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

Abstract

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.

GSK-3β Allosteric Inhibition: A Dead End or a New Pharmacological Frontier?

“…A fragment-based approach, for example, has rarely been used to identify allosteric GSK-3β inhibitors [ 108 ]. Such an approach would allow a punctual and complete pocket occupation, which is more suitable for identifying critical areas within broad and shallow pockets.…”

African Natural Products for Multitarget-Based Treatment of Alzheimer’s Disease: An In Silico Study

“…Compounds 159 forms a fourth H-bond with Asp200 while also interacting with the catalytic amino acid Lys85 through bad interaction (Lys85, Glu97 are highly conserved catalytic residues that enhance ATP interactions) [53]. Again, all of the observed interactions suggest that compound 159 could be a potential GSK-3β inhibitor [48,54]. Compound 157 on the other hand (Figure 5b), acted as a H-bond acceptor as well as a donor with Val135 forming two H-bonds.…”