Large library docking identifies positive allosteric modulators of the calcium-sensing receptor

Abstract

Positive allosteric modulator (PAM) drugs enhance the activation of the calcium-sensing receptor (CaSR) and suppress parathyroid hormone (PTH) secretion. Unfortunately, these hyperparathyroidism-treating drugs can induce hypocalcemia and arrhythmias. Seeking improved modulators, we docked libraries of 2.7 million and 1.2 billion molecules against the CaSR structure. The billion-molecule docking found PAMs with a 2.7-fold higher hit rate than the million-molecule library, with hits up to 37-fold more potent. Structure-based optimization led to nanomolar leads. In ex vivo organ assays, one of these PAMs was 100-fold more potent than the standard of care, cinacalcet, and reduced serum PTH levels in mice without the hypocalcemia typical of CaSR drugs. As determined from cryo-electron microscopy structures, the PAMs identified here promote CaSR conformations that more closely resemble the activated state than those induced by the established drugs.

Fig. 1.. Ligands identified from the in-stock and large library screens targeting the 7TM sites of CaSR.

(A) Larger-scale docking against the 7TM^B site of CaSR resulted in a higher hit rate (13.6% in the 2.7-million-molecule docking campaign versus 36.5% in the 1.2-billion-molecule docking campaign). Hit rates were defined by more than 10% BRET response compared with cinacalcet at 100 μM. The overall structure of the homodimeric CaSR is shown on the left, highlighting the binding sites for extracellular ligands—calcium (orthosteric ligand), tryptophan, and phosphate [PDB ID 7M3G (23)]. PAMs bind in transmembrane domains in different fashions. On the right, a zoomed-in view of the 7TM^B site shows evocalcet (an example of a PAM, shown in mauve) adopting a bent conformation. C, Cys. (B) BRET response (normalized to cinacalcet) of the initial hits at 100 μM (mean ± SEM of one to three biological replicates). (C) Hit-rate comparison of the 2.7-million- and 1.2-billion-molecule screens with different affinity definitions. The overall hit rate of the 1.2-billion-molecule screen is significantly better than the in-stock 2.7-million-molecule screen (p < 0.05 by z test). (D) Total docking energies of top-scoring molecules out of the large-scale docking screen compared with the in-stock screen (only molecules with DOCK scores <−35 kcal/mol are plotted). (E) Examples of the docking hits in comparison to the known PAM drugs cinacalcet and evocalcet (colors represent the different moieties fulfilling the same role). Docked poses of the new representative PAMs at the 7TM^B site are shown.

Fig. 2.. Initial hits to high-affinity analogs.

(A) Contact analysis of the initial docking hits versus cinacalcet. Hydrogen bonds (distance ≤3.2 Å, donor angle ≥120°, acceptor angle ≥90°), stacking (aromatic groups face to face or face to edge), salt bridges (electrostatic interaction, oppositely charged residues within 3.5 Å), and van der Waals interactions (attractions between atoms within a 3.5 Å cutoff) are shown. G, Gly; R, Arg; T, Thr. (B) 1.2-billion-molecule screen docking hit ‘5250 (three biological replicates) and its optimized analog ‘2021 (a diastereomer of ‘6783; three biological replicates). (C) 1.2-billion-molecule screen docking hit ‘5670 (three biological replicates) and its optimized analog ‘2460 (an enantiomer of ‘6218; four biological replicates). (D) 1.2-billion-screen docking hit ‘0522 (mean ± SEM of three biological replicates) and its optimized analog ‘5526 (three technical replicates). (E) 2.7-million-molecule screen docking hit ‘21374 (three technical replicates) and its optimized analog ‘54149 (two biological replicates). EC₅₀ was determined by monitoring G_i activation by CaSR upon compound addition at [Ca²⁺] = 0.5 mM. The efficacy of the compounds is normalized to the maximum BRET response induced by cinacalcet. The EC₅₀ of cinacalcet is 71 nM (48 to 106 nM; 12 biological replicates). In (B) to (D), error bars represent the SEM.

Fig. 3.. Structural comparison between docked and experimentally determined poses for ‘54149 and ‘6218.

(A) Close-up view of ‘6218 in the 7TM^A site, with its EM density shown (level = 0.668). Surrounding residues are in green. (B) Superposition of docked and experimentally determined poses of ‘6218 in the 7TM^A site. (C) Close-up view of ‘6218 in the 7TM^B site, with its EM density (level = 0.58). Surrounding residues are in blue. (D) Superposition of docked and experimentally determined poses of ‘6218 in the 7TM^B site. (E) Close-up view of ‘54149 in the 7TM^A site, with its EM density (level = 0.215). The surrounding residues are in green. (F) Superposition of docked and experimentally determined poses of ‘54149 in the 7TM^A site. (G) Close-up view of ‘54149 in the 7TM^B site, with its EM density (level = 0.191). The surrounding residues are in blue. (H) Super-position of docked and experimentally determined poses of ‘54149 in the 7TM^B site. In (B), (D), (F), and (H), the residues under-going conformational changes in the experimental structures are shown. Docked poses and protein residues in the docked structures are in cyan.

Fig. 4.. ‘54149 increases the TM6-TM6 interface and is more effective in suppressing PTH secretion in ex vivo parathyroid glands.

(A) From the cinacalcet-bound to the ‘54149-bound to the Gi-bound CaSR, the 7TM^A protomer undergoes a downward and rotational movement, bringing TM6 closer to the 7TM^B. Cinacalcet-bound CaSR is in gray, ‘54149-bound CaSR is in orange, ‘6218-bound CaSR is in pink, and G_i-bound CaSR is in blue. (B) Parathyroid glands of 4-week-old WT C57/B6 mice were sequentially incubated with increasing concentrations of ‘54149, cinacalcet, and evocalcet from 0.01 nM to 50 μM in the presence of 0.75 mM extracellular calcium ([Ca²⁺]_e). The median inhibitory concentrations (IC₅₀) of ‘54149, evocalcet, and cinacalcet in suppressing PTH secretion are 583 nM (122 to 4727 nM), 998 nM (412 to 4018 nM), and 53 μM, respectively. (C and D) Parathyroid glands were sequentially incubated with increasing [Ca²⁺]_e from 0.5 to 3.0 mM in the presence of vehicle [0.1% dimethyl sulfoxide (DMSO)] or 50 nM (C) or 500 nM (D) ‘54149, cinacalcet, or evocalcet. Shown at the top are changes in the rate of PTH secretion on a per-gland and per-hour basis with increasing [Ca²⁺]_e to compare the secreted PTH at a mean maximal rate (PTH-max). Shown at the bottom are the normalized PTH secretion rates (the highest rates are normalized to the basal rate at 0.5 mM [Ca²⁺]_e of the vehicle, and the lowest rates are normalized to the rate at 3.0 mM [Ca²⁺]_e) to better assess changes in the Ca²⁺ set point ([Ca²⁺]_e needed to suppress 50% of [Ca²⁺]_e-suppressible PTH secretion). Dotted vertical lines indicate Ca²⁺ set points for the corresponding treatments. In (B) to (D), error bars represent the SEM of n = 8 groups of parathyroid glands for each treatment. Dashed lines are used for visual guidance only and do not represent curve fits.

Fig. 5.. ‘54149 suppresses serum PTH at a lower dose and causes less of a hypocalcemia effect than cinacalcet and evocalcet.

(A) Pharmacokinetics of ‘54149 compared with that of cinacalcet and evocalcet after 3 mg/kg subcutaneous injection (n = 3 mice). (B) Serum PTH concentration change over 8 hours after 1 mg/kg subcutaneous injection of ‘54149, cinacalcet, or evocalcet (n = 3 mice). (C) Serum PTH concentration change over 8 hours after 10 mg/kg subcutaneous injection of ‘54149 or cinacalcet (n = 5 mice). (D) Comparison of ‘54149 and cinacalcet in regulating serum PTH at different doses (subcutaneous injection) after 30 min of injection. Each dose was administered to 10 mice, except for the injection at 10 mg/kg (n = 5 mice). P values were assessed by unpaired Student’s t test. (E) Plasma calcium concentration in mice after 3 mg/kg subcutaneous injection of ‘54149, cinacalcet, or evocalcet (n = 3 mice). (F) Serum calcium concentration after 1 mg/kg subcutaneous injection of ‘54149, cinacalcet, or evocalcet (n = 5 mice). For experiments in (B) to (D) and (F), the concentrations of evocalcet and cinacalcet are corrected for their molecular weight difference with ‘54149. Error bars represent the SEM.