Critical Cysteine Residues in Both the Calcium-Sensing Receptor and the Allosteric Activator AMG 416 Underlie the Mechanism of Action

Abstract

AMG 416 is a novel D-amino acid-containing peptide agonist of the calcium-sensing receptor (CaSR) that is being evaluated for the treatment of secondary hyperparathyroidism in chronic kidney disease patients receiving hemodialysis. The principal amino acid residues and their location in the CaSR that accommodate AMG 416 binding and mode of action have not previously been reported. Herein we establish the importance of a pair of cysteine residues, one from AMG 416 and the other from the CaSR at position 482 (Cys482), and correlate the degree of disulfide bond formation between these residues with the pharmacological activity of AMG 416. KP-2067, a form of the CaSR agonist peptide, was included to establish the role of cysteine in vivo and in disulfide exchange. Studies conducted with AMG 416 in pigs showed a complete lack of pharmacodynamic effect and provided a foundation for determining the peptide agonist interaction site within the human CaSR. Inactivity of AMG 416 on the pig CaSR resulted from a naturally occurring mutation encoding tyrosine for cysteine (Cys) at position 482 in the pig CaSR. Replacing Cys482 in the human CaSR with serine or tyrosine ablated AMG 416 activity. Decidedly, a single substitution of cysteine for tyrosine at position 482 in the native pig CaSR provided a complete gain of activity by the peptide agonist. Direct evidence for this disulfide bond formation between the peptide and receptor was demonstrated using a mass spectrometry assay. The extent of disulfide bond formation was found to correlate with the extent of receptor activation. Notwithstanding the covalent basis of this disulfide bond, the observed in vivo pharmacology of AMG 416 showed readily reversible pharmacodynamics.

Clinical Pharmacokinetics and Pharmacodynamics of Etelcalcetide, a Novel Calcimimetic for Treatment of Secondary Hyperparathyroidism in Patients With Chronic Kidney Disease on Hemodialysis

“…Etelcalcetide is a synthetic peptide with a molecular weight of 1048 Da; it is composed of a 7 d ‐amino acid residue backbone and 1 l ‐cysteine linked via a disulfide bond to a d ‐cysteine in the backbone (Figure ). Etelcalcetide activates the calcium‐sensing receptor by exchanging the l ‐cysteine for cysteine 482, an allosteric site in the extracellular domain of the calcium‐sensing receptor . This binding site is distinct from both the calcium‐activating site and from the cinacalcet‐binding site .…”

“…Etelcalcetide activates the calcium‐sensing receptor by exchanging the l ‐cysteine for cysteine 482, an allosteric site in the extracellular domain of the calcium‐sensing receptor . This binding site is distinct from both the calcium‐activating site and from the cinacalcet‐binding site . Etelcalcetide was shown to dose‐dependently suppress PTH secretion effectively within 1 hour postdose and produce prolonged but reversible reductions in PTH in normal and secondary hyperparathyroidism nonclinical models, healthy human subjects, and hemodialysis patients with CKD and secondary hyperparathyroidism …”

Population Pharmacokinetics and Pharmacodynamics of the Calcimimetic Etelcalcetide in Chronic Kidney Disease and Secondary Hyperparathyroidism Receiving Hemodialysis

“…Etelcalcetide is a novel peptide that allosterically activates the CaSR . When administered as an i.v.…”

The Calcium-Sensing Receptor and the Parathyroid: Past, Present, Future

“…Administered intravenously it has superior pharmacokinetics including effective suppression of PTH levels beyond 24 h (Walter et al, 2013) due, presumably, to its ability to form a di-sulfide with CaSR residue C482 in its extracellular domain (Alexander et al, 2015). …”