Biophysical screening for the discovery of small-molecule ligands.
Biophysical screening for the discovery of small-molecule ligands.
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DOI：
10.1007/978-1-62703-398-5_13
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Ciulli, Alessio
中科院分区：
文献类型：
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作者：
Ciulli, Alessio
发现蛋白质功能的小分子化学探针具有很大的潜力，可以阐明生物学途径，并为目标验证提供早期概念验证。因此，这种探针的发现支持了学术界和制药业中的许多化学生物学和药物发现工作。该过程通常从筛选小分子开始，以识别真正的“命中”，非共价结合到靶蛋白。本章关注的是应用生物物理和结构技术的小分子配体筛选，并与验证命中从结构（结合模式）和能量（结合亲和力）的立场。讨论的方法包括差示扫描荧光法（热位移），荧光偏振（FP），表面等离子体共振，配体观察NMR光谱，等温滴定量热法，和蛋白质X射线晶体学。这些技术的原理和用于检测大分子-配体结合的观测量的基本性质进行了简要概述。每种技术的实用性，优点和缺点进行了描述，特别是在检测弱亲和力的背景下，相关的片段筛选。详细讨论了基于双折射的方法，该方法提供了高通量和低成本的有吸引力的组合。有人认为，应用不同方法的组合提供了最强大和有效的方法来确定后续药物化学的高质量起点，并建立结构-活性关系，更好地通知高质量的有效开发，细胞活性化学探针通过基于结构的药物设计。
Discovering small-molecule chemical probes of protein function has great potential to elucidate biological pathways and to provide early-stage proof-of-concept for target validation. Discovery of such probes therefore underpins many of the chemical biology and drug discovery efforts in both academia and the pharmaceutical industry. The process generally begins with screening small molecules to identify bona fide “hits” that bind non-covalently to a target protein. This chapter is concerned with the application of biophysical and structural techniques to small-molecule ligand screening, and with the validation of hits from both structural (binding mode) and energetic (binding affinity) stand-points. The methods discussed include differential scanning fluorimetry (thermal shift), fluorescence polarization (FP), surface plasmon resonance, ligand-observed NMR spectroscopy, isothermal titration calorimetry, and protein X-ray crystallography. The principles of these techniques and the fundamental nature of the observables used to detect macromolecule-ligand binding are briefly outlined. The practicalities, advantages, and disadvantages of each technique are described, particularly in the context of detecting weak affinities, as relevant to fragment screening. Fluorescence-based methods, which offer an attractive combination of high throughput and low cost are discussed in detail. It is argued that applying a combination of different methods provides the most robust and effective way to identify high-quality starting points for follow-up medicinal chemistry and to build structure–activity relationships that better inform effective development of high-quality, cell-active chemical probes by structure-based drug design.