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

作者：D'Urzo Annalisa; De Simone Angela; Fiori Jessica; Naldi Marina; Milelli Andrea; Andrisano Vincenza^*

来源：Journal of Pharmaceutical and Biomedical Analysis, 2016, 124: 104-111.

DOI：10.1016/j.jpba.2016.02.029

• GSK-3 beta activity and inhibition
• Direct assay
• UHPLC method
• ATP and ADP separation
• Inhibitor mechanism of action

Altered GSK-3 beta activity can contribute to a number of pathological processes including Alzheimer's disease (AD). Indeed, GSK-3 beta 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-3 beta 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-3 beta, 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 (K-M and V-max) for GSM and ATP and by analyzing well known GSK-3 beta 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 (K-i). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3 beta 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.

• 出版日期2016-5-30