Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.
Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.
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基于自动 NMR 片段的筛选鉴定出 LARG/RhoA 复合物的新型界面阻断剂
DOI：
10.1371/journal.pone.0088098
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ruan K
中科院分区：
文献类型：
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作者：
Gao J;Ma R;Wang W;Wang N;Sasaki R;Snyderman D;Wu J;Ruan K
在上游鸟嘌呤交换因子的催化下，小GTP酶在无活性GDP形式和活化GTP形式之间循环。通过小分子调节这种过程已被证明是用于治疗干预以防止小GT3过度活化的富有成效的途径。在过去的十年中出现的基于片段的方法已经证明了其在发现针对这种新颖且具有挑战性的蛋白质-蛋白质相互作用的抑制剂方面的巨大潜力。关于核磁共振碎片筛选的具体步骤，目前还没有全面的报道，这限制了它的广泛应用。为了实现适用于许多目标的一致筛选，我们开发了一种高度自动化的方案，以尽可能覆盖NMR片段筛选的各个方面，包括构建小而多样的库，通过NMR测定水溶性，将具有相互分散性的化合物分组为混合物，以及基于配体的筛选光谱的自动化处理和可视化。我们举例说明了我们在单独的RhoA和小GTTR RhoA及其上游鸟嘌呤交换因子LARG的复合物中的简化筛选。从混合物中的初步筛选和对LARG/RhoA复合物的单个命中的二次筛选中确认了两个命中，而其中一个也从单独的RhoA筛选中鉴定。HSQC滴定的两个命中超过单独的RhoA和LARG，分别确定了一个化合物结合RhoA.GDP在0.11 mM的亲和力，并扰乱了在开关II区的RhoA的残基。该命中阻断了LARG/RhoA复合物的形成，这通过天然凝胶电泳以及在不存在和存在该化合物的情况下RhoA对15 N标记的LARG的滴定来验证。因此，它为我们提供了一个起点，以寻找更有效的抑制剂来抑制LARG催化的RhoA激活。
The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to 15N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.
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影响因子：
64.8
作者：
Renault, L;Guibert, B;Cherfils, J
通讯作者：
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