Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination
Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination
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DOI：
10.1073/pnas.0605224103
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发表时间：
2006-10-24
影响因子：
11.1
通讯作者：
Edwards, Aled M.
中科院分区：
文献类型：
--
作者：
Vedadi, Masoud;Niesen, Frank H.;Edwards, Aled M.
人类治疗靶点的3D结构使药物发现成为可能。然而，它们的纯化和结晶仍然是速率决定性的。在个别情况下，配体已被用于提高蛋白质纯化和结晶的成功率，但这种方法的广泛适用性是未知的。我们实施了两个筛选平台，基于荧光测定法或静态光散射法，以测量蛋白质热稳定性在与配体结合后的增加，而不需要监测酶活性。总共有221种来自人类和人类寄生虫的不同蛋白质针对两种小分子文库中的一种或两种进行了筛选。第一个文库包含不同的盐、pH条件和常见的小分子，适用于所有蛋白质。第二种包括对特别感兴趣的蛋白质家族特异的化合物（例如，蛋白激酶）。在20个案例中，包括9种独特的人类蛋白激酶，鉴定出一种小分子，它稳定了蛋白质并促进了结构确定。这些方法具有成本效益，可以在任何实验室实施，有望显著提高纯化和结晶人类蛋白质的成功率，并为这些蛋白质鉴定新的配体。
The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.