The 'allosteric modulator' SCH-202676 disrupts G protein-coupled receptor function via sulphydryl-sensitive mechanisms.
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]GTPgammaS autoradiography with rat brain cryostat sections together with classical membrane [35S]GTPgammaS binding assays to evaluate how the thiadiazole affects G protein activity mediated by various receptors linked to the Gi-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]GTPgammaS-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 A1, alpha2-adrenergic, cannabinoid CB1, lysophosphatidic acid LPA1, muscarinic M2/M4, purinergic P2Y12 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.
Lewandowicz
Anna M
AM
Department of Physiology, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland.
Vepsäläinen
Jouko
J
Laitinen
Jarmo T
JT
D000494:Allosteric Regulation; D000818:Animals; D001345:Autoradiography; D001923:Brain Chemistry / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008297:Male; D019906:Nuclear Magnetic Resonance, Biomolecular; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism*; D013830:Thiadiazoles; D013844:Thiazoles / Q000494:pharmacology*
D043562:Receptors, G-Protein-Coupled; C418425:SCH-202676; D013830:Thiadiazoles; D013844:Thiazoles; D016244:Guanosine 5'-O-(3-Thiotriphosphate)