Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Int J Mol Sci. 2022 Mar 31;23(7):3856. doi: 10.3390/ijms23073856.


Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer's disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition.

Keywords: Alzheimer’s disease; FBDD; NMR; cancer; drug discovery.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Alzheimer Disease* / metabolism
  • Binding Sites
  • Glycogen Synthase Kinase 3 beta / metabolism
  • Humans
  • Phosphorylation


  • Adenosine Triphosphate
  • Glycogen Synthase Kinase 3 beta