Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90β
Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90β
复制标题
DOI：
10.1177/1087057104265538
复制
发表时间：
2004-10-01
影响因子：
--
通讯作者：
Filipuzzi, I
中科院分区：
文献类型：
--
作者：
Schilb, A;Riou, V;Filipuzzi, I
热休克蛋白90（Hsp 90 β）的β同种型是参与对细胞外因子的生长反应以及各种细胞类型的致癌转化的关键蛋白质成熟所需的细胞伴侣。因此，抑制Hsp 90 β功能的化合物被认为具有作为新型抗癌药物的潜力。迄今为止，已知2种真菌代谢物抑制Hsp 90 β。然而，不溶性和肝毒性限制了这些分子的临床应用。鉴定新的和安全的Hsp 90 β抑制剂的限制是目前没有合适的高通量筛选测定法可用。在这里，作者提出了一种均相测定的基础上的二维荧光强度分布分析的四甲基罗丹明（塔姆拉）标记的根赤霉素结合热休克蛋白90 β的发展。此外，该测定已被证明与Evotec-Technologies的共聚焦纳米筛选平台Mark II（TM）兼容，因此可用于小型化高通量筛选。所应用的检测技术提供了关于热力学平衡下生物分子相互作用的性质的关键信息，例如结合的亲和常数和化学计量参数。该测定用于鉴定取代TAMRA-根赤霉素的小分子量化合物。这类化合物被认为是发现新型抗癌药物的重要分子。
The beta isoform of the heat shock protein 90 (Hsp90beta) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90beta are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90beta. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90beta inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90beta. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II(TM) from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.