Allosteric modulation of muscarinic receptors by the multivalent ligand THRX‐160209

Abstract

THRX-160209 is a multivalent ligand that binds simultaneously to the orthosteric binding pocket and an allosteric region of the M2 muscarinic receptor. We tested the hypothesis that THRX-160209 can modulate binding of orthosteric ligands by binding to the allosteric site on the muscarinic M2 receptor previously described to interact with obidoxime and gallamine. In kinetic studies, the dissociation rate of [3H] N-methyl scopolamine ([3H] NMS) from all five muscarinic receptors was retarded by THRX-160209, with the highest potency at the M2 receptor (pEC50diss = 6.5). These results are consistent with the actions of other known allosteric modulators. In radioligand binding assays designed to study allosteric modulators, the equilibrium dissociation constant for THRX-160209 at the unoccupied M2 receptor was determined to be 63 pM (pKA = 10.2), and the degree of negative cooperativity (α) between THRX 160209 and [3H] NMS was determined to be 2500 (pα = −3.4). Additionally, the effects of THRX-160209 on [3H] NMS dissociation rates were tested in the presence of obidoxime. Increasing concentrations of obidoxime caused a rightward shift in the concentration effect curve for THRX-160209 induced retardation of [3H] NMS dissociation rates in a manner suggestive of a competitive interaction. The “pKB” value for obidoxime in the presence of THRX-160209 was determined to be 4.0, closely matching the pEC50diss value of 4.1 determined for obidoxime alone. These data suggest that THRX-160209 can act as an allosteric modulator of muscarinic receptors binding to the same allosteric site as gallamine and obidoxime.3