Differential effects of the allosteric enhancer (2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone (PD81,723) on agonist and antagonist binding and function at the human wild-type and a mutant (T277A) adenosine A(1) receptor

The 2-amino-benzoylthiophene derivative PD81,723 [(2-amino-4,5-dimethyl-tri enyl)[3-(trifluoromethyl) phenyl]methanone] has been shown to allostericall y enhance agonist binding and function at the adenosine A(1) receptor. The aim of the present study was to elucidate the effects of PD81,723 both as a n allosteric enhancer and as an antagonist on the adenosine A(1) receptor. We investigated its effect on the human wild-type in relation to a mutant ( T277A) adenosine A(1) receptor for which agonists have a greatly diminished affinity. Binding (saturation and displacement experiments) and functional adenosine 3',5'-cyclic monophosphate studies were performed, and different ial effects of allosteric enhancer PD81,723 on agonists and antagonists wer e observed on the wild-type (wt) and mutant adenosine A(1) receptor. Our re sults showed opposite effects of PD81,723 on the binding of agonists and an tagonists. Within the concept of a simplified two-state receptor model, it is possible that the effects of PD81,723 are mainly "allosteric", enhancing the binding of adenosine A(1) agonists and inhibiting the binding of antag onists/inverse agonists. However, the suggestion that PD81,723 acts as an a llosteric inhibitor of DPCPX (1,3-dipropyl-8-cyclopentylxanthine) binding c annot be confirmed by kinetic studies, since PD81,723 does not seem to affe ct the dissociation kinetics of [H-3]DPCPX. Nevertheless, our results show that the action of PD81,723 on DPCPX binding is due to more than mere compe titive antagonistic activity, i.e. binding to the ligand-binding site and c ompeting with the binding of DPCPX, as suggested previously. The effect of PD81,723 on the mutant receptor was much less pronounced. Mutation of Thr27 7 to Ala not only decreased agonist affinity but also inhibited the effects of PD81,723. Insensitivity of the mutT277A to PD81,723 may be linked to th e fact that this mutant appears to be uncoupled from G proteins. It further supported a differential binding mode of PD81,723 compared to competitive antagonists for the adenosine A(1) receptor. (C) 2001 Elsevier Science Inc. All rights reserved.