Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor

Abstract

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α_2A-adrenergic receptor (α_2AAR), seeking new α_2AAR agonists chemotypes that lack the sedation conferred by known α_2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential G_i and G_o signaling. Experimental structures of α_2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC₅₀) of 52 nanomolar] and two analogs, '7075 and PS75 (EC₅₀ 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

Fig. 1.. Newly discovered a_2AAR agonists from ultralarge library docking.

(A) 301 million molecules were docked against the active state of a_2BAR (PDB 6K41). Lead-like molecules (pink carbons) often spilled out of the orthosteric site, whereas fragment molecules (green carbons) are well complemented by that site. Hit rates were determined with a K_i cutoff of 10 μM. E_VDW, van der Waals; E_ES, electrostatic; E_LDS, ligand desolvation. (B) The αAR pharmacophore model (9) overlaid on known α_2AAR agonists dexmedetomidine, clonidine, and norepinephrine and new agonists from docking (colors represent the different moieties fulfilling the same role). (C) G_i activation and β-arrestin-2 recruitment for norepinephrine (NorEpi), dexmedetomidine (dex), clonidine (clon), and several of the newly discovered docking agonists. (D) Docked poses of these new agonists with hydrogen bonds to key recognition residues of α_2BAR shown as black dashed lines. For (C), data are means ± SEMs of normalized results (n = 4 to 17 measurements for G_i and n = 3 to 8 measurements for β-arrestin-2). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 2.. Docking-predicted poses of ‘9087 and ‘4622 superpose well on the cryo-EM structures of the ‘9087-α_2AAR-G_oA and ‘4622-α_2AAR-G_oA complexes.

(A and C) Cryo-EM structure of the ‘9087-α_2AAR-G_oA (A) and ‘4622-α_2AAR-G_oA (C) complexes. (B) Experimental ‘9087 structure (pink carbons) superposed on the docked pose (orange carbons) (PDB 7W6P; RMSD 1.14 Å). Hydrogen bonds and ion pairs are shown with dashed black lines to F427^7.39 and D128^3.32, respectively. (D) Experimental ’4622 structure (green carbons) superposed on the docked pose (orange carbons) (PDB 7W7E; RMSD 1.14 Å). Hydrogen bond shown with dashed black lines to D128^3.32. For (B) and (D), side chains of α_2AAR residues within 4 Å of ligands are shown as sticks.

Fig. 3.. Structure-based optimization of ‘9087.

(A) Strategies for analoging ‘9087 (left). Analogs of the pyridine, exocyclic nitrogen, and lipophilic nature of the bicyclic ring revealed their importance for ‘9087 activity (middle). Sampling alternate lipophilic bicyclic rings and modifying their substituents identified eight more potent agonists (right). ${\text{EC}}_{50}$ values are shown for G_i activation. (B) G_i and β-arrestin-2 recruitment for ‘9087 and its two most potent analogs, ‘7075 and PS75. (C) Modeled poses of ‘7075 (pink carbons) and PS75 (blue carbons) based on the ‘9087-α_2AAR cryo-EM structure with substituents oriented toward open space in the orthosteric site. Hydrogen bonds and ionic interactions are shown with dashed black lines to F427^7.39 and D128^3.32, respectively. For (A), G_i and β-arrestin-2 recruitment data for analogs are shown in figs. S14 and S15 and table S8. For (B), data are means ± SEMs of normalized results (n = 7 to 17 measurements for G_i and n = 4 to 8 measurements for β-arrestin-2).

Fig. 4.. The docking-derived agonists are antinociceptive in neuropathic, inflammatory, and acute thermal pain through the α_2AAR but are not sedating.

(A to C) Effect of new α_2AAR agonists in neuropathic pain model in mice after SNI with mechanical allodynia. (A) The new agonists ‘9087 and PS75 administered in naïve mice (baseline versus ‘9087, 5 mg/kg; baseline versus PS75, 5 mg/kg; one-way ANOVA; ns, not significant; ****P < 0.0001), dose response of ‘9087 in SNI mice and analogs ‘7075 and PS75 compared with their vehicles (20% kolliphor versus all ‘9087 doses; 20% cyclodextran versus ‘7075 and PS75; one-way ANOVA; **P < 0.01; ***P < 0.001; ****P < 0.0001) with positive control dexmedetomidine (DEX), and ‘9087 administered orally (p.o.) compared with its vehicle (40% captisol versus ‘9087 doses; one-way ANOVA; ****P < 0.0001). (B) Effect of additional agonists ‘4622, ‘0172, and ‘2998 compared with their vehicles (20% kolliphor versus ‘4622, 5 mg/kg; ‘4622, 10 mg/kg; and ‘0172, 5 mg/kg; one-way ANOVA; 20% cyclodextran versus ‘2998; two-tailed t test; ns = *P < 0.05; **P < 0.01; ****P < 0.0001) and positive control DEX. (C) Administration of α₂AR antagonist atipamezole (ATPZ, 2 mg/kg i.p.) to block agonist efficacy in neuropathic pain model (‘9087 versus ‘9087 with ATPZ; ‘7075 versus ‘7075 with ATPZ; PS75 versus PS75 with ATPZ; ‘0172 versus ‘0172 with ATPZ; ‘4622 versus ‘4622 with ATPZ; ‘2998 versus ‘2998 with ATPZ; DEX versus DEX with ATPZ; two-tailed t test; *P < 0.05; **P < 0.01). (D) Diminished analgesia in α_2AAR D79N mice in the 50°C tail flick assay for acute thermal (heat) pain. The mutation does not affect morphine analgesia but substantially decreases the analgesia by DEX, ‘9087, and PS75 (baseline WT versus D79N; morphine WT versus D79N; DEX WT versus D79N; ‘9087 WT versus D79N; PS75 WT versus D79N; two-tailed t test; *P < 0.05; **P < 0.01). (E) Analgesia of ‘9087 and PS75 in 50°C tail flick assay for acute thermal (heat) pain compared with its vehicle (20% Kolliphor versus ‘9087 and PS75; one-way ANOVA; ****P < 0.0001). (F) Analgesia of ‘9087 in 55°C hot plate assay for acute thermal (heat) pain compared with its vehicle (20% Kolliphor versus ‘9087; two-tailed t test; ***P < 0.001). (G) Efficacy of newly characterized agonists in CFA-induced hyperalgesia compared with the vehicle (vehicle versus ‘9087, ‘2998, and ‘0172; one-way ANOVA; *P < 0.05; ***P < 0.001). (H) Evaluating motor impairment and sedation of newly characterized agonists in the rotarod motor test. Only ‘4622 causes slight motor impairment, whereas other agonists do not. DEX causes significant impairment and complete sedation at higher doses. All compounds compared with their vehicles (20% Kolliphor versus ‘9087, ‘0172, and ‘4622; 20% cyclodextran versus ‘2298, ‘7075, and PS75; saline versus DEX; one-way ANOVA; *P < 0.05; **P < 0.01; ****P < 0.0001). For (A) to (G), all compounds were administered s.c., unless otherwise indicated. Data are shown as individual data points and means ± SEMs (n = 4to 25).