Background

Kinases are enzymes that play a crucial role in biological processes including differentiation, cell proliferation and apoptosis, by putting in motion signaling pathways upon catalyzing the transfer of the γ-phosphate from ATP to substrate (Jia et al., 2008; Efstathiou et al., 2019). Deregulation of kinases can frequently lead to a variety of diseases (Ways and Sheetz, 2000; Cohen and Goedert, 2004; Mazitschek and Giannis, 2004; Resnick and Fennell, 2004) and therefore, they are considered one of the largest classes of drug targets (Cohen 1999; Manning et al., 2002). The first step of the lead kinase inhibitor discovery is the establishment of an in vitro kinase assay. The radioactive assays are the standard laboratory practice due to their high sensitivity (Jia et al., 2008; Lilienthal et al., 2010). However, drawbacks of kinase-based radioactivity assays include the need of special handling and the restriction in flexibility because of the short half-life of ³²P. To resolve these limitations, new non-radioactive technologies have been created that are based on fluorescence or luminescence (Jia et al., 2008). Herein, we describe a non radioactive protocol using luciferase-based ATP assay, for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s Glycogen Synthase Kinase-3 (TbGSK-3s). In the bloodstream form of T. brucei, TbGSK-3s is essential for survival (Ojo et al., 2008) and therefore it is a molecular target for the discovery of new anti-trypanosomal agents (Ojo et al., 2008; Oduor et al., 2011; Woodland et al., 2013; Urich et al., 2014; Swinney et al., 2016). Mammalian GSK-3 has been related to a wide range of diseases and thus small molecular weight GSK-3 inhibitors has been developed (Woodland et al., 2013; Gaboriaud-Kola et al., 2015; Masch and Kunick, 2015). Amongst GSK-3 inhibitors, there are the indirubins, a family of natural bis-indole derivatives (Hoessel et al., 1999, Polychronopoulos et al., 2004, Vougogiannopoulou et al., 2008, Myrianthopoulos et al., 2013). In this protocol, a luminescent kinase assay based on the Kinase-Glo^® reagent of Promega, is described. This method is straightforward, radioactive-free, fast and it doesn’t lack sensitivity. While the protocol described below is specific for the recombinant TbGSK-3s expressed in baculovirus system as described before (Efstathiou et al., 2019), it can be applicable to any kinase with the appropriate alterations for the specific kinase (substrate, ATP concentration, buffer, etc.).


Figure 1. Scheme for the luminescent kinase assay based on the Kinase-Glo^® reagent. The kinase reaction is conducted under the appropriate conditions with or without inhibitors. The remaining ATP at the time that the reagent is added, is used as a substrate by the Kinase-Glo^® Luciferase to catalyze the mono-oxygenation of luciferin. The luciferase reaction produces one photon of light per turnover. Luminescence is inversely related to kinase activity (Promega).