Investigating the interaction of McN-A-343 with the M2 muscarinic receptor using its nitrogen mustard derivative

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We investigated whether the aziridinium ion formed from a nitrogen mustard derivative (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; BR384) structurally related to McN-A-343 (4-(trimethyl-amino)-2-butynyl N-(3-chlorophenyl)carbamate) interacts allosterically or orthosterically with the M2 muscarinic receptor. Chinese hamster ovary cells expressing the human M2 muscarinic receptor were incubated with the aziridinium ion of BR384 in combination with McN-A-343 or other known orthosteric and allosteric ligands for various incubation times. After removing unreacted ligands, we measured the binding of [3H]N-methylscopolamine to residual unalkylated receptors. Affinity constants, rate constants for alkylation, and cooperativity constants were estimated for the interacting ligands using a mathematical model. Receptor alkylation by BR384 was consistent with a two-step process. After rapidly equilibrating with the receptor (step one), the aziridinium ion-receptor complex became covalently linked with a first order rate constant of about 0.95 min-1 (step two). McN-A-343, acetylcholine and N-methylscopolamine competitively protected the M2 receptor from irreversible alkylation by BR384. In contrast, the allosteric modulators, gallamine and WIN 51,708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]pyrimido[1,2-α]benzimidazole), allosterically inhibited or had no effect on, respectively, receptor alkylation by BR384. There was good agreement between affinity constants estimated from the kinetics of receptor alkylation and by displacement of [3H]N-methylscopolamine binding. Our results suggest that BR384 covalently binds to the orthosteric site of the M2 receptor and that McN-A-343 binds reversibly at the same locus. Our method of analyzing allosteric interactions does not suffer from the limitations of more conventional approaches and can be adapted to detect allosteric interactions at receptors other than the muscarinic subtypes.

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