Investigating the interaction of McN‐A‐343 with the M 1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

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.