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.

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Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes ␣ and ␤. They share high similarity in their catalytic domains but differ in their N-and C-terminal regions, with GSK-3␣ having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3␣ N-terminal region. We found that unlike GSK-3␤, which shuttles between the nucleus and cytoplasm, GSK-3␣ was excluded from the nucleus. Deletion of the N-terminal region of GSK-3␣ resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3␣ accumulation in the nucleus. GSK-3␣ rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3␣. Rather, we show that calcium-induced GSK-3␣ nuclear accumulation was governed by GSK-3␣ binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3␣ was likely an exclusive characteristic of mammalian GSK-3␣. Finally, we show that nuclear localization of GSK-3␣ reduced the nuclear pool of ␤-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3␣ is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3␣ nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3␣-mediated inhibition of the canonical Wnt/␤-catenin pathway.

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