1. Targeting tau with GSK-3β
inhibitors in the fight against
Alzheimer’s disease.
Presented by: Helen Turner
Student ID: A3791765

2. Introduction
• Alzheimer’s disease (AD) is characterised by two types of lesion in the brain:
• Extracellular senile plaques, formed from aggregated β-amyloid protein
• Intracellular neurofibrillary tangles (NFTs), formed from aggregated hyperphosphorylated
tau protein.
• Drugs currently licensed to treat AD only treat the symptoms and are not disease-modifying.
• Most of the effort to find disease-modifying drugs has focused on the amyloid cascade; however
there has been a high failure rate.
• Taupathies, where the usually unfolded tau forms aggregated structures of hyperphosphorylated
fibrillar tau are present in a number of neurodegenerative diseases including AD.
• There is a strong correlation between loss of cognitive function and NFTs [1] making tau a
potential drug target.
• This presentation examines the use of the GSK-3β inhibitor Tideglusib in the treatment of AD.

3. What is tau?
• Tau is a microtubule-associated protein (MAP) that regulates the assembly, dynamic behaviour
and spatial organisation of microtubules by binding to their surface [1].
• Neuronal tau is located predominantly in the axons in a highly soluble phosphoprotein form [1].
• Abnormal levels of intracellular tau are found are AD patients [4].
• Overexpression of tau compromises microtubule function [4].
• Tau pathologies are present in a number of neurodegenerative diseases .
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Figure 1. Formation of neurofibrillary tangles
from hyperphosphorylated tau [2]. Tau
phosphorylation precedes its aggregation into
NFTs. Initially the NFTs were thought to toxic
however there is some evidence that the soluble,
hyperphosphorylated tau is toxic and NFTs to be
neuroprotective [2].

4. Tau phosphorylation
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• Tau has 85 potential phosphorylation sites of which approximately 10 are phosphorylated in
normal brain tau whereas approximately 45 different sites are phosphorylated in AD brain tau
(Figure 2) [3].
• Tau supports microtubule assembly better in a more unphosphorylated state [4].
Hyperphosphorylation of tau in AD is believed to cause it to dissociate from microtubules
impairing its microtubule and axonal transport functions which leads to neuronal loss [1].
• When hyperphosphorylated the normally unfolded tau assembles into paired helical filaments
(PHF) which aggregates into NFTs [1].
• Kinases involved in the abnormal phosphorylation of tau include GSK-3β and CDK5 [5].
Figure 2. Phosphorylation sites on tau from AD
brain are predominantly in the proline-rich
domain and the area around the microtubule-
binding domains (M1-M4) [3].

5. Glycogen synthase kinase-3 beta (GSK-3β)
• GSK-3β is a serine/threonine kinase that influences the
phosphorylation of a variety of substrates. It affects multiple
physiological pathways and is essential for life [5].
• Two isoforms exist (GSK-3α and GSK-3β). GSK-3β is predominant in
neuronal tissue and is involved in regulation of tau
phosphorylation.
• GSK3β is constitutively active with its basal activity regulated
through different mechanisms [5]
• GSK-3β is upregulated in AD brains and causes tau
hyperphosphorylation when over expressed [5].
• It has been proposed that GSK-3β links the amyloid and tau
pathways of AD [6].
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Figure 3. Structure of GSK-3β (PDB ID: 1I09). There is a β-strand
domain at the N-terminal and an α-helix domain at the C-
terminal with the ATP binding site at their interface [7].

6. GSK-3β inhibitors
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Figure 4. Molecular structure of tideglusib (also known as
NP-12 or NP031112), a thiadiazolidinedione (TDZD).
• GSK-3β inhibitors include lithium, thiadiazolidinediones (TDZDs), paullones, maleimides,
indurubines and natural alkaloids derived from marine sponges [7].
• As GSK-3β is involved in many pathways caution should be taken that its inhibition does not
prevent essential biochemical processes occurring.
• To treat AD GSK-3β inhibitors should decrease activity by no more than 25% - this level of
inhibition should counter the upregulation of the enzyme in the pathological condition, whilst
levels of other physiological pathways can be restored through compensatory mechanisms [5].
• To be useful against AD GSK-3β inhibitors must be lipophilic enough to cross the blood-brain
barrier and hydrophilic enough to be administered orally [5].

7. Tideglusib mechanism
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Figure 5 (Copied from [6]). Showing that tideglusib is an
irreversible inhibitor of GSK-3β as GSK-3β activity is not
restored after the removal of the unbound drug. Shaded bars
show GSK-3β activity before the removal of the unbound
compounds; clear bars show GSK-3β activity after removal of
bound drug by two filtration-dilution cycles. Ctrl = control,
untreated GSK-3β; Tid = GSK-3β treated with 1μM tideglusib
GSK-3β treated with 1μM; SB = GSK-3β treated with SB-415286
(known reversible inhibitor). The very low dissociation
constant (not significantly different from zero) also indicates
that tideglusib is an irreversible inhibitor of GSK-3β [6].
Most GSK-3β inhibitors are ATP-competitive however tideglusib is non-ATP competitive irreversible
inhibitor of GSK-3β. Advantages of this are [5];
• May be more potent than ATP-competitive inhibitors as not competing with endogenous ATP
• May be more selective for specific kinase as binding outside the conserved ATP binding site
• Should have lower IC50 therefore have lower effective dose
• Irreversible inhibitors are less susceptible to drug resistance [6].

8. Proposed binding of tideglusib to GSK-3β
It is proposed that TDZDs, including tideglusib, bind to the primed phosphate substrate binding site
of GSK-3β with its main binding interactions with the amino acids Arg 96, Lys 205 and Tyr 216 [7].
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Hydrogen bond with Arg 96
Hydrogen bond with Lys 205
Pi-pi stacking interaction
with Tyr 216
Figure 6. Left: TDZD showing the 1,3-dicarbonyl linked by a nitrogen atom (highlighted in yellow) in a
pentagonal ring that is important for the inhibition of GSK-3β [5]. Right: tideglusib showing the proposed main
interactions with GSK-3β [7].

9. Tideglusib in animal models of AD
• Tideglusib was effective in treatment of animal models of AD leading to decreased tau
phosphorylation, decreased amyloid deposits, decreased plaque-associated astrocyte
proliferation and improved spatial memory [6].
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Clinical Trials and the future of GSK-3β inhibitors in the treatment of
AD
• Tideglusib has undergone 3 clinical trials, 2 for the treatment of AD where its safety and efficacy
were assessed [8].
• In an initial phase 2 clinical trial tideglusib was administered orally to 30 mild-moderate AD
patients to assess its safety and tolerability. It was well tolerated and showed positive effects in
cognitive tests; however these effects were not statistically significant [1].
• In a larger phase 2b clinical trial tideglusib was administered orally to over 300 mild-moderate AD
patients to assess its efficacy compared to placebo. It did not meet the primary and two
secondary endpoints; therefore suggesting that it was not clinically beneficial [1].

10. Conclusions
• Tau pathology is a key feature of AD and other neurodegenerative diseases.
• Tau is essential for microtubule function and its hyperphosphorylation leads to impaired
microtubule function.
• GSK-3β is one of many enzymes involved in tau phosphorylation and particularly linked to aberrant
phosphorylation seen in AD.
• GSK-3β inhibitors can reduce tau phosphorylation and are therefore of interest in the treatment of
AD.
• Tideglusib is a non-ATP competitive, irreversible GSK-3β inhibitor which has shown promising
results in animal models of AD.
• In clinical trials tideglusib was well tolerated however did not meet required endpoints.
• Further research into GSK-3β inhibitors is required.
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11. References
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http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3256883&tool=pmcentrez&rendertype=abstract.
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8. Del Ser T, Steinwachs KC, Gertz HJ, et al. Treatment of Alzheimer’s disease with the GSK-3 inhibitor tideglusib: a pilot study. J. Alzheimers. Dis.
[Internet]. 33(1), 205–15 (2013). Available from: http://www.ncbi.nlm.nih.gov/pubmed/22936007.
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