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 Ca ²⁺ -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 Ca ²⁺ /Fluo-4 assay. Nb4 and Nb10 also displayed comparable PAM properties at a stable CaSR-HEK293 cell line in a Ca ²⁺ /Fura-2 imaging assay, but surprisingly Nb45 was completely inactive at this cell line in both the Ca ²⁺ /Fura-2 and Ca ²⁺ /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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)