McN-A 343

The pharmacology of McN-A-343.[Pubmed:22643681]

Pharmacol Ther. 2012 Aug;135(2):216-45.

The unusual pharmacology of McN-A-343 was first described by Roszowski in 1961. The agonist appeared to be a selective stimulant of muscarinic receptors in sympathetic ganglia, now known to be the muscarinic M(1) receptor subtype. However, subsequent research demonstrated that McN-A-343 is a partial agonist with similar affinity at all five muscarinic acetylcholine receptor subtypes and its relative selectivity depends on a higher efficacy at the M(1) (and M(4)) subtypes. Being a partial agonist its action is also dependent on factors, such as receptor density and coupling efficacy between receptor activation and tissue response. Nevertheless, the relatively high efficacy at M(1) receptors led to its widespread use as an aid to distinguish responses mediated through M(1) receptors from those utilizing M(2) or M(3) muscarinic receptor subtypes, especially in the CNS. There is also evidence that it has an allosteric action at some receptor subtypes. Recently, it was demonstrated that McN-A-343 can bind to an allosteric site on the M(2) receptor as well as to the orthosteric site and has thus been termed a "bitopic agonist". This allosteric site differs from that occupied by allosteric modulators, such as gallamine. Comparison of comparable mutagenic changes in M(2) and M(4) receptors also suggests that McN-A-343 utilizes different regions of the two receptors for ERK1/2 activation. McN-A-343 has a number of non-muscarinic actions. These include activation of some types of nicotinic acetylcholine receptors, antagonism of serotonin 5-HT(3) and 5-HT(4) receptor subtypes, inhibition of the uptake mechanism and a local anesthetic action.

Investigating the interaction of McN-A-343 with the M muscarinic receptor using its nitrogen mustard derivative and ACh mustard.[Pubmed:20590642]

Br J Pharmacol. 2010 Jul;160(6):1534-49.

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.

Effects of asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors on the irreversible binding of nitrogen mustard analogs of acetylcholine and McN-A-343.[Pubmed:23826889]

Biochemistry. 2013 Jul 23;52(29):4914-28.

We investigated how asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2-M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to that of wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of the M1 and M3 receptors but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1-M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself.

Mutagenesis of nucleophilic residues near the orthosteric binding pocket of M1 and M2 muscarinic receptors: effect on the binding of nitrogen mustard analogs of acetylcholine and McN-A-343.[Pubmed:20643905]

Mol Pharmacol. 2010 Oct;78(4):745-55.

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors.[Pubmed:11341365]

J Pharm Pharmacol. 2001 Apr;53(4):487-96.

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.

Functional determination of McN-A-343 affinity for M1 muscarinic receptors.[Pubmed:1691785]

J Pharmacol Exp Ther. 1990 Apr;253(1):310-4.

The affinity (KA) of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343), acting as an agonist of M1 muscarinic receptors, has been estimated by means of fractional receptor inactivation, employing the irreversible muscarinic antagonist propylbenzylcholine mustard. Two M1-mediated responses elicited by McN-A-343 were studied: relaxation of the isolated rat duodenum and inhibition of twitch contractions in rabbit was deferens. A comparison was made with the affinity of McN-A-343 as an antagonist (KB) of acetylcholine-induced contraction in rat duodenum. Results showed that McN-A-343 displayed similar affinities as an agonist and as an antagonist: -log KA were 4.68 and 5.17 in duodenum and vas deferens, respectively, vs. -log KB of 4.96 in duodenum, indicating that the ability of McN-A-343 to selectively stimulate M1 receptors is not based on a greater affinity for this subtype. In both preparations examined, McN-A-343 reached maximum effect through occupation of a fraction of the total available receptors (approximately 30% in duodenum, approximately 80% in vas deferens), implying that occupation of M1 receptors is translated into effect in a highly efficient way.