The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway.
The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway.
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MALDI-TOF E2/E3连接酶分析是泛素途径中药物发现的通用工具。
DOI：
10.1016/j.chembiol.2018.06.004
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发表时间：
2018-09-20
影响因子：
8.6
通讯作者：
Trost M
中科院分区：
文献类型：
--
作者：
De Cesare V;Johnson C;Barlow V;Hastie J;Knebel A;Trost M
由于其在许多疾病中的作用，泛素系统的酶最近已成为有趣的药物靶点。尽管做出了努力，但针对E2酶和E3连接酶的化合物文库的初步筛选一直受到缺乏可靠和快速的高通量分析的强烈限制。在这里，我们报告了一种基于MALDI-TOF质谱学的无标记高通量筛选泛素E2结合酶和E3连接酶的方法。MALDI-TOF E2/E3试验允许测试E2酶和E3连接酶的泛素转移活性，鉴定E2/E3活性对、抑制剂效力和特异性，以及在没有化学或荧光探针的情况下在体外筛选化合物文库。我们证明，MALDI-TOF E2/E3试验是泛素途径药物发现筛选的通用工具，因为它适用于所有E3连接酶家族，并且与标准生化分析相比，需要更少的试剂。我们开发了一种针对E2/E3酶的高通量MALDI-TOF方法它允许在没有化学或荧光探针的情况下筛选化合物文库我们测试了三种与疾病相关的E3连接酶：MDM2、ITCH和HOIP我们对1,430种化合物进行了概念验证高通量筛选由于它们在许多疾病中的作用，因此需要在稳健和高通量分析中确定泛素系统的酶的候选药物。在这里，我们报告了基于MALDI-TOF质谱学的泛素E2结合酶和E3连接酶的无标记高通量筛选方法。
Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays. We have developed a high-throughput MALDI-TOF assay for E2/E3 enzymes It allows screening compound libraries without chemical or fluorescent probes We tested the screen on three disease-relevant E3 ligases: MDM2, ITCH, and HOIP We performed a proof-of-concept high-throughput screen against 1,430 compounds Due to their role in many diseases, there is a need to identify drug candidates for enzymes of the ubiquitin system in robust and high-throughput assays. Here we report as label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry.
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DOI：
10.1177/2472555217717473
发表时间：
2017-12
期刊：
SLAS discovery : advancing life sciences R & D
影响因子：
--
作者：
Heap RE;Hope AG;Pearson LA;Reyskens KMSE;McElroy SP;Hastie CJ;Porter DW;Arthur JSC;Gray DW;Trost M
通讯作者：
Trost M
MDM2/X 靶向治疗的临床概述。
影响因子：
4.7
作者：
Burgess A;Chia KM;Haupt S;Thomas D;Haupt Y;Lim E
通讯作者：
Lim E
影响因子：
16
作者：
Brownell, James E.;Sintchak, Michael D.;Dick, Lawrence R.
通讯作者：
Dick, Lawrence R.
影响因子：
64.5
作者：
Cohen, Philip;Tcherpakov, Marianna
通讯作者：
Tcherpakov, Marianna
影响因子：
64.8
作者：
Ikeda, Fumiyo;Deribe, Yonathan Lissanu;Skanland, Sigrid S.;Stieglitz, Benjamin;Grabbe, Caroline;Franz-Wachtel, Mirita;van Wijk, Sjoerd J. L.;Goswami, Panchali;Nagy, Vanja;Terzic, Janos;Tokunaga, Fuminori;Androulidaki, Ariadne;Nakagawa, Tomoko;Pasparakis, Manolis;Iwai, Kazuhiro;Sundberg, John P.;Schaefer, Liliana;Rittinger, Katrin;Macek, Boris;Dikic, Ivan
通讯作者：
Dikic, Ivan