The Second Extracellular Loop of the Adenosine A₁ Receptor Mediates Activity of Allosteric Enhancers

Abstract

Allosteric enhancers of the adenosine A 1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A 1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A 1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated 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.

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors

“…Figure b shows the top‐ranked consensus binding pose for 3 and (Supporting Information Figure 2) shows this pose in comparison to four other representative “hot‐spots” on the extracellular receptor surface, where other low‐energy poses and clusters of poses were identified. One of the hot‐spots shown highlighted with an orange circle (Supporting Information Figure 2) is in the proximity to a ECL2 binding mode for 2 and structurally related PAMs that were recently predicted by Abagyan and coworkers . The hot‐spot circled in purple is in reasonable agreement with the model for 6 presented by the authors of the bitopic ligand study .…”

“…While this manuscript was in review, to comprehensively investigate the differences between our docking modes and that of Kennedy et al, we performed additional targeted flexible docking for 3 and 4 into A 1 R in an attempt to recapitulate similar induced‐fit low energy binding modes . While we were able to obtain a similar induced binding pocket in the ECL2 region using a flexible receptor approach, this binding mode was predicted to have less‐favorable energy compared to flexible docking poses in the extracellular region that were similar to those obtained with a rigid docking methodology (Supporting Information Figure 5).…”

“…The salt bridge described in A 1 R (H264, E172) and A 2A R (H264, E169) does not exist in the A 3 R structure, due to glutamate being replaced by valine. This is one of many sequence differences that result in a more hydrophobic extracellular receptor surface for A 3 R. As A 2A R residue H264 becomes E258 in the A 3 R models, this E258 residue extends out into solvent rather than being buried in the exact location of the H264 residue in A 2A R. This was the case for the E258 conformation in all of the models built by Modeler from all template pdb files (2YDO.pdb, 3QAK.pdb, 4UG2.pdb, 3EML.pdb) . This can be rationalized in two ways.…”

“…Although several previous computational investigations of the binding mode of GPCR PAMs have utilized advanced methodologies in which the receptor can be fully flexible and sample various conformational states, such methodologies may not be necessary at a first approximation. Based on the recent crystal structure of M 2 R PAM, it is possible that PAMs may bind to and stabilize the active conformation of the extracellular receptor surface.…”

“…However, numerous small‐molecule PAMs of adenosine receptors have been characterized that compete with radioligand agonists and likely bind to an extracellular surface binding site in proximity to the orthosteric adenosine binding site . Previous A 1 R studies have found that residues on ECL2 are implicated in PAM activity and have also suggested that the allosteric binding site may be located in a pocket formed by the ECL2 loop bounded by residues S150 and M162 . It is possible that Class A GPCR extracellular surface allosteric modulators share a common binding site or common molecular binding mechanisms.…”