Figure 7
From: Pharmacological characterisation of novel adenosine A3 receptor antagonists
Pharmacological characterisation of K series of compounds at the rat A3R. (A) Comparison of the residues of the orthosteric binding area in human and rat A3Rs. (B) Saturation binding experiment with AV039 with a KD of 102 ± 7.59 nM. (C) Inhibition of BRET between Nluc and AV039 at the rat A3R by MRS 1220 and K compounds. HEK293 cells stably expressing Nluc-rat A3R were treated with 100 nM AV039 and increasing concentrations of unlabelled compound. The resulting concentration dependent decrease in BRET ratio at 5 minutes was taken to calculate pKi through fitting the Cheng-Prusoff equation59. Each data point represents mean ± SEM of n (n = 5 for MRS 1220, K1, K20, K23 and K25, n = 3 for K10, K17, K18 and K32) experiments, performed in duplicate. (D) Top and side (E) views of Rat A3R in complex with K18. Starting pose (carbons of the ligand in green), after 100 ns MD simulation (carbons of the ligand in orange). Light blue sticks show residues conserved with human A3R. M2647.34 most likely hampers K18 binding due to steric hindrance of the dichloro-phenyl group. (F) Top and side views (G) of the average structure of rat A3R in complex with K25 from 100 ns MD simulations (carbons of the ligand are shown in orange sticks and light blue sticks show residues in contact with K25). K25 was docked into the orthosteric site of the rat A3R using the GoldScore scoring function and the highest scoring pose was inserted in a hydrated POPE bilayer. The complexes were subjected to MD simulations with Amber14ff. and K25 adopts a potential binding pose within the orthosteric binding area.
