G protein ß4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex.

Adenosine A2A receptors (A2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A2AAR in a complex with full-length Gs α and Gß4γ2 (A2AAR-Gs α:ß4γ2). The orthosteric binding site of A2AAR-Gs α:ß4γ2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gß4 versus the closest analog, A2AAR-miniGs α:ß1γ2. Consequently, there are fewer interactions between the switch II in Gs α and the Gß4 torus. In reconstitution experiments Gß4γ2 displayed a ten-fold higher efficiency over Gß1γ2 in catalyzing A2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A2AAR-Gs α:ß4γ2 accounts for this increased efficiency. These results suggest that Gß4 functions as a positive allosteric enhancer versus Gß1.

Unlocking antitumor immunity with adenosine receptor blockers.

Tumors survive by creating a tumor microenvironment (TME) that suppresses antitumor immunity. The TME suppresses the immune system by limiting antigen presentation, inhibiting lymphocyte and natural killer (NK) cell activation, and facilitating T cell exhaustion. Checkpoint inhibitors like anti-PD-1 and anti-CTLA4 are immunostimulatory antibodies, and their blockade extends the survival of some but not all cancer patients. Extracellular adenosine triphosphate (ATP) is abundant in inflamed tumors, and its metabolite, adenosine (ADO), is a driver of immunosuppression mediated by adenosine A2A receptors (A2AR) and adenosine A2B receptors (A2BR) found on tumor-associated lymphoid and myeloid cells. This review will focus on adenosine as a key checkpoint inhibitor-like immunosuppressive player in the TME and how reducing adenosine production or blocking A2AR and A2BR enhances antitumor immunity.

1,3-Dialkylxanthine Derivatives Having High Potency as Antagonists at Human A2B Adenosine Receptors.

The structure-activity relationships (SAR) of alkylxanthine derivatives as antagonists at the recombinant human adenosine receptors were explored in order to identify selective antagonists of A2B receptors. The effects of lengthening alkyl substituents from methyl to butyl at 1- and 3-positions and additional substitution at the 7- and 8-positions were probed. Ki values, determined in competition binding in membranes of HEK-293 cells expressing A2B receptors using 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine), were approximately 10 to 100 nM for 8-phenylxanthine functionalized congeners. Xanthines containing 8-aryl, 8-alkyl, and 8-cycloalkyl substituents, derivatives of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) and XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-1,3-dipropylxanthine), containing various ester and amide groups, including L- and D-amino acid conjugates, were included. Enprofylline was 2-fold more potent than theophylline in A2B receptor binding, and the 2-thio modification was not tolerated. Among the most potent derivatives examined were XCC, its hydrazide and aminoethyl and fluoroethyl amide derivatives, XAC, N-hydroxyethyl-XAC, and the L-citrulline and D-p-aminophenylalanine conjugates of XAC. An N-hydroxysuccinimide ester of XCC (XCC-NHS, MRS 1204) bound to A2B receptors with a Ki of 9.75 nM and was the most selective (at least 20-fold) in this series. In a functional assay of recombinant human A2B receptors, four of these potent xanthines were shown to fully antagonize the effects of NECA-induced stimulation of cyclic AMP accumulation.