title: A covalent antagonist for the human adenosine A2Areceptor.
abstract: The structure of the human A2Aadenosine 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 hA2Areceptor 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 A2Areceptor 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.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, Jiangsu, 221004, China.
authors.forename: Guo
authors.initials: G
authors.lastname: Dong
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.
authors.forename: Thomas J M
authors.initials: TJM
authors.lastname: Michiels
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.
authors.forename: Eelke B
authors.initials: EB
authors.lastname: Lenselink
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.
authors.forename: Laura
authors.initials: L
authors.lastname: Heitman
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.
authors.forename: Julien
authors.initials: J
authors.lastname: Louvel
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands. ijzerman@lacdr.leidenuniv.nl.
authors.forename: Ad P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D058917:Adenosine A2 Receptor Antagonists / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D006801:Humans; D043705:Receptor, Adenosine A2A / Q000187:drug effects / Q000378:metabolism*; D014227:Triazines / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D014230:Triazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D058917:Adenosine A2 Receptor Antagonists; C000626539:LUF7445; D043705:Receptor, Adenosine A2A; D014227:Triazines; D014230:Triazoles
keywords: A2A adenosine receptor; Adenosine; Covalent antagonist; G protein-coupled receptors; Radioligand binding