Molecular Mechanisms of Adenosine A1 Receptor Allosteric Enhancers

Allosteric enhancers of the adenosine A1 receptor amplify signaling of orthosteric agonist ligands. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied, thereby conferring specificity to stressed or injured tissues that produce adenosine.
In chapter 2, we explore the mechanism of allosteric enhancer activity. We examine AE activity on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants and (4) site-specific mutants. These findings are combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of docked allosteric enhancers correlate with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site, and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.
In chapter 3, we describe several observations that characterize the mechanisms by which AEs function: (1) Reducing agents such as dithiothreitol (DTT), reduced glutathione (GSSG) and tris(2-carboxyethyl)phosphine (TCEP) can completely block and slowly (t½ = 10 min) reverse AE activity without chemically modifying AEs;
(2) Mutations occluding an A1R disulfide bond pocket (C80-C169) reduce AE activity; (3) Hydrogen peroxide elicits a resistance to GTPγS-indiced decoupling, similar to AEs; (4) compound screening of disulfide oxidizing agents revealed that aryl disulfides have AE activity; and (5) mutations rendering the disulfide more accessible introduce engineered AE sensitivity to the AE-insensitive A2AR. Evaluation of protein structures reveals this disulfide region may be dynamic upon ligand binding. AE binding may prevent this change in conformational states. Chapter 2 identifies an AE binding pocket in ECL2. Chapter 3 suggests that AE activity is derived from a second, independent site: a pocket near the C80-C169 disulfide bond connecting ECL1 and ECL2.