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A multivalent ligand that bridges the orthosteric and allosteric sites of the muscarinic M 2 receptor
Author(s) -
Steinfeld Tod,
Mammen Mathai,
Smith Jacqueline A. M.,
Wilson Richard R,
Jasper Jeffrey R.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1118-c
Subject(s) - allosteric regulation , chemistry , muscarinic acetylcholine receptor , binding site , receptor , dissociation (chemistry) , ligand (biochemistry) , biophysics , muscarinic acetylcholine receptor m2 , stereochemistry , receptor–ligand kinetics , biochemistry , biology
To design potent, M 2 selective antagonists, we employed a multivalent strategy, simultaneously targeting the orthosteric site as well as a nearby allosteric site. The novel ligand, THRX‐160209, exhibited a much higher affinity for the M 2 receptor subtypes compared to the M 1 , M 3 and M 5 receptors. The binding affinity of multivalent ligand for the M 2 receptor (K I = 0.31 nM) was >10,000‐fold higher than that of its monovalent components. When incubated together, the binding affinity of these fragments was at least 17‐fold lower than the affinity of THRX‐160209; the combination of fragments merely bound according to an additive interaction model. Dissociation kinetics of [3H] N‐methyl scopolamine ([ 3 H] NMS) and [ 3 H] THRX‐160209 were determined via an “infinite” dilution kinetic assay. The dissociation rate of [ 3 H] THRX‐160209 (k 1 = 0.056 min 1), but not [ 3 H] NMS, from the M 2 receptor was accelerated 30‐fold by atropine. Similar accelerations were observed with other muscarinic ligands known to interact with either the orthosteric site of the receptor or an allosteric site. The accelerated dissociation rate could be explained by displacement of THRX‐160209 from one of its binding sites, resulting in a lower affinity and a more rapid dissociation. Therefore, THRX‐160209 binds in a multivalent manner to the M 2 receptor, simultaneously occupying orthosteric and allosteric sites. Through simultaneous occupancy of discrete binding sites, THRX‐160209 exhibits substantial increases in receptor affinity and specificity, demonstrating the utility of multivalent ligands for muscarinic receptors.