Efforts to take down the activity of glycogen synthase kinase 3 (GSK3)—a pivotal kinase that promotes pathology in Alzheimer’s disease—have come up short. Now, a new class of inhibitor may provide a fresh offensive. As described in the November 15 Science Signaling, a short peptide dubbed L807 blocked GSK3 by slipping into the kinase’s substrate binding pocket and becoming phosphorylated. L807 then wedges tightly into the pocket, preventing GSK3 from modifying other substrates, including tau and β-catenin. Unlike a so-called “suicide inhibitor,” which permanently modifies its target through covalent bonds, the new type of inhibitor resisted dissociation from its target but was technically reversible. When given to mice intranasally, the inhibitor entered the brain, where it prevented tau phosphorylation and ramped up lysosomal function and autophagy, both controlled by GSK3 activity. L807 also reduced plaque load and dampened memory and social problems in an AD mouse model. The researchers, led by Hagit Eldar-Finkelman of Tel Aviv University, hope to move this inhibitor into the clinic.

“This exciting work reveals a unique mode of inhibition of GSK3,” commented Stephen Haggarty of Massachusetts General Hospital in Boston. He added that this novel approach has potential for treating AD and related neuropsychiatric disorders.

When it comes to involvement in neurodegenerative disease, GSK3 seems to have its fingers in every pie (see Llorens-Marítin et al., 2014). The kinase, which exists in two closely related forms (GSK3α and GSK3β) phosphorylates tau, transforming it into a tangle-forming toxin (see Hanger et al., 1992). It is both activated by and promotes the accumulation of Aβ, the latter possibly a result of GSK3’s stifling of lysosome activity. What’s more, GSK3 phosphorylates and destabilizes β-catenin, a molecule that plays an important role in the Wnt signaling pathway, which in turn plays a role in neurodegeneration when it is turned down (see Marzo et al., 2016). Researchers have made many attempts to block GSK3. However, most inhibitors they found worked by blocking the ATP binding site of the kinase and, since all kinases use ATP, these types of inhibitors generally lacked specificity.

First author Avital Licht-Murava and colleagues set out to design a highly selective GSK3 inhibitor. They aimed to target the enzyme’s substrate binding site, rather than its ATP binding site, using a peptide modeled after a sequence in the known GSK3 substrate heat shock factor-1 (HSP-1). Eldar-Finkelman previously described such an inhibitor, dubbed L803, which reduced amyloid pathology and restored lysosomal function in an AD mouse model, albeit with weak potency (see Avrahami et al., 2013). 

In their attempts to improve the potency of L803, the researchers uncovered the new inhibitor—L807. This 11-amino-acid peptide contains serine residues at positions 6 and 10. Serine-6 corresponds to the residue in HSF-1 that is normally phosphorylated by GSK3, while serine-10—known as the priming site—corresponds to a residue that is normally phosphorylated by other kinases prior to GSK3 binding. The previous inhibitor, L803, contained a synthetically phosphorylated serine-10, but unlike L807, it had an alanine at position 6 instead of the serine.

The researchers found that GSK3 phosphorylated L807 at serine-6. The enzyme’s activity subsequently plummeted by more than 80 percent. L807 topped L803’s potency by about 20-fold. Reasoning that this reflected phosphorylation at serine-6, the researchers synthesized a version of L807 that was phosphorylated at both the GSK3 site and the priming site. Unexpectedly, this peptide was a dud. That’s when the researchers realized that GSK3 had to phosphorylate L807 itself in order to unleash the peptide’s inhibitory potential. This unique relationship between inhibitor and target also boosted L807’s specificity, as the peptide failed to block a panel of 139 other protein kinases.

The researchers ran a series of molecular dynamics simulations to nail down the molecular acrobatics underpinning L807’s unique function. They suggested that upon phosphorylation of L807 by GSK3, the peptide forms a hairpin turn that allows it to squeeze deeper into GSK3’s binding pocket. Strong interactions between hydrophobic residues in L807 and GSK3 then hold the inhibitor firmly in place, preventing it from dissociating. Eldar-Finkelman told Alzforum that the researchers cannot speculate on how often the inhibitor dissociates from its target; however, theoretically it could do so since it is not held in place by covalent bonds.

Catalytic Contortion.

Simulations revealed a proposed sequence of events that ultimately wedge L807 into the GSK3 binding pocket. The peptide slips into the pocket (A), then phosphorylation at serine-6 allows it to bend further into the cavity (B, top panel). Further hydrophobic interactions allow L807 to curl in more snugly (bottom panels). [Courtesy of Licht-Murava et al., Science Signaling 2016.]

The researchers tested the activity of the inhibitor in cell lines and animal models. For these experiments, they used a myristoylated version of the peptide—L807mts—that facilitates its entry into cells. In human neuroblastoma cells, 5uM of L807mts boosted the abundance of β-catenin (which is normally destroyed after phosphorylation by GSK3). The inhibitor enhanced lysosomal acidification in primary hippocampal neurons, and restored lysosomal pH to normal levels in mouse embryonic fibroblasts lacking presenilins, which promote lysosomal acidification. When the scientists gave two daily doses of the inhibitor to mice intranasally, then dissected their hippocampi four hours later, they found increased β-catenin, as well as a reduction in phosphorylation of tau. The inhibitor also knocked down phosphorylation of human P301S tau in transgenic mice by more than half. 

In mice, the inhibitor remained detectable in the blood for 12 hours, with a half-life of five to six hours. In the brain, the inhibitor’s concentration peaked four hours after administration, and disappeared by 12 hours. High doses of L807mts triggered no noticeable toxic effects or neurological symptoms.

Finally, Licht-Murava and colleagues tested the inhibitor in the 5xFAD mouse model of AD. Starting at two months old, the mice received 60μg intranasal doses (or placebo) every other day until they reached six months of age. Compared to untreated 5xFAD mice, treated mice performed better on a contextual fear conditioning task, meaning they spent a longer time freezing in response to frightening stimuli they had experienced previously. The treated mice preferred to explore novel objects as opposed to familiar ones, and favored the company of “stranger” mice over familiar cage-mates. Collectively, these findings suggested that L807mts improved memory and social behavior in 5xFAD mice.

A reduction in AD pathology underpinned this cognitive improvement. Probing brain sections with Aβ antibodies and Congo Red indicated the treated 5xFAD mice had a two- to threefold lower amyloid load than their untreated brethren. They had less neuroinflammation, as assessed by the number of active astrocytes in their hippocampi. In keeping with GSK3’s role in slowing down lysosomal and autophagic function, the researchers also found markers of elevated autophagic flux in the treated animals’ brains.

Lars Ittner of University of New South Wales and Neuroscience Australia in Sydney commented that while peptide inhibitors may achieve greater specificity than some small-molecule inhibitors have, their use is usually considered a tough trade-off due to their vulnerability to proteases and their larger size. “However, Avital Licht-Murava and colleagues successfully developed a specific inhibitor of GSK-3 that has good drug properties,” he said. Ittner said that beyond pathology and cognition in 5xFAD mice, he would be interested to see whether the inhibitor improves memory and motor function in P301S mice, which only have mutations in tau.

Because the 5xFAD mice have no tauopathy, it is likely that L807mts improved symptoms in the mice via enhanced clearance of Aβ via autophagy, rather than by preventing tau phosphorylation, Eldar-Finkelman told Alzforum. In humans, tau-related effects could also occur. She is searching for industry partners to develop L807mts.—Jessica Shugart

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  1. This exciting work from Licht-Murava and colleagues reveals a unique mode of inhibition of GSK3, using a peptide scaffold that acts as both a substrate and inhibitor through a kinase activity-dependent, “dissociation-resistant” conversion.

    Besides elucidating the mechanism of action of L807mts both in vitro and in cellular assays, an impressive aspect of the study is the in vivo data further demonstrating efficacy of selectively targeting GSK-3 in the context of preclinical models of neurodegeneration. This is a longstanding interest in the field that has been hampered by suboptimal pharmacological agents. 

    Further optimization of the pharmacological properties of L807mts to address potency and brain exposure has tremendous potential to advance novel therapeutic agents for Alzheimer’s and related neuropsychiatric disorders. One wonders if this method could be used for obtaining selective GSK3-α versus GSK3-β inhibitors, as well as inhibitors that show cell- or circuit-specific functional selectivity due to variation in the endogenous substrate concentration.

    Overall, these findings have important implications for designing selective probes of a number of kinases through the concept of substrate-to-inhibitor conversion mechanism.

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References

Research Models Citations

  1. hTau.P301S
  2. 5xFAD

Paper Citations

  1. . GSK-3β, a pivotal kinase in Alzheimer disease. Front Mol Neurosci. 2014;7:46. Epub 2014 May 21 PubMed.
  2. . Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci Lett. 1992 Nov 23;147(1):58-62. PubMed.
  3. . Reversal of Synapse Degeneration by Restoring Wnt Signaling in the Adult Hippocampus. Curr Biol. 2016 Oct 10;26(19):2551-2561. Epub 2016 Sep 1 PubMed.
  4. . Inhibition of Glycogen Synthase Kinase-3 Ameliorates β-Amyloid Pathology and Restores Lysosomal Acidification and Mammalian Target of Rapamycin Activity in the Alzheimer Disease Mouse Model: IN VIVO AND IN VITRO STUDIES. J Biol Chem. 2013 Jan 11;288(2):1295-306. PubMed.

Further Reading

Papers

  1. . GSK-3 inhibition: achieving moderate efficacy with high selectivity. Biochim Biophys Acta. 2013 Jul;1834(7):1410-4. Epub 2013 Jan 29 PubMed.

Primary Papers

  1. . A unique type of GSK-3 inhibitor brings new opportunities to the clinic. Sci Signal. 2016 Nov 15;9(454):ra110. PubMed.