Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1


Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z? scores of 0.62 (fluorescein–Nec-1) and 0.57 (fluorescein–Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.



Necrostatin(Nec), Receptor, interactoin protein 1(RIP1), Kinase, Fluorescence polarization(FP), competition assays, high-throughput screening (HTS), Ligand docking


Copyright 2012 Analytical Biochemistry. This is a post-print version of a published paper that is available at: https://www.sciencedirect.com/science/article/pii/S0003269712002886. Recommended citation: Maki, Jenny L., Elizabeth E. Smith, Xin Teng, Soumya S. Ray, Gregory D. Cuny, and Alexei Degterev. "Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1." Analytical biochemistry 427, no. 2 (2012): 164-174. doi: 10.1016/j.ab.2012.05.019. This item has been deposited in accordance with publisher copyright and licensing and with the author's permission.