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Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM 1-5 ) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM 1 , hM 2 and hM 3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM 2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M 1 -M 5 (hM 1 -hM 5 ) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [ 3 H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 μM of MB ranging from none in hM 3 to 4.6-fold in hM 2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [ 3 H]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: logK I (± S.E.) hM 3  = 8.71 ± 0.15, hM 1  = 8.68 ± 0.14, hM 5  = 8.58 ± 0.07, hM 2  = 8.27 ± 0.07 to hM 4  = 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 logK B : hM 1  = 9.53 ± 0.05, hM 4  = 9.33 ± 0.05, hM 5  = 8.80 ± 0.05, hM 2  = 8,79 ± 0.06, to hM 3  = 8.43 ± 0.04. In conclusion, MB, below 1 μM, binds competitively and non-selectively (except for the difference between hM 3 vs. hM 4 ) 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 hM 1 and hM 4 .