Iperoxo/dequalinium and W84/dequalinium hybrid ligands: synthesis, pharmacological and computational investigation at M2 mAChRs

[PO 032] The use of bitopic ligands to study the properties of Muscarinic Acetylcholine Receptors (mAChRs) is a very active field of research [1,2,3]. In this regard, bipharmacophoric molecular probes were found to switch between two different binding orientations in the M2 subtype, resulting in both active and inactive populations of receptors bound by a given ligand, a behavior that has been termed dynamic ligand binding [2]. Continuing our interest in this field, we focused our attention on the properties of Dequalinium chloride, a bis-pyridinium quaternary ammonium compound, which was recently reported to act as a potent muscarinic allosteric modulator, showing an overall selectivity towards the M2 subtype [4]. Starting from this observation, we designed and synthesized two series of novel hybrid ligands incorporating in their molecular skeleton the allosteric moiety of Dequalinium (Figure 1). The first series is based on the combination of Dequalinium with the orthosteric superagonist Iperoxo and aimed at novel chemical probes interacting cooperatively with both the orthosteric and the allosteric binding site. In the second series, Dequalinium was hybridized with the allosteric modulator W-84, affording new putative allo/allosteric ligands. In this study, the newly synthetized compounds have been characterized through competitive binding assays at the five mAChR subtypes. The activity on the allosteric site has been investigated by measuring the affinity (logKocc) at the [3H]-NMS-occupied muscarinic hM2 subtype. Moreover, docking simulations have been performed to highlight the interactions within the ortho/allosteric binding pockets of the hM2 crystal structure addressing the role of linker length. The synthetic approach and the details of the pharmacological and computational investigations will be illustrated and discussed. References 1. Holze, J.; Bermudez, M.; Pfeil, E.M.; Matera, C.; Dallanoce, C.; De Amici, M. et al. ACS Pharmacol. Transl. Sci. 2020, 3, 859–867. 2. Bock, A.; Bermudez, M.; Krebs, F.; Matera, C.; Chirinda, B.; Sydow, D.; Dallanoce, C. et al. J. Biol. Chem. 2016, 291, 16375. 3. Bock, A.; Merten, N.; Schrage, R.; Dallanoce C.; Bätz J.; Klöckner J.; Schmitz J.; Matera C. et al. Nat. Commun. 2012, 2, 1044. 4. Mazzolari, A.; Gervasoni, S.; Pedretti, A.; Fumagalli, L.; Matucci, R.; Vistoli, G. Int. J. Mol. Sci. 2020, 21, 5961.