Species Differences and Mechanism of Action of A Adenosine Receptor Allosteric Modulators

Activity of the A 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 AAR species homologs used in preclinical drug development. In guanosine 5'-[γ-[S]thio]triphosphate ([S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant AARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit AARs but provided only weak activity at mouse AARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the AAR agonist 2-chloro-N -(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 -(4-amino-3-[I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([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 AAR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that AAR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.