Small vs. Large Library Docking for Positive Allosteric Modulators of the Calcium Sensing Receptor

Abstract

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism.

Fig. 1.. Novel 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 results in higher hit rate (13.6% in 2.7-million docking campaign versus 36.5% in 1.2-billion docking campaign). Hit rates were defined by over 10% BRET response compared to cinacalcet at 100 μM. E_VDW: van der Waals; E_ES: electrostatic; E_LDS: ligand desolvation. Cinacalcet is in gold and evocalcet is in pink for illustration of the binding sites (PDB: 7MCF). (B) BRET response (normalized to cinacalcet) of the initial hits at 100 μM. (C) Hit rate comparison between 2.7 million and 1.2 billion screens with different affinity definitions. The overall hit rate of the 1.2 billion screen is significantly better than the in-stock 2.7 million screen (P < 0.01 by z-test). (D) Total docking energies of top-scoring molecules out of LSD compared to in-stock screen (only molecules with DOCK score < −35 kcal mol⁻¹ 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 novel PAM representatives at two 7TM sites are shown.

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

(A) Contact analyses of the initial docking hits versus cinacalcet. (B) Docking hit ‘5250 and its optimized analog ‘2021 (a diastereomer of ‘6783). (C) Docking hit ‘5670 and its optimized analog ‘2460 (an enantiomer of ‘6218). (D) Docking hit ‘7909 and its optimized analog ‘3161. (E) In-stock docking hit ‘21374 and its optimized analog ‘54149. EC₅₀ was determined by monitoring Gi 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. Data represent means and SEMs of 3–27 replicates.

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. Surrounding residues are in green. (B) Superposition of docked and experimentally determined pose of ‘6218 in the 7TM^A site. (C) Close-up view of ‘6218 in the 7TM^B site, with its EM density. Surrounding residues are shown in blue. The docked pose and its surrounding residues are in silver. (D) Superposition of docked and experimentally determined pose of ‘6218 in the 7TM^B site. (E) Close-up view of ‘54149 in the 7TM^A site, with its EM density. The surrounding residues are in green. (F) Superposition of docked and experimentally determined pose of ‘54149 in the 7TM^A site. (G) Close-up view of ‘54149 in the 7TM^B site, with its EM density. The surrounding residues are in blue. (H) Superposition of docked and experimentally determined pose of ‘54149 in the 7TM^B site. (B, D, F, H) The residues undergoing 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) The 7TM^A protomer undergoes a downward and rotational movement bringing TM6 closer to the 7TM^B from cinacalcet-bound to ‘54149-bound structure to Gi-bound CaSR. Cinacalcet-bound CaSR is in grey, ‘54149-bound CaSR is in orange, ‘6218-bound CaSR is in pink and Gi-bound CaSR is in blue. (B) Parathyroid glands of 4-week-old C57/B6 wild-type (B6:Wt) 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 [Ca²⁺]_e. The IC₅₀s of ‘54149, evocalcet and cinacalcet in suppressing PTH secretion are 583 nM [122 –4727 nM], 998 nM [412 – 4018 nM] and 53 μM respectively. (C-D) Parathyroid glands were sequentially incubated with increasing [Ca²⁺]_e from 0.5 mM to 3.0 mM in the presence of vehicle (0.1% DMSO), 50 nM (C) or 500 nM (D) of ‘54149, cinacalcet or evocalcet. Top panels show changes in the rate of PTH secretion on a per-gland and per-hour basis with raising [Ca²⁺]_e to compare the PTH-max. Bottom panels show normalized PTH secretion rate (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. Mean ± SEM of n = 8 groups of PTGs for each treatment.

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

‘ (A) Pharmacokinetics of ‘54149 compared to cinacalcet and evocalcet after 3 mg/kg subcutaneous injection. (B) Serum PTH concentration change over 8 hours after 1 mg/kg subcutaneous injection of ‘54149, cinacalcet or evocalcet. (C) Serum PTH concentration change over 8 hours after 10 mg/kg subcutaneous injection ‘54149 or cinacalcet (n = 5). (D) Comparison of ‘54149 to cinacalcet in regulating serum PTH at different doses (subcutaneous injection) after 30min of injection. Each dose consists of n = 10 mice except injection at 10 mg/kg (n = 5). 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. (F) Serum calcium concentration after 1 mg/kg subcutaneous injection of ‘54149, cinacalcet or evocalcet. For experiments in panel B-D, F, the concentrations of evocalcet and cinacalcet are corrected for their molecular weight difference with ‘54149.