Towards a structural understanding of allosteric drugs at the human calcium-sensing receptor

Abstract

Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an "enriched" picture of structure-function requirements for interaction between Ca(2+)o and allosteric modulators within the CaSR's 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca(2+)o-binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators with distinct and potentially pathway-biased pharmacological effects.

Figure 1

A strategy for enriching allosteric modulator structure-function studies. (A) Mutagenesis was coupled with allosteric analytical modeling to determine amino acid residues that contributed to PAM and NAM binding and allosteric modulation. Mapping important residues onto molecular models of the CaSR's 7TM domains enabled prediction of additional residues to mutate. (B) Structures of PAMs (cinacalcet and AC-265347) and the NAM (NPS-2143) investigated in the current study. (C) Snake diagram of the primary CaSR 7TM domain sequence, indicating residues that were mutated in the current study. Mutations at residues predicted to be important for the binding of the modulators from earlier CaSR molecular models^,,,, are shown in blue. Mutations at homologous CaSR residues that line the mGlu₁ or mGlu₅ allosteric binding site are shown in red. All engineered mutations were substituted with Ala, with the exception of A615^1.42, A772^5.39, A844^7.39 and A840^7.35, which were substituted to Val and E837^7.32 that was also mutated to Asp and Ile. The residue highlighted in orange indicates where a naturally occurring mutation alters the activity of some PAMs. Thick black arrows point to the X.50 conserved class C amino acid residues based on the modified Ballesteros-Weinstein numbering system suggested in.

Figure 2

7TM domain mutations alter the activity of PAMs and NAMs at the CaSR. Concentration-response curves to Ca²⁺_o in the absence and the presence of modulator were determined in Ca²⁺_i mobilization assays to identify mutations that altered PAM and NAM activity. Representative mutants are shown that caused a loss in modulator pK_B (NPS-2143 at E837^7.32I), a gain in modulator pK_B (AC-265347 at A844^7.39V), a loss in modulator cooperativity (cinacalcet at L848^7.43A) or a loss in modulator affinity with a gain in cooperativity (AC-265347 at F688^3.40A). Data are mean ± SEM determined in at least three separate experiments performed in duplicate. Curves through the data points are the best fit of the data to equation 1.

Figure 3

7TM domain mutations alter the pK_B and cooperativity of PAMs and NAMs at the CaSR. Concentration-response curves to Ca²⁺_o in the absence and the presence of modulator determined in Ca²⁺_i mobilization assays were fitted to an operational model of allosterism (equation 1) to determine the change in pK_B (ΔpK_B) and cooperativity (ΔLogαβ) of allosteric modulators at the mutant CaSRs in comparison to the WT receptor. Only data for mutations that caused a significant decrease in pK_B (red bars), increase in pK_B (yellow bars), decrease in Logαβ (orange bars) or increase in Logαβ (turquoise bars) for one or more modulators are shown. White bars represent no significant change in pK_B or Logαβ. Bars that sit above and below zero represent an increase or decrease in pK_B or αβ, respectively. NA, no modulator activity. ^*Significant difference in comparison to the WT (P < 0.05, one-way ANOVA with Dunnett's multiple comparisons post test).

Figure 4

Phenylalkylamines and AC-265347 bind to the CaSR in a distinct manner. (A) Homology model of the CaSR 7TM domains based on the mGlu₅ receptor (grey ribbon representation), bound to cinacalcet (purple), NPS-2143 (blue) and AC-265347 (green). For clarity, TM4 has been cropped from the images in this panel. Functional effects of mutations were determined by fitting Ca²⁺_o concentration-response curves in the absence and the presence of the modulators (Supplementary information, Figures S1-S3) to equation 1 (described in Materials and Methods), and the corresponding amino acid residues where mutations cause a significant decrease or increase in modulator pK_B are shown in ball and stick representation and are colored red and yellow, respectively. Note that although E837^7.32 is shown in red for cinacalcet because substitution with Ala and Ile reduce cinacalcet pK_B, Asp substitution increases cinacalcet pK_B. Hydrogen bonds between E767^ECL2, E837^7.32 and cinacalcet, or R680^3.32, E837^7.32 and NPS-2143 are shown as dashed lines. (B) Cross-sectional view through CaSR homology models based on the mGlu₅ receptor, highlighting the position of cinacalcet (purple), NPS-2143 (blue) and AC-265347 (green). E767^ECL2 and E837^7.32 are shown in CPK coloring, where hydrogen is white, carbon is black, nitrogen is blue and oxygen is red.

Figure 5

Phenylalkylamine modulators bind to an extended cavity in the CaSR. Comparison between the known binding site location of class A (M₂, CCR5 and GPR40), B (CRF₁R), C (mGlu₁, mGlu₅ and CaSR) and F (SMO) GPCR allosteric modulators. Crystal structures (M₂ PDB: 4MQT; CCR5 PDB: 4MBS; GPR40 PDB: 4PHU; CRF₁ PDB: 4K5Y; SMO PDB: 4O9R; mGlu₁ PDB: 4OR2 and mGlu₅ PDB: 4OO9) and CaSR homology models were superimposed using Cα atoms of the TM helices and ligands are shown in surface representation, with solved ligand positions represented in red and predicted ligand positions represented in blue. An overlay of the 7TM domains of each of the solved structures with their ligands is shown as a reference (left). Blue spheres shown on the overlay represent the polar side-chain nitrogens of Arg and Lys residues, which tend to cluster at the membrane boundary. Dashed lines indicate the boundaries of the 7TM domains across different GPCR classes. This figure is based on those illustrated in^,.

Figure 6

Distinct amino acids transmit cooperativity mediated by different modulators. Homology models of the 7TM domains and ECLs of the CaSR based on the mGlu₅ receptor and the predicted binding pose of each modulator. For clarity, TM4 has been cropped from the images. Residues whose mutations cause a significant decrease or increase in the cooperativity of each modulator are shown in ball and stick representation and are colored orange and turquoise, respectively.

Figure 7

Distinct amino acids contribute to biased allosteric modulation by PAMs and NAMs. White bars depict ΔpK_B or ΔLogαβ between WT and mutants determined in Ca²⁺_i mobilization assays, and black bars depict those determined in pERK1/2 assays. Asterisks mark those mutants where there was a significant difference in the ΔpKB or ΔLogαβ for one mutant between Ca²⁺_i mobilization and pERK1/2 assays (P < 0.05, multiple t-tests).