Efstathiou Antonia, Smirlis Despina
Molecular Parasitology Lab, Dpt of Microbiology, 127 Bas. Sofias Ave., Hellenic Pasteur Institute, 11521 Athens, Greece.
Bio Protoc. 2020 Jan 20;10(2):e3493. doi: 10.21769/BioProtoc.3493.
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.
鉴定对特定靶激酶具有抑制活性的小分子是药物发现过程的第一步。在此，我们描述了一种基于荧光素酶的ATP检测的非放射性方案，用于鉴定糖原合酶激酶-3（GSK-3s）短异构体的抑制剂。GSK-3s是寄生虫生存所必需的，代表了一个潜在的药物靶点。我们研究中使用的小分子是在双吲哚骨架不同位置具有取代基的靛玉红类似物。目前，激酶检测的标准实验室方法是掺入来自[γ-P]ATP的放射性标记磷酸盐，因为开发非放射性检测方法（基于ELISA的检测方法、荧光猝灭检测方法等）存在局限性，如灵敏度不足或广泛应用受限。该方案可以作为先导化合物发现的有用起点，因为它克服了放射性激酶检测的缺点，并且能够对GSK-3s的激酶抑制进行相对灵敏的测量。
