“Cleavable Trifunctional” Approach to Receptor Affinity Labeling: Chemical Regeneration of Binding to A₁-Adenosine Receptors

A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionallyregenerated, receptor protein that also may contain a reporter group. The ligand recognition site of -adenosine receptors in bovine brain membranes was probed to demonstrate the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand [³H]CPX was preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with an IC₅₀ of 0.29 M. Hydroxylamine displaced specific agonist ([³H]PIA) binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-[(carboxymethyl)-oxylphenyl]-1, 3-dipropylxanthine) and shown to bind irreversibly to-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g., DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or an ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to Ai-receptors was inhibited by these reactive xanthines in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [³H]CPX in Arreceptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.