The 'allosteric modulator' SCH-202676 disrupts G protein-coupled receptor function via sulphydryl-sensitive mechanisms

Abstract

1 Previous studies suggest that the thiadiazole compound SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine) acts as an allosteric modulator of a variety of structurally distinct G protein-coupled receptors (GPCRs). It was postulated that SCH-202676 would directly bind a structural motif in the receptor molecule common to divergent members of the GPCR family. The molecular mechanisms of such a promiscuous action, however, remain obscure. 2 To clarify the mechanism of SCH-202676 action, we used the functional approach of [ 35S]GTPγS autoradiography with rat brain cryostat sections together with classical membrane [ 35S] GTPγS binding assays to evaluate how the thiadiazole affects G protein activity mediated by various receptors linked to the G i-family of G proteins. 3 We found that in the absence of dithiotreitol (DTT), SCH-202676 (10 -7-10 -5 M) elicits nonspecific effects in the [ 35S]GTPγS-based G protein activation assays, thereby severely compromising interpretations on the compounds ability to allosterically inhibit receptor-mediated G protein activity. Such a nonspecific behaviour was fully reversed upon addition of DTT (1 mM), revealing thiol-based mechanism of action. 4 In routine incubations containing DTT, SCH-202676 had no effect on receptor-driven G protein activity, as assessed for adenosine A 1, α 2-adrenergic, cannabinoid CB 1, lysophosphatidic acid LPA 1, muscarinic M 2/M 4, purinergic P2Y 12 or sphingosine 1-phosphate receptors, suggesting that the thiadiazole does not act as an allosteric modulator of GPCR function. 5 1H NMR analysis indicated that SCH-202676 underwent structural changes after incubation with the reducing agent DTT or with brain tissue. 6 We conclude that SCH-202676 modulates GPCRs via thiol modification rather than via true allosteric mechanisms. © 2006 Nature Publishing Group All rights reserved.