A Radioactive-free Kinase Inhibitor Discovery Assay Against the Glycogen Synthase Kinase-3 short (GSK-3s).

The identification of small molecules possessing inhibitory activity , against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the 's Glycogen Synthase Kinase-3 (GSK-3s). GSK-3s represents a potential drug target as it is essential for parasite survival. Small molecules used in our study are indirubin analogues possessing substitutions in different positions in the bis-indole backbone. Presently, the standard laboratory practice for the kinase assays is the incorporation of radiolabeled phosphate from [gamma-P]ATP as the efforts for developing non-radioactive assays (ELISA-based assays, fluorescence quenching assays, ) exhibit limitations such as lack in sensitivity or limitations for broad applications. This protocol can be a useful starting point for lead discovery, as it surpasses the drawbacks of radioactive kinase assays and it allows for relatively sensitive measurements of kinase inhibition for GSK-3s.