GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins

Abstract

Gastropod mollusks have been used for over 2500 years to produce the "Tyrian purple" dye made famous by the Phoenicians. This dye is constituted of mixed bromine-substituted indigo and indirubin isomers. Among these, the new natural product 6-bromoindirubin and its synthetic, cell-permeable derivative, 6-bromoindirubin-3'-oxime (BIO), display remarkable selective inhibition of glycogen synthase kinase-3 (GSK-3). Cocrystal structure of GSK-3beta/BIO and CDK5/p25/indirubin-3'-oxime were resolved, providing a detailed view of indirubins' interactions within the ATP binding pocket of these kinases. BIO but not 1-methyl-BIO, its kinase inactive analog, also inhibited the phosphorylation on Tyr276/216, a GSK-3alpha/beta activation site. BIO but not 1-methyl-BIO reduced beta-catenin phosphorylation on a GSK-3-specific site in cellular models. BIO but not 1-methyl-BIO closely mimicked Wnt signaling in Xenopus embryos. 6-bromoindirubins thus provide a new scaffold for the development of selective and potent pharmacological inhibitors of GSK-3.

Controlled levels of canonical Wnt signaling are required for neural crest migration

“…To analyze the role of canonical Wnt signaling in migration of cranial NC cells, 6-bromoindirubin-3'-oxime (BIO), an inhibitor of GSK3ß was used (Meijer et al, 2003;Myers et al, 2014) Similarly to treatments at premigratory stages, the separation of the cranial NC cells into branches in this phenotype was not disrupted, but a reduction of the Twist-expressing cells was again observed. In addition, the embryos treated at migratory stages also showed the 'merged branches'…”

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

“…In addition, whether the HEK 293-TOP cells specifically responded to canonical Wnt/β-catenin signaling was also examined in this study. Two GSK-3β inhibitors LiCl and BIO, which are able to increase β-catenin stability and induce β-catenin nuclear translocation (26), were used in this study. LiCl competes for the cofactor Mg 2+ and blocks GSK-3β kinase activity, while BIO selectively binds to the ATP binding pocket of the kinase and inhibits enzyme activity (26).…”

“…Two GSK-3β inhibitors LiCl and BIO, which are able to increase β-catenin stability and induce β-catenin nuclear translocation (26), were used in this study. LiCl competes for the cofactor Mg 2+ and blocks GSK-3β kinase activity, while BIO selectively binds to the ATP binding pocket of the kinase and inhibits enzyme activity (26). Our results with HEK 293-TOP showed that luciferase activity increased in the presence of BIO or LiCl.…”

Xenopus: An ideal system for chemical genetics

“…Although not ideal, the ease of knockdown and overexpression in Xenopus often provides a good correlation with specificity. As a result, Xenopus assays are frequently used to validate compounds that affect Wnt and other pathways (Liu et al, 2005;Meijer et al, 2003). The drug-protein interaction will also ideally be rapid and reversible; in some cases, competitive inhibitors can be used to test both speed and reversibility, as with small molecule effectors of Hedgehog signaling (FrankKamenetsky et al, 2002).…”