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.
Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, USA.
Jenny L
JL
Maki
Elizabeth E
EE
Smith
Xin
X
Teng
Soumya S
SS
Ray
Gregory D
GD
Cuny
Alexei
A
Degterev
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016367:Baculoviridae; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D019793:Fluorescein; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays; D006801:Humans; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology; D007211:Indoles / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009336:Necrosis / Q000517:prevention & control; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000031:analogs & derivatives* / Q000494:pharmacology; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera; D013194:Staining and Labeling
D007093:Imidazoles; D007211:Indoles; D008024:Ligands; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C507699:necrostatin-1; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D019793:Fluorescein