Crystal structure of the adenosine A _2A receptor bound to an antagonist reveals a potential allosteric pocket

Abstract

The adenosine A 2A receptor (A 2A R) has long been implicated in cardiovascular disorders. As more selective A 2A R ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A 2A R antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A 2A receptor bound to compound 1 (Cmpd-1), a novel A 2A R/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A 2A receptor with a cytochrome b562-RIL (BRIL) fusion (A 2A R-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A 2A R-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A 2A R-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A 2A R structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr9 1.35 and Tyr271 7.36 , which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A 2A R structures, highlighting flexibility in the binding pocket that may facilitate the development of A 2A R-selective compounds for the treatment of Parkinson's disease.T he adenosine A 2A receptor (A 2A R) is one of the four subtypes of adenosine receptors (A 1 R, A 2A R, A 2B R, A 3 R). As a member of the G protein-coupled receptor (GPCR) family, the A 2A receptor couples to stimulatory G protein G s and elevates intracellular cAMP upon activation by endogenous adenosine. The A 2A R has been an attractive drug target due to its role in cardiovascular and immune system function, as well as in the central nervous system as a potential therapeutic target for Parkinson's disease (PD) (1-4). PD is a neurodegenerative disease that affects more than 1% of the population over 65 years old. Currently, major treatments target the restoration of dopamine signaling, which is impaired in PD patients, by dopamine-replacing agents. Although these treatments effectively address PD-related motor disturbances, the long-term use of dopamine-replacing agents is associated with the development of motor complications; therefore, there is a need for nondopaminergic drugs (2). It is known that A 2A R signaling regulates dopaminergic neurotransmission, and A 2A R antagonists have been shown to enhance D2 dopamine receptor signaling, which has been found to improve PD symptoms in animal models and patients, without the side effects common to dopaminereplacing agents, such...

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

“…Ye et al utilized an XAC ligand affinity column as an additional chromatography step during purification (Ye et al, 2016) that we did not include in this study. Our purification is similar to what has been published in structural studies of A 2A R, following a general purification scheme of IMAC followed by size exclusion chromatography prior to crystallization in detergent (Hino et al, 2012;Carpenter et al, 2016;Sun et al, 2017) or lipidic cubic phase (Jaakola et al, 2008;Liu et al, 2012b;Xu et al, 2011). These structures contain A 2A R bound to agonists, antagonists, or in complex with an engineered 'mini-Gs' protein.…”

The Aminotriazole Antagonist Cmpd‐1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor

“…[1] Theadenosine 2A receptor (A 2A R) represents ap romising target for Parkinsonsd isease [2] and drug development efforts led to the discovery of Cmpd-1, ad ual antagonist to the A 2A Ra nd the N-methyl d-aspartate receptor subtype 2B (NR2B). [3] Them ethylphenyl and aminotriazole rings of Cmpd-1 occupy adeeply buried position similar to the widely- Figure 1. [3] Them ethylphenyl and aminotriazole rings of Cmpd-1 occupy adeeply buried position similar to the widely- Figure 1.…”

“…Cmpd-1, composed of three rings including ac entral aminotriazole,amethylphenyl and amethoxyphenyl, was crystallised in complex with the A 2A R (Figure 1). [4] B,C) Superimposing A 2A Rbound to ZM 241385 (5OLG, [5] grey,StaR2, cytochrome b562-RIL (BRIL) in ICL3 -o mitted for clarity,and 3PWH, [4] light green, StaR2, no ICL3 insert), Cmpd-1 (5UIG, [3] orange, wild type with BRIL in ICL3) and caffeine (3RFM, [4] blue, StaR2, no ICL3 insert) reveals the unusual outward movement of helix Vwith Cmpd-1. A) Depending on the construct used, the phenyl moiety of ZM 241385 either points towards ECL2 (grey) or aligns with the methoxyphenyl moiety of Cmpd-1 pointing down towards helix I(light green (thermostabilised StaR2) with Cmpd-1 in orange).…”

“…This is consistent with the published rationalisation of the 19 Fc hemical shift on V229 6.31 Cs ite where increased electronic shielding was linked to solvent exposure. [3] It is generally accepted that GPCR antagonists function by stabilising an inactive form of the receptor, through ac ombination of ligand-receptor interactions and intra-receptor contacts (e.g.the ionic lock). [3] It is generally accepted that GPCR antagonists function by stabilising an inactive form of the receptor, through ac ombination of ligand-receptor interactions and intra-receptor contacts (e.g.the ionic lock).…”

“…[16] Cmpd-1 exchanges between equally populated MS4 and 5and thus occupies an extended orthosteric site and am etastable allosteric site with equal probability. [3] However,t he binding to the extended orthosteric site, although important, might be only part of the binding picture. [3] However,t he binding to the extended orthosteric site, although important, might be only part of the binding picture.…”