Detailed functional characterization of four nanobodies as positive allosteric modulators of the human calcium-sensing receptor

The calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and small-molecule and peptide positive allosteric modulators (PAMs) of CaSR, so-called calcimimetics, are used in the treatment of hyperparathyroidism and hypocalcemic disorders. In this study, four monovalent nanobodies - representing four distinct nanobody families with CaSR PAM activity - were subjected to elaborate pharmacological profiling at the receptor. While Nb5 displayed negligible PAM activity at CaSR in all assays, Nb4, Nb10 and Nb45 all potently potentiated Ca²⁺-evoked signalling through a myc epitope-tagged CaSR expressed in HEK293 or HEK293T cells in Gα_q and Gα_i1 protein activation assays and in a Ca²⁺/Fluo-4 assay. Nb4 and Nb10 also displayed comparable PAM properties at a stable CaSR-HEK293 cell line in a Ca²⁺/Fura-2 imaging assay, but surprisingly Nb45 was completely inactive at this cell line in both the Ca²⁺/Fura-2 and Ca²⁺/Fluo-4 assays. Investigations into this binary difference in Nb45 activity revealed that the nanobody only possesses modulatory activity at CaSRs tagged N-terminally with various epitopes (myc, HA, Flag-SNAP), whereas it is inactive at the untagged wild-type receptor. In conclusion, overall each of the four nanobodies exhibit similar CaSR PAM properties in a range of assays, and thus none of them display pathway bias as modulators. However, of the four nanobodies Nb4 and Nb10 would be applicable as pharmacological tools for the wild-type CaSR, whereas the complete inactivity of Nb45 at the untagged CaSR serves as an reminder that epitope-tagging of a receptor, even if deemed functionally silent, can have profound implications for ligand discovery efforts.