Abstract
The identification of small molecules possessing inhibitory activity in vitro, 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 Trypanosoma brucei's Glycogen Synthase Kinase-3 (TbGSK-3s). TbGSK-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-32P]ATP as the efforts for developing non-radioactive assays (ELISA-based assays, fluorescence quenching assays, etc.) 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 TbGSK-3s.