Species differences and mechanism of action of A3 adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR 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-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]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 A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.