Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Department of Experimental and Clinical Pharmacology University of Minnesota College of Pharmacy Minneapolis MN 55455 USA

Institute of Physiology Czech Academy of Sciences 14220 Prague Czech Republic

Zobrazit více v PubMed

Bonner T. I. The molecular basis of muscarinic receptor diversity. Trends Neurosci 12, 148–51 (1989). PubMed

Eglen R. M. Overview of Muscarinic Receptor Subtypes In Handb exp pharmacol (eds Fryer A. D., Christopoulos A. & Nathanson N. M.) pp. 3–28, Springer (2012). PubMed

Jakubík J. et al.. Outline of therapeutic interventions with muscarinic receptor-mediated transmission. Physiol Res 63 Suppl 1, S177–89 (2014). PubMed

Kruse A. C. et al.. Muscarinic acetylcholine receptors: novel opportunities for drug development. Nat Rev Drug Discov 13, 549–60 (2014). PubMed PMC

Haga K. et al.. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547–51 (2012). PubMed PMC

Kruse A. C. et al.. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–6 (2012). PubMed PMC

Leppik R. A., Miller R. C., Eck M. & Paquet J. L. Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor. Mol Pharmacol 45, 983–90 (1994). PubMed

Gnagey A. L., Seidenberg M. & Ellis J. Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors. Mol Pharmacol 56, 1245–53 (1999). PubMed

Krejčí A. & Tuček S. Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors. Mol Pharmacol 60, 761–7 (2001). PubMed

Voigtländer U. et al.. Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupie. Mol Pharmacol 64, 21–31 (2003). PubMed

Jakubík J., Krejčí A. & Doležal V. Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators. J Pharmacol Exp Ther 313, 688–96 (2005). PubMed

Jäger D. et al.. Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation. J Biol Chem 282, 34968–76 (2007). PubMed

Kruse A. C. et al.. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–6 (2013). PubMed PMC

Abdul-Ridha A. et al.. Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor. J Biol Chem 289, 6067–79 (2014). PubMed PMC

Gregory K. J., Hall N. E., Tobin A. B., Sexton P. M. & Christopoulos A. Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias. J Biol Chem 285, 7459–74 (2010). PubMed PMC

Valant C. et al.. A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand. J Biol Chem 283, 29312–21 (2008). PubMed PMC

Jakubík J., Zimčík P., Randáková A., Fuksová K., El-Fakahany E. E. & Doležal V. Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors. Mol Pharmacol 86, 180–92 (2014). PubMed

Keov P. et al.. Molecular mechanisms of bitopic ligand engagement with the M1 muscarinic acetylcholine receptor. J Biol Chem 289, 23817–37 (2014). PubMed PMC

Thorsen T. S., Matt R., Weis W. I. & Kobilka B. K. Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis. Structure 22, 1657–64 (2014). PubMed PMC

Thal D. M. et al.. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335–340 (2016). PubMed PMC

Järv J., Hedlund B. & Bartfai T. Isomerization of the muscarinic receptor. antagonist complex. J Biol Chem 254, 5595–8 (1979). PubMed

Järv J., Hedlund B. & Bartfai T. Kinetic studies on muscarinic antagonist-agonist competition. J Biol Chem 255, 2649–51 (1980). PubMed

Jakubík J., El-Fakahany E. E. & Tuček S. Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors. J Biol Chem 275, 18836–44 (2000). PubMed

Redka D. S., Pisterzi L. F. & Wells J. W. Binding of orthosteric ligands to the allosteric site of the M(2) muscarinic cholinergic receptor. Mol Pharmacol 74, 834–43 (2008). PubMed

Goodwin J. A., Hulme E. C., Langmead C. J. & Tehan B. G. Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands. Mol Pharmacol 72, 1484–96 (2007). PubMed

Scarselli M., Li B., Kim S. & Wess J. Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation. J Biol Chem 282, 7385–96 (2007). PubMed

Warne T. et al.. Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454, 486–91 (2008). PubMed PMC

Dror R. O. et al.. Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc Natl Acad Sci USA 108, 13118–23 (2011). PubMed PMC

Guo D. et al.. Molecular Basis of Ligand Dissociation from the Adenosine A2A Receptor. Mol Pharmacol 89, 485–91 (2016). PubMed

Ballesteros J. & Weinstein H. Integrated methods for the construction of three dimensional models and computational probing of structure function relations in G protein-coupled receptors. In Methods in neurosciences (eds Sealfon S. & Conn P.) pp. 366–428, Academic Press (1995).

Burgisser E., Lefkowitz R. J. & DeLean A. Alternative explanation for the apparent “two-step” binding kinetics of high-affinity racemic antagonist radioligands. Mol Pharmacol 19, 509–12 (1981). PubMed

Waelbroeck M., Tastenoy M., Camus J. & Christophe J. Binding kinetics of quinuclidinyl benzilate and methyl-quinuclidinyl benzilate enantiomers at neuronal (M1), cardiac (M2), and pancreatic (M3) muscarinic receptors. Mol Pharmacol 40, 413–20 (1991). PubMed

Lazareno S. & Birdsall N. J. Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. Mol Pharmacol 48, 362–78 (1995). PubMed

Dror R. O. et al.. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature 503, 295–9 (2013). PubMed

Guo D., Hillger J. M., IJzerman A. P. & Heitman L. H. Drug-target residence time–a case for G protein-coupled receptors. Med Res Rev 34, 856–92 (2014). PubMed

Van Durme J. et al.. A graphical interface for the FoldX forcefield. Bioinformatics 27, 1711–2 (2011). PubMed

Jakubík J., El-Fakahany E. E. & Doležal V. Towards predictive docking at aminergic G-protein coupled receptors. J Mol Model 21, 284 (2015). PubMed

Bowers K. J. et al.. Scalable algorithms for molecular dynamics simulations on commodity clusters. SC06: International Conference for High Performance Computing, Networking, Storage and Analysis, November 11–17, 2006, Tampa, FL. In Proceedings of the ACM/IEEE conference on supercomputing. p. 84, ACM Press New York (2006).

Krieger E., Koraimann G. & Vriend G. Increasing the precision of comparative models with YASARA NOVA–a self-parameterizing force field. Proteins 47, 393–402 (2002). PubMed

Current Advances in Allosteric Modulation of Muscarinic Receptors

Novel long-acting antagonists of muscarinic ACh receptors