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

Glycogen synthase kinase 3 beta (GSK-3 beta) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer's disease and cancer. Even though GSK-3 beta 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-3 beta complete inhibition which translates into the impairment of the plethora of pathways GSK-3 beta is involved in. Starting from a 1D F-19 NMR fragment screening, we set up several biophysical assays for the identification of GSK-3 beta 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-3 beta in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3 beta, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3 beta inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3 beta activity without leading to its complete inhibition.