Article date: February 2006
By: Anna M Lewandowicz, Jouko Vepsäläinen, Jarmo T Laitinen in Volume 147, Issue 4, pages 422-429
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.
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 Gi‐family of G proteins.
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.
In routine incubations containing DTT, SCH‐202676 had no effect on receptor‐driven G protein activity, as assessed for adenosine A1, α2‐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.
1H NMR analysis indicated that SCH‐202676 underwent structural changes after incubation with the reducing agent DTT or with brain tissue.
We conclude that SCH‐202676 modulates GPCRs via thiol modification rather than via true allosteric mechanisms.
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.
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 Gi‐family of G proteins.
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.
In routine incubations containing DTT, SCH‐202676 had no effect on receptor‐driven G protein activity, as assessed for adenosine A1, α2‐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.
1H NMR analysis indicated that SCH‐202676 underwent structural changes after incubation with the reducing agent DTT or with brain tissue.
We conclude that SCH‐202676 modulates GPCRs via thiol modification rather than via true allosteric mechanisms.
British Journal of Pharmacology (2006) 147, 422–429. doi:10.1038/sj.bjp.0706624
DOI: 10.1038/sj.bjp.0706624
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