Applications of Biophysics in High-Throughput Screening Hit Validation
Applications of Biophysics in High-Throughput Screening Hit Validation
复制标题
DOI：
10.1177/1087057114529462
复制
发表时间：
2014-06-01
影响因子：
--
通讯作者：
Ottl, Johannes
中科院分区：
文献类型：
--
作者：
Genick, Christine Clougherty;Barlier, Danielle;Ottl, Johannes
近十年来，生物物理方法已被用于验证从高通量筛选（HTS）活动中选择的阳性命中，目的是使用无标记测定法验证结合相互作用。通过应用无标记读数，发现由复合干涉和荧光产生的屏幕伪影，从而进一步表征命中的目标特异性和选择性。需要使用几种生物物理方法来提取这种类型的高含量信息，以防止将假阳性提升到下一级命中验证，并选择最佳候选者进行进一步的化学优化。在这一领域应用的典型技术包括动态光散射、浊度测定、共振波导、表面等离子体共振、差示扫描荧光法、质谱法等。每种技术都可以提供不同类型的信息，以实现绑定相互作用的表征。因此，这些技术不仅可以根据药物化学家所期望的化学物质的概况，而且可以以与目标蛋白质对筛选格式的适应性一致的方式纳入命中验证策略。在这里，我们介绍了生物物理学筛选策略的结果，目的是评估这些方法，讨论优势和挑战，并总结参考先导物质发现的好处。总之，这里展示的生物物理筛选显示了不同的命中率，这些命中率来自于类似2000个预选的、经过IC50验证的HTS命中（IC50是观察到50%活性抑制的抑制剂浓度）。本文将讨论从这些生物物理筛选中获得的一些经验教训。
For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein's amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of similar to 2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article.