Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, USA.
Anal Biochem. 2012 Aug 15;427(2):164-74. doi: 10.1016/j.ab.2012.05.019. Epub 2012 May 29.
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.
细胞坏死普遍存在于许多不同的病理疾病状态和创伤中。细胞坏死是一种源自特定信号通路的坏死形式，其关键调节因子是受体相互作用蛋白 1（RIP1），一种丝氨酸/苏氨酸激酶。RIP1 的特异性抑制剂，称为坏死抑制剂，是细胞坏死的有效抑制剂。坏死抑制剂在结构上彼此不同，但仍具有抑制 RIP1 激酶活性的能力。为了进一步了解各种坏死抑制剂与 RIP1 的结合差异，并开发一种强大的高通量筛选（HTS）测定法，该测定法可用于鉴定新类别的 RIP1 抑制剂，我们合成了 Necrostatin-1（ Nec-1）和 Nec-3 的荧光素衍生物。这些化合物用于建立荧光偏振（FP）测定法，以直接测量坏死抑制剂与 RIP1 激酶的结合。荧光素标记的化合物非常适合 HTS，因为测定法在二甲基亚砜（DMSO）中的容忍度高达 5%，并且 Z'分数为 0.62（荧光素-Nec-1）和 0.57（荧光素-Nec-3）。此外，FP 测定法和配体对接研究的结果提供了对 Nec-1、Nec-3 和 Nec-4 假定结合位点的深入了解。
