Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity.
Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity.
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DOI：
10.1093/nar/gkq1082
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发表时间：
2011-01
影响因子：
14.9
通讯作者：
Simeonov A
中科院分区：
文献类型：
--
作者：
Dorjsuren D;Kim D;Maloney DJ;Wilson DM 3rd;Simeonov A
FEN 1是DNA复制和修复的关键参与者，是识别和切割皮瓣DNA结构的主要人皮瓣核酸内切酶。FEN 1功能缺陷或fen 1基因缺失具有深远的生物学效应，包括抑制由各种遗传毒性剂作用引起的DNA损伤的修复。鉴于FEN 1在解析异常DNA结构中的重要性，该酶的抑制剂具有作为DNA相互作用抗癌药物的增强剂的潜力。为了促进FEN 1活性的研究和寻找新的抑制剂，我们开发了一对互补读出均相检测，利用荧光供体/淬灭剂和AlphaScreen化学发光策略。先前报道的FEN 1抑制剂3-羟基-5-甲基-1-苯基噻吩并[2，3-d]嘧啶-2，4（1H，3 H）-二酮在新试验中显示出相同的效力，与其公布的IC 50一致。该试验被优化为低4 μl体积，并用于研究一组小分子，从而鉴定出先前未报道的FEN 1抑制剂，其中金精三羧酸和NSC-13755（一种芳基锑衍生物）显示出亚微摩尔效力（平均IC 50分别为0.59和0.93 μM）。这些简单的互补测定的可用性消除了对不期望的基于放射性示踪剂的测定的需要，并且应该促进开发针对该关键生物靶点的新型抑制剂的努力。
FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC50. The assays were optimized to a low 4 µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC50 of 0.59 and 0.93 µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target.
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DOI：
10.1016/0006-291x(73)91198-4
发表时间：
1973-01-01
影响因子：
3.1
作者：
BLUMENTHAL, T;LANDERS, TA
通讯作者：
LANDERS, TA
影响因子：
4.8
作者：
Kao, KI;Henricksen, LA;Bambara, RA
通讯作者：
Bambara, RA
影响因子：
64.5
作者：
Chapados, BR;Hosfield, DJ;Tainer, JA
通讯作者：
Tainer, JA
影响因子：
4.8
作者：
HARRINGTON, JJ;LIEBER, MR
通讯作者：
LIEBER, MR
影响因子：
4.8
作者：
Kaiser, MW;Lyamicheva, N;Lyamichev, VI
通讯作者：
Lyamichev, VI