic with Differential Effects on Parathyroid Hormone and Calcitonin Secretion

“…Global fitting of the entire family of curves to an operational model of allosterism/agonism yielded the estimates of orthosteric agonist potency (LogEC 50 ), modulator affinity (LogK B ), modulator efficacy (Log B ), and cooperativity (Log␣␤) shown in Table 2. Because of the large number of parameters associated with the model, it was necessary to estimate the cooperativity (␣) and efficacy modulation (␤) as a composite parameter, ␣␤ (Lu et al, 2009). We found that both R-568 and calcimimetic B are allosteric modulators as well as partial agonists because their B values were greater than 0.…”

“…3D). It is noteworthy that affinity modulation (␣) cannot be distinguished from efficacy modulation (␤) when the orthosteric agonist (Ca 2ϩ in this case) is a full agonist (Lu et al, 2009).…”

“…Positive cooperativity (␣ Ͼ 1) results in an increase in affinity of the orthosteric ligand when the receptor is occupied by the allosteric ligand and vice versa. We define ␤ as a measure of the allosteric effect of the modulator on the signaling efficacy of the orthosteric agonist over and above any effects the modulator has on the binding affinity of the agonist (see Leach et al, 2007;Aurelio et al, 2009;Lu et al, 2009). For ␤ Ͼ 1, cellular response increases in the presence of modulator and orthosteric ligand relative to the orthosteric ligand alone, whereas ␤ Ͻ 1 results in reduced responsiveness.…”

“…Ca 2ϩ flux (FLIPR) Ϫ7.2 Ϯ 0.14 (n ϭ 9) Ϫ8.1 Ϯ 0.35 (n ϭ 37)* Ca 2ϩ flux (aequorin) Ϫ6.4 Ϯ 0.10 (n ϭ 3) Ϫ7.8 Ϯ 0.14 (n ϭ 9)* IP accumulation Ϫ6.4 Ϯ 0.13 (n ϭ 33) Ϫ7.7 Ϯ 0.15 (n ϭ 32)* Activation and modulation of the CaSR by R-568 and calcimimetic B is a composite of 1) the affinity of the compound at the receptor denoted K B , 2) the intrinsic efficacy denoted B , 3) the ability of the compound to modulate the affinity of Ca 2ϩ to the CaSR denoted ␣ (cooperativity), and 4) the ability of the compound to modulate the coupling of the Ca 2ϩ occupied CaSR to downstream signaling pathways denoted ␤ (efficacy modulation). These parameters were quantitated using an operational model of allosteric modulation and allosteric agonism (Leach et al, 2007;Aurelio et al, 2009;Lu et al, 2009). Shown in Fig.…”

“…4 was performed using the following form of an operational model of allosterism/agonism (see Fig. 3; also see Lu et al, 2009):…”