Ronzetti Michael H, Baljinnyam Bolormaa, Itkin Zina, Jain Sankalp, Rai Ganesha, Zakharov Alexey V, Pal Utpal, Simeonov Anton
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, United States.
Department of Veterinary Medicine, College of Agriculture and Natural Resources, University of Maryland, College Park, MD, United States.
Front Pharmacol. 2022 Nov 24;13:1040039. doi: 10.3389/fphar.2022.1040039. eCollection 2022.
Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of T values with an average coefficient of variation of 0.51% and median coefficient of variation of compound T of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.
差示扫描荧光法是一种快速且经济的生物物理技术，用于监测热梯度过程中蛋白质结构的扰动，通常是通过环境敏感荧光团检测蛋白质的解折叠事件。通过使用对组氨酸标签蛋白附近结构变化敏感的NTA复合荧光团，建立了一种具有基于亲和标签系统特异性的强大且灵敏的差示扫描荧光法（DSF）检测方法。我们以伯氏高温需求A蛋白酶（BbHtrA）作为工作流程的概念验证，将这种组氨酸标签DSF检测方法（HIS-DSF）开发、优化并小型化为一个1536孔高通量生物物理平台。BbHtrA HIS-DSF检测方法的一次生产运行显示，阴性对照组T值分布紧密，平均变异系数为0.51%，化合物T的中位变异系数为0.26%。HIS-DSF平台将为未来的药物发现活动提供一个额外的检测平台，可应用于缓冲液筛选和优化、靶点结合筛选以及其他生物物理检测工作。
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