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Abstract
The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.
Highlights
G protein-coupled receptors (GPCRs), all membrane-bound proteins, represent one of the largest classes of drug targets and are the anchor point for approx. one third of all marketed drugs [1]
We describe our efforts to obtain a covalent antagonist probe for the hA2AAR, as a logical extension of our previous research on long residence time antagonists, i.e., compounds that dissociate only slowly from the receptor [19]
Our research group has explored series of triazolotriazine derivatives based on the reference adenosine A2A antagonist ZM241385, 4-(2-(7-amino-2-- [1, 2, 4]triazolo[1,5-a] [1, 3]triazin-5ylamino)ethyl)phenol (Fig. 1), to investigate their structure-activity and structure-kinetics relationships (SAR and SKR) [22, 29]
Summary
G protein-coupled receptors (GPCRs), all membrane-bound proteins, represent one of the largest classes of drug targets and are the anchor point for approx. one third of all marketed drugs [1]. A combination of technological advances has allowed the structure elucidation of an increasing number of these important drug targets [2,3,4] In this context, covalent modification of the receptor with ligands is emerging as a useful way to investigate ligand-receptor binding domains in membrane proteins, because such covalent ligands, acting as pharmacological chaperones, tend to stabilize the otherwise fragile receptor proteins. Covalent modification of the receptor with ligands is emerging as a useful way to investigate ligand-receptor binding domains in membrane proteins, because such covalent ligands, acting as pharmacological chaperones, tend to stabilize the otherwise fragile receptor proteins Covalent binding of both agonists and antagonists to adenosine receptors has known a long history in purinoceptor research. One existing example is the para-fluorosulfonyl derivative of Purinergic Signalling (2017) 13:191–201
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