Calcimimetics with potent and selective activity on the parathyroid calcium receptor

Abstract

Parathyroid hormone (PTH) secretion is regulated by a cell surface Ca2+ receptor that detects small changes in the level of plasma Ca2+. Because this G protein-coupled receptor conceivably provides a distinct molecular target for drugs useful in treating bone and mineral-related disorders, we sought to design small organic molecules that act on the Ca2+ receptor. We discovered that certain phenylalkylamine compounds, typified by NPS R-568 and its deschloro derivative NPS R-467, increased the concentration of cytoplasmic Ca2+ ([Ca2+]i) in bovine parathyroid cells and inhibited PTH secretion at nanomolar concentrations. These effects were stereoselective and the R enantiomers were 10- to 100-fold more potent than the S enantiomers. NPS R-568 potentiated the effects of extracellular Ca2+ on [Ca2+]i and PTH secretion but was without effect in the absence of extracellular Ca2+. Both compounds shifted the concentration-response curves for extracellular Ca2+ to the left. Presumably, these compounds act as positive allosteric modulators to increase the sensitivity of the Ca2+ receptor to activation by extracellular Ca2+. Both NPS R-467 and NPS R-568 increased [Ca2+]i in HEK 293 cells expressing the human parathyroid Ca2+ receptor but were without effect in wild-type HEK 293 cells. Neither compound affected the cytoplasmic Ca2+ responses elicited by several other G protein-coupled receptors in HEK 293 cells or in bovine parathyroid cells. Significantly, these compounds did not affect responses elicited by the homologous metabotropic glutamate receptors, mGluR1a, mGluR2, or mGluR8. These compounds therefore act selectively on the Ca2+ receptor. Compounds that mimic or potentiate the effects of extracellular Ca2+ at the Ca2+ receptor are termed calcimimetics. The discovery of calcimimetic compounds with potent and selective activity enables a pharmacological approach to regulating plasma levels of PTH. Calcimimetic compounds could conceivably provide a specific medical therapy for primary hyperparathyroidism.

Figure 1

Structures of NPS 467 and NPS 568 shown as the R enantiomer.

Figure 2

NPS 467 and NPS 568 increase [Ca²⁺]_i in a concentration-dependent and stereoselective manner. Bovine parathyroid cells loaded with fura-2 (A) were suspended in PCB containing 0.5 mM CaCl₂ and treated with the indicated concentration of the R or S enantiomer of NPS 467. HEK 293 cells expressing the human parathyroid cell Ca²⁺ receptor and loaded with fluo-3 (B) were suspended in medium containing 1 mM CaCl₂ and treated with the indicated concentration of NPS R-568 or NPS S-568. For both cell types, the peak transient increase in [Ca²⁺]_i evoked by NPS 467 or NPS 568 is plotted. Each point is the mean ± SEM of three or four experiments.

Figure 3

Increases in [Ca²⁺]_i elicited by NPS R-568 are refractory to inhibition by La³⁺ but are blocked by removal of extracellular Ca²⁺. Bovine parathyroid cells were suspended in medium containing 0.5 mM CaCl₂. In all traces, the final concentration of NPS R-568 was 1 μM. La³⁺ (1 μM, final), EGTA (1 mM, final), or Mg²⁺ (3 mM, final) was added as indicated. The numbers accompanying each trace are estimates of [Ca²⁺]_i in nM. Each trace is from a single preparation of cells and is representative of the pattern seen in three other experiments.

Figure 4

NPS R-568 potentiates the effects of extracellular Ca²⁺ on [Ca²⁺]_i and PTH secretion. In A, bovine parathyroid cells loaded with fura-2 and suspended in buffer containing 0.5 mM CaCl₂ were pretreated with 100 nM (⧫) or 10 nM (▪) NPS R-568 or vehicle control (○) for 1 min before extracellular Ca²⁺ was increased to the final concentration indicated. In B, bovine parathyroid cells were incubated in buffer containing 0.5 mM CaCl₂ for 30 min in the presence or absence of the indicated NPS R-568 (▪) or NPS S-568 (⧫). For comparison, the effects of increasing the concentration of CaCl₂ to 2 mM (○) are shown. In C, bovine parathyroid cells were incubated for 30 min in buffer containing the indicated concentration of CaCl₂ in the absence (○) or presence of 10 nM (⧫) or 100 nM (⧫) NPS R-568. Each point is the mean ± SEM of three experiments.

Figure 5

NPS R-467 and NPS R-568 act selectively on the parathyroid Ca²⁺ receptor. All traces are from either wild-type HEK 293 cells (A and B) or HEK 293 cells expressing the mGluR1a receptor (C and D).

Figure 6

NPS R-467 does not affect responses mediated by mGluR2 (A and C) or mGluR8 (B and D). Oocytes were equilibrated in HK25 buffer for 3–5 min before the application of NPS R-467 (10 μM) or (R,S)-α-cyclopropyl-4-phosphonophenylglycine (CPPG; 10 μM) and l-glutamate (3 or 10 μM) as indicated by the horizontal bars above each trace.