Affinity Mass Spectrometry-Based Fragment Screening Identified a New Negative Allosteric Modulator of the Adenosine A_2A Receptor Targeting the Sodium Ion Pocket

Abstract

Allosteric ligands provide new opportunities to modulate G protein-coupled receptor (GPCR) function and present therapeutic benefits over orthosteric molecules. Negative allosteric modulators (NAMs) can inhibit the activation of a receptor and downstream signal transduction. Screening NAMs for a GPCR target is particularly challenging because of the difficulty in distinguishing NAMs from antagonists bound to the orthosteric site as they both show inhibitory effects in receptor signaling assays. Here we report an affinity mass spectrometry (MS)-based approach tailored to screening potential NAMs of a GPCR target especially from fragment libraries. Compared to regular surface plasmon resonance or NMR-based methods for fragment screening, our approach features a reduction of the protein and compound consumption by 2−4 orders of magnitude and an increase in the data acquisition speed by 2−3 orders of magnitude. Our affinity MS-based fragment screening led to the identification of a new NAM of the adenosine A 2A receptor (A 2A AR) bearing an unprecedented azetidine moiety predicted to occupy the allosteric sodium binding site. Molecular dynamics simulations, ligand structure−activity relationship (SAR) studies, and in-solution NMR analyses further revealed the unique binding mode and antagonistic property of this compound that differs considerably from HMA (5-(N,N-hexamethylene)amiloride), a wellcharacterized NAM of A 2A AR. Taken together, our work would facilitate fragment-based screening of allosteric modulators, as well as guide the design of novel NAMs acting at the sodium ion pocket of class A GPCRs.

Allosteric Modulation of Adenosine A2A Receptors as a New Therapeutic Avenue

“…A new approach specifically targeting the sodium ion pocket, known as fragment-screening based on affinity mass spectrometry, led to the discovery of fragment Fg754 as a new A 2A R NAM carrying a novel azetidine moiety and exhibiting inhibitory potency comparable to HMA. Subsequent simulations of the molecular dynamics, structureactivity relationship studies of the ligand, and nuclear magnetic resonance analyses in solution revealed the unique binding mode and antagonistic properties of Fg754, which is distinctly different from HMA [72]. In addition, cholesterol is reported to be a weak PAM of A 2A Rs [73].…”

“…Increased the dissociation rate of the antagonist ZM-241,385 at rat A2ARs [71]. Increased the dissociation rate of the agonist CGS21680 at A 2A Rs expressing HEK-293 cells [72].…”

“…Moreover, all-atom simulations of molecular dynamics have shown that Fg754 can steadily enter the transmembrane domain core and form contacts with transmembrane helices 2, 3, 6, and 7, and extracellular loop 2. Particularly, the azetidine moiety of Fg754 may occupy the sodium ion-binding site by forming a salt bridge [72]. Another molecular dynamics simulation study described the allosteric effects of a mini-Gs protein on A 2A Rs [87].…”

“…Unknown MGCMA; 5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride Allosteric modulator Increased the dissociation rate of the antagonist ZM-241,385 at rat A2ARs [71]. Unknown MIBA; 5-(N-methyl-N-isobutyl)amiloride Allosteric modulator Increased the dissociation rate of the antagonist ZM-241,385 at rat A2ARs [methyl-7-[(methyl-amino)carbonyl]oxy}-2H-1-benzopyran-2-one} Allosteric modulatorEnhanced agonist radioligand binding to rat striatal A2ARs without functional enhancement[18,91].Unknown Fg754 Allosteric modulatorIncreased the dissociation rate of the agonist CGS21680 at A2ARs expressing HEK-293 cells[72]. binding to rat striatal A 2A Rs without functional enhancement[18,91].Int.…”

The Impact of the Secondary Binding Pocket on the Pharmacology of Class A GPCRs

“…Based on molecular dynamics (MD) simulations, it may overlap with the orthosteric binding site, probably acting in a mixed mode. The compound could thus be a new starting point for the development of allosteric modulators or bitopic compounds ( Yan Lu et al, 2021 ). The A 1 R and A 3 R preferentially bind to G i/o proteins to inhibit adenylate cyclase activity, while the A 2A R and A 2B R preferentially bind to G s proteins to stimulate adenylate cyclase activity.…”