Investigation of the Interaction of a Putative Allosteric Modulator, N-(2,3-Diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) Methanamine Hydrobromide (SCH-202676), with M1 Muscarinic Acetylcholine Receptors

  1. Alfred Lanzafame and
  2. Arthur Christopoulos
  1. Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia
  1. Address correspondence to:
    Dr. Arthur Christopoulos, Department of Pharmacology, University of Melbourne, Grattan St., Parkville, 3010, Victoria, Australia. E-mail: arthurc1{at}unimelb.edu.au

Abstract

The interaction between a novel G protein-coupled receptor modulator, N-(2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide (SCH-202676), and the M1 muscarinic acetylcholine receptor (mAChR) was investigated. In contrast to the prototypical mAChR allosteric modulator, heptane 1,7-bis-(dimethyl-3′-phthalimidopropyl)-ammonium bromide (C7/3-phth), SCH-202676 had no effect on the dissociation kinetics of [3H]N-methylscopolamine ([3H]NMS) at M1 mAChRs stably expressed in Chinese hamster ovary (CHO) cell membranes. However, SCH-202676 completely inhibited the binding of [3H]NMS in membrane preparations, with a Hill slope significantly greater than unity, indicative of positive cooperativity in the binding of the inhibitor. Moreover, SCH-202676 caused dextral shifts of the [3H]NMS saturation binding curve that were greater than expected for a competitive interaction. The addition of C7/3-phth (100 μM) had no significant effect on the inhibitory potency of SCH-202676. In contrast to the findings in cell membranes, the interaction between SCH-202676 and [3H]NMS in intact M1 CHO cells yielded saturation and inhibition isotherms that were compatible with the predictions for a competitive interaction. Intact cell assays of acetylcholine-mediated phosphoinositide hydrolysis in the absence or presence of SCH-202676 revealed a mixed competitive/noncompetitive mode of interaction that was dependent on the concentration of SCH-202676. These data reveal that the nature of the interaction between SCH-202676 and the M1 mAChR is dependent on whether it is studied using intact versus broken cell preparations. It is proposed that SCH-202676 uses a dual mode of ligand-receptor interaction involving both extra- and intracellular attachment points on the M1 mAChR that are distinct from the allosteric binding site recognized by prototypical mAChR modulators such as C7/3-phth.

Footnotes

  • This work was supported by National Health and Medical Research Council of Australia Grant 251538. A.C. is a Senior Research Fellow of the National Health and Medical Research Council. A.L. is the recipient of a National Health and Medical Research Council Dora Lush Biomedical Postgraduate Scholarship.

  • ABBREVIATIONS: GPCR, G protein-coupled receptor; TCM, ternary complex model; mAChR, muscarinic acetylcholine receptor; SCH-202676, N-(2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide; NMS, N-methylscopolamine; C7/3-phth, heptane, 1,7-bis-(dimethyl-3′-phthalimidopropyl)-ammonium bromide; DMEM, Dulbecco's modified Eagle's medium; PI, phosphoinositide; ACh, acetylcholine; C-R, concentration-response.

  • DOI: 10.1124/jpet.103.060590.

    • Received September 28, 2003.
    • Accepted November 4, 2003.
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  1. Published online before print November 14, 2003, doi: 10.1124/jpet.103.060590 JPET March 2004 vol. 308 no. 3 830-837
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