Binding affinities on all scales from fragment screening to intrinsically disordered proteins with isothermal spectral shift technology

生物分子相互作用的精确表征，它们的结合常数和化学计量，是理解细胞和疾病事件的核心。此外，新药的发现和开发通常需要筛选含有数百种（如果不是数千种）化合物的化学文库。在发现少量命中化合物后，通过精确了解它们与其生物靶标的结合，极大地帮助了对有前景的先导化合物的优化。在许多不同的筛选技术中，精确测量结合强度（结合常数Kd）和复合物组成（化学计量）的生物物理技术通常被认为是金标准。然而，许多治疗目标是具有挑战性的，有些仍然完全在当前生物物理技术的范围内。这些包括许多不能以所需量或浓度产生的膜蛋白。NanoTemper Technologies的Dianthus基于一种新的光谱偏移技术，代表了灵敏度的巨大飞跃，能够在非常小的体积（小于5微升）和非常低的浓度（纳摩尔）下精确测量结合常数。因此，分析最具挑战性的蛋白质目标，如经常需要洗涤剂的膜蛋白和即使在中等浓度下也倾向于沉淀的内在无序蛋白质，变得可行。此外，将该技术与高通量方法相结合，可以在不到一小时的时间内并行测量384个威尔斯孔，从而可以筛选具有数千种化合物的整个化学文库。重要的是，石竹系统将成为达勒姆大学生物物理科学研究所生物物理筛选平台的核心部分，该平台包括各种补充仪器。结合技术和科学专业知识，该平台为学术和工业用户社区提供了一套独特的仪器。

The precise characterisation of biomolecular interactions, their binding constants and stoichiometries, lie at the heart of understanding events in cells and disease. Furthermore, the discovery and development of new drugs often requires the screening of chemical libraries containing hundreds if not many thousands of chemical compounds. After a small number of hit compounds are discovered, the optimisation towards a promising lead compound is greatly aided by a precise knowledge of their binding to their biological targets. Among the many different screening technologies, biophysical techniques that precisely measure the strength of the binding (binding constant Kd) and the composition of the complex (stoichiometry) are often considered the gold standard. However, many therapeutic targets are challenging and some are still entirely within the reach of current biophysical techniques. These include many membrane proteins that cannot be produced at the required amount or concentration. The Dianthus by NanoTemper Technologies, based on a novel spectral shift technology, represents a huge leap in sensitivity which enables the precise measurement of binding constants in very small volumes (less than five microliter) and at very low (nanomolar) concentrations. Hence, analyses of the most challenging protein targets, such as membrane proteins that often require detergents and intrinsically disordered proteins that tend to precipitate even at moderate concentrations, become feasible. Furthermore, combining this technology with high-throughput methods allows the measurements of 384 wells in parallel in less than one hour, which allows the screening of entire chemical libraries with thousands of compounds. Importantly, the Dianthus system will be the centre piece of the biophysical screening platform at the Biophysical Sciences Institute at Durham University which includes a wide range of complementary instrumentation. Taken together with the technical and scientific expertise, the platform offers a unique set of instruments to the academic and industrial user community.