Direct determination of GSK-3β activity and inhibition by UHPLC-UV–vis diode arrays detector (DAD)

•A new and very fast liquid chromatographic analysis for the direct determination of GSK 3β activity and inhibition has been developed and validated.•One of the last generation of UHPLC hyphenated with UV has been employed.•Kinetic constants of the enzymatic reaction have been determined through the analysis of ADP as cofactor.•Inhibitors potency and mechanism of action have been determined depending on the binding site and competition with either substrate or ATP.•This method can be used for the very fast screening and characterization of GSK 3β inhibitors in Alzheimer’s disease drug discovery.

Altered GSK-3β activity can contribute to a number of pathological processes including Alzheimer’s disease (AD). Indeed, GSK-3β catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment.Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3β kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery.The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3β, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3β inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3β in an ATP competitive manner.This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide