Activity of LUF6000 and LUF6096 as positive allosteric modulators (PAMs) for the A3 adenosine receptor (AR) is species-dependent

Adenosine is increased in ischemic tissues where it serves a protective role by activating adenosine receptors (ARs), including the A3 AR subtype. We investigated the effect of N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarboxamide (LUF6096), a positive allosteric modulator of the A3 AR, on infarct size in a barbital-anesthetized dog model of myocardial ischemia/reperfusion injury. Dogs were subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Infarct size was assessed by macrohistochemical staining. Three experimental groups were included in the study. Groups I and II received two doses of vehicle or LUF6096 (0.5 mg/kg i.v. bolus), one administered before ischemia and the other immediately before reperfusion. Group III received a single dose of LUF6096 (1 mg/kg i.v. bolus) immediately before reperfusion. In preliminary in vitro studies, LUF6096 was found to exert potent enhancing activity (EC50 114.3 ± 15.9 nM) with the canine A3 AR in a guanosine 5′-[γ-[35S]thio]triphosphate binding assay. LUF6096 increased the maximal efficacy of the partial A3 AR agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide and the native agonist adenosine more than 2-fold while producing a slight decrease in potency. In the dog studies, administration of LUF6096 had no effect on any hemodynamic parameter measured. Pretreatment with LUF6096 before coronary occlusion and during reperfusion in group II dogs produced a marked reduction in infarct size (~50% reduction) compared with group I vehicle-treated dogs. An equivalent reduction in infarct size was observed when LUF6096 was administered immediately before reperfusion in group III dogs. This is the first study to demonstrate efficacy of an A3 AR allosteric enhancer in an in vivo model of infarction.Medicinal Chemistr

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)-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 A(3)AR species homologs used in preclinical drug development. In guanosine 5'-[gamma-[S-35]thio]triphosphate ([S-35]GTP gamma S) 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-[I-125]iodobenzyl)adenosine-5'-N-methylcarboxamide ([I-125]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 [I-125]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.Medicinal Chemistr