Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization
Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization
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DOI：
10.1016/j.ab.2004.05.055
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发表时间：
2004-09-15
影响因子：
2.9
通讯作者：
Wang, SM
中科院分区：
文献类型：
--
作者：
Nikolovska-Coleska, Z;Wang, RX;Wang, SM
X连锁凋亡抑制剂（XIAP）是一种有效的细胞凋亡抑制剂。设计针对 XIAP BIR3 结构域的小分子抑制剂，其中 Smac/DIABLO（第二个线粒体来源的 caspase 激活剂/低 pI 的直接 IAP 结合蛋白）和 caspase-9 结合，是抑制 XIAP 抗凋亡活性和克服 XIAP 介导的癌细胞凋亡抵抗的有前途的策略。在此，我们报告了基于荧光偏振的同质高通量测定的开发，用于测量小分子抑制剂与 XIAP BIR3 结构域的结合亲和力。在测试的四种荧光探针中，起始的 N 末端 Smac 肽 (AbuRPFK-(5-Fam)-NH2) 显示出最高的亲和力 (K-d = 17.92 nM) 和较大的动态范围 (DeltamP = 231 +/- 0.9)，并被选为最适合结合测定的探针。已研究了结合条件（DMSO 耐受性和稳定性）。在优化条件下，96 孔板的 Z' 因子达到 0.88，用于高通量筛选。结果发现，在基于 FP 的竞争性结合测定条件下，流行的 Cheng-Prusoff 方程对于计算抑制剂的竞争性抑制常数（K-i 值）无效。因此，开发、验证了一个新的数学方程，并用于计算 Ki 值。为此任务还开发了一个相关的基于网络的计算机程序。在测定条件下对几种已知的高亲和力和低亲和力的Smac肽进行了评估，获得的结果表明基于FP的竞争性结合测定按照设计正确执行：它可以定量、准确地测定具有广泛亲和力的基于Smac的肽抑制剂的结合亲和力，并且适用于与XIAP BIR3结构域结合的抑制剂的高通量筛选。 (C) 2004 Elsevier Inc. 保留所有权利。
The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a initiated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH2) showed the highest affinity (K-d = 17.92 nM) and a large dynamic range (DeltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (K-i values) for inhibitors in the FP-based competitive binding assay conditions.. and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain. (C) 2004 Elsevier Inc. All rights reserved.