Glycogen synthase kinase-3 (GSK-3) was shown to be a key factor in attenuation of the cellular action of insulin. We speculated that inhibition of GSK-3 might have a potential therapeutic value in treatment of insulin resistance and type 2 diabetes. Here, we present a novel class of specific phosphorylated peptides inhibitors of GSK-3, which in sharp contrast to other protein kinase inhibitors that are ATP analogs, are substrate-competitive. We show that the GSK-3 peptide inhibitor activated glycogen synthase activity 2.5-fold in human embryonic kidney 293 cells, and increased glucose uptake in primary mouse adipocytes in the absence or presence of insulin compared with cells treated with two respective peptide controls. In addition, an i.p. administration of GSK-3 peptide inhibitor to normal or insulin-resistant obese C57BL/6J mice, improved their performance on glucose tolerance tests compared with control-treated animals. We present here a novel rational strategy for developing specific GSK-3 inhibitors and point toward GSK-3 as a promising therapeutic target in insulin resistance and type-2 diabetes.

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Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate-competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase-3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer's disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of β-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

Lithium, a known mood-stabilizer frequently used in treatment of bipolar disorders, is an effective glycogen synthase kinase-3beta (GSK-3beta) inhibitor. This led to the idea that GSK-3beta is an in vivo target directly inhibited by lithium. As lithium is a weak in vitro inhibitor of GSK-3beta (IC50=2 mM), however, we speculated that it inhibits GSK-3beta via an indirect, yet unknown, mechanism. The present studies show that lithium increased the phosphorylation of a key inhibitory site of GSK-3beta, serine-9 (Ser-9), in HEK293 cells and in PC12 cells. This phosphorylation was significantly reduced by protein kinase C (PKC) inhibitors GF109203X and Ro31-8425, as well as GO6976, an effective inhibitor toward conventional PKC isoforms (cPKC). Consistent with these results, lithium increased PKC-alpha activity approximately twofold in both cell lines. Because PI3 kinase is a potential upstream regulator of cPKC, its inhibition by wortmannin or LY294002 also abolished the lithium-induced serine phosphorylation of GSK-3beta in HEK293 and PC12 cells. Moreover, lithium did not activate PKB, and in addition, its activity was not dependent on the presence of medium inositol nor did it affect the autophosphorylation activity of GSK-3beta. Finally, intracerebroventricular injection of lithium increased GSK-3beta Ser-9 phosphorylation and enhanced PKC-alpha activity 1.8-fold in mouse hippocampus, confirming this lithium response in vivo. Our studies propose a new mechanism by which lithium indirectly inhibits GSK-3beta via phosphatidylinositol 3 kinase- dependent activation of PKC-alpha.