title: Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors.
abstract: The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006μM; Lower Limit of Quantification (LLOQ): 0.02μM; linearity r20.9951. The kinetic constants (KMand vmax) of the GSK-3β catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the Kidetermination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: De Simone
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Bologna, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Fiori
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Bologna, Italy; Center for Applied Biomedical Research (C.R.B.A.), S. Orsola-Malpighi Hospital, Via Massarenti 9, 40138 Bologna, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Naldi
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: D'Urzo
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Tumiatti
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Milelli
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy. Electronic address: vincenza.andrisano@unibo.it.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
mesh_terms: D000071679:Glycogen Synthase Kinase 3 beta / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D047428:Protein Kinase Inhibitors; D000071679:Glycogen Synthase Kinase 3 beta
keywords: ESI-QTOF; GSK-3β inhibitors characterization.; GSK-3β kinase assay; GSM phosphorylation