Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A₃ Receptor

Abstract

The development of covalent ligands for G protein-coupled receptors (GPCRs) is not a trivial process. Here, we report a streamlined workflow thereto from synthesis to validation, exemplified by the discovery of a covalent antagonist for the human adenosine A3 receptor (hA3AR). Based on the 1H,3H-pyrido[2,1-f]purine-2,4-dione scaffold, a series of ligands bearing a fluorosulfonyl warhead and a varying linker was synthesized. This series was subjected to an affinity screen, revealing compound 17b as the most potent antagonist. In addition, a nonreactive methylsulfonyl derivative 19 was developed as a reversible control compound. A series of assays, comprising time-dependent affinity determination, washout experiments, and [35S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hA3AR-homology model and site-directed mutagenesis study was performed to demonstrate that amino acid residue Y2657.36 was the unique anchor point of the covalent interaction. This workflow might be applied to other GPCRs to guide the discovery of covalent ligands.

Covalent Inhibition of the Histamine H3 Receptor

“…Determination of washout resistance by radioligand binding is a frequently used method for the validation of a postulated irreversible interaction between a potential covalent ligand and the targeted receptor [15][16][17][18][19][20][21][22][23]. In this experimental design, the bound ligand 44 was washed out after the preincubation with hH 3 R expressing cell homogenate and showed a concentration-dependent labeling of the receptor, while the non-covalent control 42 and reference ligands recovered radioligand binding to vehicle levels.…”

“…In the field of kinases the use of TCIs has resulted in marketed drugs in past years [12], while the progress in covalent GPCR ligands is still limited in comparison [13]. Nonetheless, aided by advances in GPCR crystallography, covalent ligands targeting the orthosteric binding site of GPCRs have been disclosed [14][15][16][17][18][19][20][21][22][23]. The targeted GPCR residue is mostly a cysteine with diverse warheads being used, such as a disulfide [17,19,23], isothiocyanate [18], or Michael acceptor [15].…”

“…The targeted GPCR residue is mostly a cysteine with diverse warheads being used, such as a disulfide [17,19,23], isothiocyanate [18], or Michael acceptor [15]. Other residues have been targeted as well with various warheads, i.e., lysine with activated ester [16] or arylsulfonyl fluoride [20], tyrosine with an arylsulfonyl fluoride [22], or asparagine with an aziridinium ion [17]. Indeed, recent extensions of the diversity in warheads give an opportunity for the fine-tuning of the reactivity [24,25].…”

Chemical Probes for the Adenosine Receptors

“…Also in the non-xanthine family, several examples of irreversible AR antagonists have been reported [56][57][58][59].…”

“…A quite similar approach has been utilized for developing covalent human A 3 AR antagonists, leading to a derivative named LUF7602 (4-((3(8-methoxy-2,4-dioxo-3-propyl-3,4-dihydropyrido [2,1-f ]purin-1(2H)yl)propyl)carbamoyl)benzenesulfonyl fluoride, 25) [57] and a pyrazolo-triazolopyrimidine compound (26) [59]. The first one showed that tyrosine 265 was involved in the covalent binding, while compound 26, through the help of computational studies, suggested that serine 247 or cysteine 251, both in TM6, could be responsible for covalent binding.…”