Dual Action Calcium-Sensing Receptor Modulator Unmasks Novel Mode-Switching Mechanism

Abstract

Negative allosteric modulators (NAMs) of the human calciumsensing receptor (CaSR) have previously failed to show efficacy in human osteoporosis clinical trials, but there is now significant interest in repurposing these drugs for hypocalcemic disorders and inflammatory lung diseases. However, little is known about how CaSR NAMs inhibit the response to endogenous activators. An improved understanding of CaSR negative allosteric modulation may afford the opportunity to develop therapeutically superior CaSR-targeting drugs. In an attempt to elucidate the mechanistic and structural basis of allosteric modulation mediated by the previously reported NAM, calhex231, we herein demonstrate that calhex231 actually potentiates or inhibits the activity of multiple CaSR agonists depending on whether it occupies one or both protomers in a CaSR dimer. These findings reveal a novel mechanism of mode-switching at a Class C G protein-coupled receptor that has implications for drug discovery and potential clinical utility.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function

“…This approach provides a measure of modulator potency, which is a composite value of affinity, cooperativity (the magnitude and direction of modulator potentiation or inhibition of the orthosteric agonist), and efficacy (i.e., agonism or inverse agonism). Although potency measurements facilitate drug comparisons in a series when in vitro assays are performed under identical conditions, they can be misleading when different assay conditions are 564 employed (e.g., different orthosteric agonist concentrations, different signaling outputs) (Gregory et al, 2018). Therefore, more recent work has quantified PAM and NAM affinity, cooperativity, and efficacy values as separate parameters using an operational model of allosterism or an allosteric ternary complex model (Davey et al, 2012;Leach et al, 2013Leach et al, , 2016Cook et al, 2015;Diepenhorst et al, 2018;Gregory et al, 2018Gregory et al, , 2020.…”

Negative allosteric modulators of the human calcium‐sensing receptor bind to overlapping and distinct sites within the 7‐transmembrane domain

“…Similarly, ronacaleret and BMS compound 1 cooperativities are threefold to sixfold lower than Pfizer compound 1 and ATF936, respectively. Although the failure of ronacaleret in the clinic has been attributed to its poor pharmacokinetic profile, these data suggest that its affinity and cooperativity also (Gregory et al, 2018;Keller et al, 2018;Leach et al, 2016).…”

“…The distinction between affinity and cooperativity facilitates identification of residues important for modulator binding. This is invaluable when interpreting SAR or molecular modelling studies, whereby CaS receptor residues important for modulator cooperativity are not necessarily the same as those that contribute to modulator affinity (Gregory et al, ; Keller et al, ; Leach et al, ). Thus, we probed the effects of 7TM and ECL amino acid mutations on NAM affinity and cooperativity.…”

“…While surprising, this finding is not without precedence. Our homology modelling is based on a single CaS receptor 7TM domain and, while outside the scope of this study, dimer formation is important for CaS receptor function, harbouring potential additional modulator binding sites (Gregory et al, 2018;Jacobsen, Gether, & Bräuner-Osborne, 2017). Further, alternative allosteric sites have been reported for other class C GPCRs (Chen, Goudet, Pin, & Conn, 2008;Noetzel et al, 2013), and the interaction between BMS compound 1 and NPS2143 appears to be allosteric in nature.…”

Dual modulation of formyl peptide receptor 2 by aspirin‐triggered lipoxin contributes to its anti‐inflammatory activity

“…Biphasic dose curves have been seen in other GPCRs. For example, allosteric modulation of the calcium‐sensing receptor by different concentrations of calhex231 results in an atypical dose curve due to the formation of receptor dimers 56 . The two‐site mechanism has also been proposed to explain the bell‐shape dose response curve of the protease‐activated receptor in response to pepducins 57 …”