Cell-based assay for the discovery of glycogen synthase kinase inhibitors

This cell-based assay is applicable to diverse biological systems for discovery of glycogen synthase kinase inhibitors. The inhibition of GSK-3b has dramatic morphogenic effects during the early development of numerous organisms, including humans, Dictyostelium, sea urchins, zebrafish, and Xenopus. In each case, components of the assay mixture (described below) are provided exogenously to a cell by microinjection and scored for their respective phenotypes.

The invention is a simple and efficient cell-based assay that allows for quick and easy determination of novel GSK-3b inhibitors. In brief, a mixture comprising GSK-3, a source of phosphate, GSK-3 substrate, and assay buffer in the presence or absence of the test compound is measured for the level of phosphorylation of a known GSK-3 substrate. A lower level of phosphorylation of the GSK-3 substrate in the presence of the test compound compared with the level of phosphorylation of the GSK-3 substrate in the absence of the test compound indicates that the test compound is a GSK-3 inhibitor.

The system utilizes diverse biological readouts in order to monitor a test compounds activity against GSK-3b. Those compounds exhibiting the expected phenotype are then tested in an in vitro assay for their ability to inhibit GSK-3b phosphorylation on a known substrate. The cell lysates from these assays are readily tested and quantified using any GSK-3b immunoassay kits.


A next generation method has been invented for the discovery of lead compounds in the treatment of GSK-3b-related disorders, such as Alzheimer's disease.

Lithium exhibits numerous physiological effects in animals. For example, lithium mimics insulin action by stimulating glycogen synthesis and is remarkably effective for the treatment of mania in many human patients. Research over the years has demonstrated that lithium administers these effects, in part, through the inhibition of glycogen synthase kinase 3 beta (GSK-3b), an enzyme involved in the regulation of glycogen synthesis and cell fate determination in diverse organisms such as humans, Dictyostelium, Drosophila melanogaster, and Xenopus laevis. But lithium treatment in humans is also accompanied by several serious side effects, such as tremors, renal dysfunction, thyroid abnormalities, and birth defects. Additionally, lithium is not administered to patients having significant renal or cardiovascular disease, severe debilitation or dehydration, and sodium depletion due to a risk of disease exasperations. Given these problems, it is imperative to discover other drugs that can inhibit GSK3 in the same manner as lithium but without the toxic side effects.

Intellectual Property Status: Patent(s) Pending