Species differences and mechanism of action of A 3 adenosine receptor allosteric modulators

Activity of the A 3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl- N -(3,4-dichlorophenyl)-1 H -imidazo [4,5-c]quinolin-4-amine) and LUF6096 ( N -{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A 3 AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[ 35 S]thio]triphosphate ([ 35 S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A 3 ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A 3 ARs but provided only weak activity at mouse A 3 ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A 3 AR agonist 2-chloro- N 6 -(3-iodobenzyl)adenosine-5′- N -methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6 -(4-amino-3-[ 125 I]iodobenzyl)adenosine-5′- N -methylcarboxamide ([ 125 I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A 3 AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [ 125 I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A 3 AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.