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

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.