Insights into PARP Inhibitors’ Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays
Insights into PARP Inhibitors’ Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays
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使用荧光偏振和表面等离子共振结合测定深入了解 PARP 抑制剂的选择性
DOI：
10.1177/1087057114538319
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发表时间：
2014
影响因子：
--
通讯作者：
R. Lupi
中科院分区：
文献类型：
--
作者：
G. Papeo;N. Avanzi;Serena Bettoni;A. Leone;M. Paolucci;R. Perego;F. Quartieri;Federico Riccardi;Sandrine Thieffine;A. Montagnoli;R. Lupi
PARP 抑制剂是一类令人兴奋的新型抗肿瘤药物，已被证明作为单一药物治疗具有固有 DNA 修复缺陷的癌症，以及与 DNA 损伤性化疗药物联合使用均有效。目前，它们被设计针对 PARP-1 的催化结构域，PARP-1 是该家族中研究最多的成员，在 DNA 损伤修复过程中发挥着关键作用。由于 PARP 抑制剂是底物 (NAD+) 竞争者，因此需要更深入地了解它们的交叉反应性。这与 PARP-2（PARP-1 最接近的同源物）尤其相关，已在双敲除小鼠中观察到其胚胎致死表型。在这项研究中，我们描述了基于荧光偏振（FP）和表面等离子共振（SPR）技术的结合测定的开发和验证。 PARP-1、PARP-2、PARP-3 和 TNKS-1 FP 置换测定是通过使用特设合成探针进行的。这些测定适用于高通量筛选 (HTS) 和选择性分析，从而可以鉴定 NAD+ 结合位点选择性抑制剂。 PARP-1 和 PARP-2 互补 SPR 结合测定证实了位移数据和深入的抑制剂表征。此外，这些格式有可能广泛适用于 PARP 家族的其他成员。
PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD+) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD+ binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.
影响因子：
10.6
作者：
Curtin, Nicola J.;Szabo, Csaba
通讯作者：
Szabo, Csaba
影响因子：
2.9
作者：
Kuzmic, P
通讯作者：
Kuzmic, P