Allosteric Modulation of the A3 Adenosine Receptor

We first screened for potential enhancing activity of LUF6000 and LUF6096 with the A3 adenosine receptor (A3AR) from animal species commonly used in preclinical drug research, i.e., dog, rabbit, and mouse. We found that both LUF6000 and LUF6096 exerted substantial enhancing activity in assays with the human, canine and rabbit, but not with the mouse A3AR. Lack of activity was also observed in limited studies with the rat A 3AR. In responsive species, both modulators increased the maximal efficacy (Emax) of the A3AR agonist Cl-IB-MECA as well as the native ligand adenosine more than twofold. A slight (5-6 fold) decrease in orthosteric ligand potency was detected with the human and canine (but not the rabbit) A3AR, explained by an effect of the modulators to reduce orthosteric ligand binding affinity observed in equilibrium displacement binding assays with the orthosteric agonist radioligand [125I]I-AB-MECA. Further evidence for a negative effect of the enhancers on orthosteric ligand binding was observed when the displacement radioligand binding data were analyzed using the allosteric ternary complex model and when association/dissociation rates were determined in kinetic radioligand binding assays. Collectively, these results revealed that, similar to orthosteric pharmacology, allosteric pharmacology of the A3AR is highly species-dependent. The currently available A3AR allosteric modulators are limited by their lack of activity with rodent receptors and by their negative modulatory effects in some species on orthosteric ligand potency.;Comparison of the primary amino acid sequence of species that were responsive to the modulators versus non-responding species identified marked differences in the first extracellular loop (EL1), a small region of GPCRs (5-10 amino acids) that links the 2nd and 3rd transmembrane regions and provides rigidity necessary for formation of the orthosteric ligand binding pocket and orthosteric ligand-induced receptor activation. We therefore focused on examining the potential role of the EL1 in mediating enhancing activity of the quinolone A3AR allosteric modulator LUF6096 using a mutagenesis approach. We observed that mutation of EL1 of the human A 3AR (responding species - GITIH) to the mouse sequence (non-responding species - QVKMH) did not diminish the ability of LUF6096 to increase maximal functional efficacy of Cl-IB-MECA in [35S]GTPγS binding assays. However, the potency of LUF6096 to produce functional enhancement was decreased markedly (100-fold). In radioligand binding assays with the EL1-mutated receptor, LUF6096 produced a greater negative modulatory effect on orthosteric ligand binding, although its potency was similarly reduced. Mutation of EL1 of the mouse A3AR to the human sequence did not restore responsiveness of the receptor to LUF6096. These studies provided evidence that the EL1 region plays an important role in allosteric modulation of the A3AR. Specifically, the data suggest that the first extracellular loop mediates negative cooperative interaction between the allosteric and orthosteric sites and likely participates in forming the allosteric ligand binding pocket.;We finally investigated the effect of LUF6096 on infarct size in a barbitalanesthetized dog model of myocardial ischemia/reperfusion injury. The dog model was chosen because of its similarity to the human condition of acute myocardial infarction and because the dog A3AR is sensitive to the enhancing activity of LUF6096. 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 prior to ischemia and the other immediately before reperfusion. Group III received a single dose of LUF6096 (1 mg/kg i.v. bolus) immediately before reperfusion. We observed that pretreatment with LUF6096 before coronary occlusion and during reperfusion in Group II dogs produced a marked reduction in infarct size (~50% reduction) as compared to Group I vehicle-treated dogs. An equivalent reduction in infarct size was observed when LUF6096 was administered immediately before reperfusion in Group III dogs. Importantly, administration of LUF6096 at either dose did not produce any adverse hemodynamic effects including changes in heart rate, arterial blood pressure, left ventricular pressure, and coronary blood flow. Plasma samples were collected at various times during the reperfusion period in Group III dogs to detect the plasma concentration of LUF6096 using a newly developed mass spectrometric procedure. The plasma measurements confirmed that LUF6096 was present at a concentration capable of producing A3AR enhancing activity. (Abstract shortened by UMI.).