Development of high-throughput assays to screen inhibitors of Tau phosphorylation
Alzheimer’s disease (AD) is a progressive cognitive deterioration of the brain preceded by years of developing pathologies, which include amyloid-beta plaques and neurofibrillary Tau tangles (NFTs). DYRK1A protein phosphorylates Tau, and it is highly expressed in post-mortem brains of AD patients. Hyper-phosphorylation of Tau (pTau) leads to clumping, build-up, and loss of the protein’s function. Our lab’s previous experiments showed that when the drug compound DYR219 inhibits DYRK1A, pTau is reduced, along with plaques and NFTs (in AD-modeled mice). Our experiment helped start the process of screening a newly developed inhibitor collection, related to the well-defined DYR219, to find even more effective versions. First, we measured DYR219 binding affinity to Dyrk1A with Promega’s nanoBRET assay, paired with a DYRK1A-Luciferase fusion protein. Next, we implemented a new cell culture model to measure Tau protein changes after over-expression of DYRK1A and treatment with DYR219. Our first experiment, the drug binding assay (nanoBRET), was informative and reliable; therefore, the lab will proceed with screening the other untested inhibitors. On the other hand, we had perplexing results from our HEK cell experiment compared to previous findings. We saw that pTau protein increased with higher concentrations of DYR219. The lab will revisit this assay to troubleshoot and test additional factors that may explain the rise in pTau under these specific conditions.