Gsk3BetaUseCase

= Glycogen Synthase Kinase 3 beta =

STORY

Glycogen synthase kinase 3 beta (GSK3β) has been implicated as a key player in several diseases: diabetes type 2, Alzheimer's, Huntington's, and possibly depression. Based on these relations, its potential market value as a possible drug target is obviously quite significant. From a patent perspective, GSK3β could offer novel drug classes for unique therapies in several disease areas where there are serious unmet medical needs; in the case of non-insulin dependent diabetes (NIDDM), a major problem of current treatment is insulin desensitization, whereby simply treating with insulin injections has decreasing effect over time. Current compounds besides insulin that are used to treat diabetes, such as Metformin and PPAR agonists have many unwelcome side-effects. It offers novel therapeutic strategies for CNS related diseases as well: Glycogen synthase kinase 3: a drug target for CNS therapies.

GSK3β (Mus musculus, Homo sapiens) is a protein kinase that works by further phosphorylating proteins already containing primed phosphorylation at serine sites (Homology modeling and molecular dynamics study of GSK3/SHAGGY-like kinase). Often there are several sites available on a single protein, each requiring the preceding site to be already primed. The resulting “phosphorylation cascade” enables GSK3β to reliably and quickly modify many sites on a protein target when conditions are met: one being the initial protein phosphorylation priming event, often performed by another specific kinases; and the other being the signal that regulates GSK3β’s own active state, via it own phosphorylation as well as the complex it is bound to (Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3.)

Unfortunately, not enough is known about what downstream targets are regulated by GSK3β to make it a well-characterized target for drug discovery (TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling.). GSK3β may have several roles in different processes that are tissue dependent (The glamour and gloom of glycogen synthase kinase-3,Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53), so future research is still required to better identify its therapeutic value as a drug target.

PATHWAYS

GSK3β works through several different pathway systems:

1)Insulin Receptor Signal: When activated by the insulin signal, via PIP3 and PKB, GSK3β phosphorylates primed substrates such as glycogen synthase, a key enzyme in glucose storage. Insulin Pathway Signaling-Gateway, STKE.

Insulin -> IR -> IRS1/2 -> PI3K -> PIP3 -> PDK1, PKB -| GSK3β -| GlycogenSynthase_Active -> Glycogen Synthesis

2)WNT Pathway: The WNT signal (through Disheveled and FRAT) releases beta-catenin from its phosphorylated complex with GSK3β (where it is typically degraded via proteasomes after being ubiquinated), to then become dephosphorylated and pass through the nucleus where it turns on gene-specific transcription. Signaling-Gateway , STKE

WNT -> DVL/FRAT -> {GSK3β::Axin-P::APC-P::b-Catenin-P(degrd) -> GSK3β::FRAT::DVL , Axin(degrd), APC, β-Catenin } ->nuclear { β-Catenin::TCF}

2a) [Joanne Luciano]

I created a BioPAX OWL version of the Xenopus Wnt-b-Catenin Pathway and posted it to:

http://www.biopathways.org/semweb/RDF_WG/

There you'll find the Xenopus Wnt pathway in BioPAX OWL and in N3. The reference for this pathway is: STKE:http://stke.sciencemag.org/cgi/cm/stkecm;CMP_6031

NOTE: This pathway is a BioPAX hack because signal transduction pathways are not supported in BioPAX Level 1. They are planned for Level 3. Metabolic Pathways are supported in BioPAX level one, so I posted the BioPAX OWL and N3 versions of the glycolysis pathway from EcoCyc there too.

3)CNS Processes: In CNS tissues, GSK3 promotes amyloid Aβ formation by phosphorylating gamma-secretase complex, the last cleavage step for amyloid Aβ production. GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides. In addition, GSK3 promotes Tau filament formation by phosphorylating the Tau subunits. PS1 activates PI3K thus inhibiting GSK-3 activity and tau over-phosphorylation: effects of FAD mutations.

• GSK3 -> gamma-secretase complex-P -> {C99 -> Aβ }
• GSK3 -> Tau-P -> Tau filaments

NOTE: GSK3β is also involved in developmental and proto-oncogenic processes, and therefore has to be carefully assessed for any possible interactions that may complicate drug development.The significance of the Wnt pathway in the pathology of human cancers

COMPOUNDS

Several selective compounds have already been created and identified that function as GSK3β antagonists :

• CHIR99021, CHIR98014 – Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo.
• ARA014418 AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. [1]
• SB216763 Insulin and Wnt1 Pathways Cooperate to Induce Reserve Cell Activation in Differentiation and Myotube Hypertrophy.
• SB415286 Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element
• Azakenpaullone 1-Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3beta
• bis-7-indolylmaleimide Design, synthesis, and biological evaluation of novel 7-azaindolyl-heteroaryl-maleimides as potent and selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors
• More inhibitors can be found at theMerck Biosciences site

These inhibitors work on GSK3β by binding either competitively to the ATP-site (Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418) or non-competitively to the primer recognition site (a-4-Dibromoacetophenone, TDZD-8, OTDZT, 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone), thereby avoiding the typical non-selective problems associated with ATP-binding site antagonists In this regard, GSK3β is unique as a kinase target since new classes of drugs may be developed that avoid kinase ATP-site side effects, making them potentially quite selective. Some of the known inhibitors interact very little or not at all with CDK or PKA kinases. Interestingly, lithium also appears to inhibit via the priming site of GSK3β, and may explain some of the CNS related benefits from lithium-based treatments. Lithium can therefore be used in a selective cellular assay to confirm GSK3β specificity.

Evidence: The potential use of GSK3β inhibitors has already been noted in animal studies: Effects of a Novel Glycogen Synthase Kinase-3 Inhibitor on Insulin-Stimulated Glucose Metabolism in Zucker Diabetic Fatty (fa/fa) Rats

GSK3β involvement in CNS functions is also under investigation: Glycogen synthase kinase-3 and Axin function in a beta-catenin-independent pathway that regulates neurite outgrowth in neuroblastoma cells

ASSAYS

• in vitro:
  • GSK3β IC50 assay
  • knowledge-based virtual kinase screening.
  • kinase anti-panel development (CDKs, PKA)
  • lithium –based selectivity assays
  • Human liver cell lines HEPG2, HuH6
  • Human embryonic kidney cells HEK
  • Animal cell lines: CHO-APP, COS-7, murine nerve cells,
• in vivo:
  • C57BL/6J mice
  • ZDF rats (blood, liver, muscle)
  • Oral Glucose Tolerance Tests (OGTT)

GOALS

• Find compounds that have excellent selective GSK3β IC50, with low binding to CDK's and other PK's, good permeability, and no tox metabolite.
• Be sure to characterize mechanisms of action as well as possible for each of the disease areas with regard to GSK3β inhibition.
• Compile different tox-response signatures on lead compounds by applying toxicogenomics.

Also assess the activities across different therapeutic spaces, keeping in mind different NOAEL (no-observed-adverse-effect-level) and age group (20-70, 40+, 60+) dependencies for different disease areas, e.g., cancer vs. diabetes. This will have an impact on the allowable levels and variance of toxicity for later stages of drug development.

STRATEGY

Since more knowledge is required about GSK3β’s role in different processes and tissues, we need to not only evaluate compounds based on binding affinity (IC50) to GSK3β, but to develop a profiling strategy to assess as many facets of GSK3β druggability for each of the therapeutic areas. Without this knowledge, many avenues of drug development may end in irremediable adverse effects and low efficacy.

Initial studies with the current set of inhibitors should first be performed using animal models to assess tissue and gene-specific effects under a few disease related conditions. This will serve to better understand the full set of responses and effects of GSK3β targeting. This will require a thorough comparison of animal GSK3β effects to human, in order to validate the animal models(pharmacophylogenomics). A system to assemble (aggregate) mouse and human GSK3β facts via sources such as Entrez Gene, should be developed and used as an aggregator system to automatically match which features are constant between species and which may not be. In addition, pharmacogenetic information may also help identify new indications as well as segment the population effectively for trials.

• A glycogen synthase kinase 3-beta promoter gene single nucleotide polymorphism is associated with age at onset and response to total sleep deprivation in bipolar depression.
• Identification of sequence variants and analysis of the role of the glycogen synthase kinase 3 beta gene and promoter in late onset Alzheimer's disease.

Generate series of compounds based on literature-based inhibitors and newly constructed libraries. Focus on developing and enhancing high-throughput assays to measure non-competitive interactions with priming binding sites. Develop additional efficient assays for glycogen synthase phosphorylation, gluconeogenesis inhibition (PEPCK suppression), and β-catenin release/activation. Develop assays for optimization stages that pinpoint direct GSK3β effects as well as off-target side effects.

It will also be important to develop improved pathway models that can be augmented/adjusted based on inclusion of new scientific findings (agent-based aggregation). Use pathway models to deconvolute evidence from compound optimization assays, in order to classify interaction profile of each compound in lead series.

The strategy should also include multiple indication evaluations throughout the stages, in order to effectively decide which therapeutic branch to pursue further. Since GSK3β is implicated in developmental and proto-oncogenic pathways, it is critical to develop and perform assays that will help avoid any tumorigenic/oncogenic activities at the earliest points possible.