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CELLULAR AND MOLECULAR

ZM241385, DPCPX, MRS1706 Are Inverse Agonists with Different Relative Intrinsic Efficacies on Constitutively Active Mutants of the Human Adenosine A_2B Receptor

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research (Q.L., K.Y., C.C.B., A.P.I., M.W.B.) and Division of Molecular Genetics (H.d.D., J.B.), Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

The human adenosine A_2B receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A_2B receptors were identified from a random mutation bank. In the current study, three known A_2B receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A_2B receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A_2B receptor because they were able to fully reverse the basal activity of four low-level constitutively active A_2B receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A_2B receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 ± 0.03, 0.35 ± 0.03, and 0.31 ± 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A_2B receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.

Address correspondence to: Dr. Margot Beukers, Division of Medicinal Chemistry, LACDR, Gorlaeus Laboratories, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: beukers{at}chem.leidenuniv.nl

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