Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 μM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0 nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79 ± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1 μM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.
Keywords: Allosteric; Hyperhidrosis axillaris; Methanthelinium bromide; Muscarinic receptor; NMS; Orthosteric.