Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of μ and δ opioid receptors with SNpys-containing enkephalins. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala²,Cys(Npys)⁸] dynorphin A-(1-9) amide (1) and Co-Ala²,Cys(Npys)¹²] dynorphin A-(1-13) amide (2). When rat (μ and δ) or guinea pig (χ) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (μ), deltorphin II (δ), and U-69593 (χ), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)- containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label μ receptors (EC₅₀ = 27-33 nM), but also label δ receptors fairly well (160-180 nM). However, for χ receptors they showed drastically different potencies as to affinity labeling; i.e., EC₅₀ = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled χ receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in μ, δ, and χ receptors, whereas analog 2 only labels the Cys residues conserved in μ, and δ receptors.