Identification of specific inhibitors of human RAD51 recombinase using high-throughput screening.
Identification of specific inhibitors of human RAD51 recombinase using high-throughput screening.
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DOI：
10.1021/cb100428c
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发表时间：
2011-06-17
影响因子：
4
通讯作者：
Mazin, Alexander V.
中科院分区：
文献类型：
--
作者：
Huang, Fei;Motlekar, Nuzhat A.;Burgwin, Chelsea M.;Napper, Andrew D.;Diamond, Scott L.;Mazin, Alexander V.
RAD51是同源重组的关键蛋白，在DNA双链断裂（DSB）和链间交联（ICL）修复中起关键作用。为了更好地了解人类RAD51的细胞功能，我们建议开发特异性的RAD51抑制剂。RAD51抑制剂也可能有助于提高通过诱导dsb或icl起作用的抗癌药物的效力，例如顺铂或电离辐射。在体外，RAD51促进同源的ss-和dsDNA之间的DNA链交换。在这里，我们开发了一种基于荧光共振能量转移的DNA链交换试验，并利用该试验通过高通量筛选NIH小分子库（bbb20万个化合物）来鉴定RAD51抑制剂。鉴定了17种RAD51抑制剂，并使用额外的非荧光dna检测分析其选择性。结果，我们鉴定出一种化合物（B02）特异性抑制人类RAD51 (IC50 = 27.4 μM)，但不抑制其大肠杆菌同源物RecA （IC50 = 250 μM）。另外两种化合物（A03和A10）对RAD51和RecA均有抑制作用，但对结构无关的RAD54蛋白没有抑制作用。结构-活性关系（SAR）分析使我们能够确定B02的结构成分，这些结构成分对RAD51抑制至关重要。所描述的方法可用于鉴定在DNA修复中起重要作用的其他人类蛋白质的特异性抑制剂，例如RAD54或布鲁姆综合征解旋酶。
RAD51 is a key protein of homologous recombination that plays a critical role in the repair of DNA double-strand breaks (DSB) and interstrand cross links (ICL). To better understand the cellular function(s) of human RAD51, we propose to develop specific RAD51 inhibitors. RAD51 inhibitors may also help to increase the potency of anticancer drugs that act by inducing DSBs or ICLs, e.g., cisplatin or ionizing radiation. In vitro, RAD51 promotes DNA strand exchange between homologous ss- and dsDNA. Here, we developed a DNA strand exchange assay based on fluorescence resonance energy transfer and used this assay to identify RAD51 inhibitors by high throughput screening of the NIH Small Molecule Repository (>200,000 compounds). Seventeen RAD51 inhibitors were identified and analyzed for selectivity using additional non-fluorescent DNA-based assays. As a result, we identified a compound (B02) that specifically inhibited human RAD51 (IC50 = 27.4 μM), but not its E. coli homologue RecA (IC50 > 250 μM). Two other compounds (A03 and A10) were identified that inhibited both RAD51 and RecA, but not the structurally unrelated RAD54 protein. The structure-activity relationship (SAR) analysis allowed us to identify the structural components of B02 that are critical for RAD51 inhibition. The described approach can be used for identification of specific inhibitors of other human proteins that play an important role in DNA repair, e.g., RAD54 or Bloom’s syndrome helicase.
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影响因子：
4.8
作者：
Mazina, Olga M.;Rossi, Matthew J.;Mazin, Alexander V.
通讯作者：
Mazin, Alexander V.
影响因子：
3.9
作者：
Brendel, V;Brocchieri, L;Karlin, S
通讯作者：
Karlin, S
影响因子：
11.4
作者：
Mazin, AV;Zaitseva, E;Kowalczykowski, SC
通讯作者：
Kowalczykowski, SC
影响因子：
4.8
作者：
Mazina, OM;Mazin, AV
通讯作者：
Mazin, AV
影响因子：
56.9
作者：
OGAWA, T;YU, X;EGELMAN, EH
通讯作者：
EGELMAN, EH