Figueroa_2010_Br.J.Pharmacol_160_1534

BACKGROUND AND PURPOSE: We investigated how McN-A-343 inhibited the alkylation of the M(1) muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically. EXPERIMENTAL APPROACH: We incubated the M(1) muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [(3)H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model. KEY
RESULTS: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [(3)H]N-methylscopolamine binding. CONCLUSIONS AND IMPLICATIONS: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M(1) muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.