High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay.
High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay.
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DOI：
10.1177/1087057108328761
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发表时间：
2009-02
影响因子：
--
通讯作者：
Neubig RR
中科院分区：
文献类型：
--
作者：
Evelyn CR;Ferng T;Rojas RJ;Larsen MJ;Sondek J;Neubig RR
鸟嘌呤核苷酸交换因子(GEF)刺激细胞因子、酪氨酸激酶、黏附、整合素和G蛋白偶联受体(GPCRs)上的细胞外刺激，刺激鸟嘌呤核苷酸交换和随后的Rho家族蛋白的激活。在Rho激活后，会发生几个下游事件，如形态和细胞骨架的变化、运动、生长、存活和基因转录。Rhogef白血病相关基因(LARG)是G蛋白信号同源结构域(RH)家族的一员，最初被发现是急性髓系白血病染色体易位的结果。使用一种新的基于BODIPY-德州红-GTPγS(BODIPY-TR-GTPγS)的新型荧光偏振鸟嘌呤核苷酸结合分析方法，我们对LARG刺激的RhoA核苷酸结合抑制剂进行了一万个化合物的高通量筛选。从高通量筛选中鉴定的5个化合物在非荧光放射性鸟嘌呤核苷酸结合实验中得到证实，该实验测量了LARG刺激的[35S]GTPγS与RhoA的结合，从而排除了非特异性荧光效应。5个化合物均选择性地抑制LARG刺激的RhoA[35S]GTPγ与S的结合，而对RhoA或Gαo[35S]GTPγS的结合几乎没有影响。因此，这五个化合物应该成为开发LARG介导的核苷酸交换小分子抑制剂作为药理工具和治疗药物的有希望的起点。此外，本文所述的荧光偏振鸟嘌呤核苷酸结合试验对于高通量筛选和一般生物学应用都是一种有用的方法。
Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
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影响因子：
4.8
作者：
Rossman, KL;Cheng, L;Sondek, J
通讯作者：
Sondek, J
影响因子：
2.9
作者：
McEwen, DP;Gee, KR;Neubig, RR
通讯作者：
Neubig, RR
影响因子：
56.9
作者：
Lutz, Susanne;Shankaranarayanan, Aruna;Tesmer, John J. G.
通讯作者：
Tesmer, John J. G.
影响因子：
6.5
作者：
Rujkijyanont, Piya;Beyene, Joseph;Dror, Yigal
通讯作者：
Dror, Yigal
影响因子：
4.8
作者：
Peterson, YK;Kelly, P;Casey, PJ
通讯作者：
Casey, PJ