Novel Scaffold Agonists of the α2A Adrenergic Receptor Identified via Ensemble-Based Strategy

Abstract

The α_2A adrenergic receptor (α_2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α_2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α_2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α_2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 μM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α_2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were -45.93 and -71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics.

Keywords: bitopic agonist; ensemble-based screening; molecular dynamics simulation; α2A-AR.

Figure 1

Chemical structures of α_2A-AR agonists and antagonists.

Figure 2

(A) Workflow of ensemble-based screening strategy for novel α_2A-AR agonists; (B) RMSD of protein backbone during MD simulation; (C) conformations of 10 clusters and crystal structures; (D) conformational differences of key binding site residues for 10 clusters.

Figure 3

Structure-based virtual screening for novel α_2A-AR agonists. (A) Workflow of virtual screening in Chemdiv library; (B) LogP, molecular weight and PSA comparison of candidate compounds (pink) with known α_2A-AR ligands (green); (C) docking scores of 20 molecules for each cluster.

Figure 4

Biological evaluation for candidate compounds based on PKA and cAMP assays. (A) Effect of 20 compounds in PKA redistribution assay; (B) preliminarily biological evaluation on cAMP assay in vitro for candidate compounds; (C) the dose–effect of SY-17 and SY-15 in regulating cAMP levels.

Figure 5

Chemical structures and binding modes of α_2A-AR ligands. (A) Interactions between α_2A-AR and DMED, with the imidazole ring binding to the orthosteric site with a slat bridge and hydrogen bond; (B–D) binding mode of molecules SY-12, SY-15, and SY-17 with α_2A-AR; (E) exosite and orthosteric site of α_2A-AR.

Figure 6

MD simulation of SY-17 and α_2A-AR complex. (A) RMSD plot of α_2A-AR with ligand SY-17. The ligand was aligned to the protein and then calculated RMSD; (B) RMSF plot of α_2A-AR, the green lines illustrated certain residues which contacted with ligand; (C) fraction of simulation time of specific residue interactions during 1000 ns simulation shown with L-P plot. The displayed residue interacted with the ligand for at least 10% of the simulation time; (D) interaction fractions of α_2A-AR active residues with SY-17 (hydrogen bonds are shown with green bars; ionic and water bridges are shown with pink and blue color bars; hydrophobic are shown with purple); (E) plots of protein–ligand contacts and interactions during 1000 ns simulation.

Figure 7

(A) Decomposed binding free energy of SY-15 (residues located in the orthosteric site were colored blue, while in exosites were red); (B) binding free energy of SY-17.

Figure 8

(A) α-AR receptor family sequence conservation in orthosteric sites; (B) sequence conservation of exosites.