Charvat Annemarie F, Mason-Chalmers Kayleigh, Grabinska-Rogala Aneta, Shivakumar Shloka, Gale-Day Zachary, Wu Taiasean, Millbern Zoe, Grimm Jonathan B, Carroll Emma C, Nilsson K Peter R, Lavis Luke D, Vinueza Nelson R, Gestwicki Jason E
Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USA.
Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA.
SLAS Discov. 2025 Sep;35:100259. doi: 10.1016/j.slasd.2025.100259. Epub 2025 Aug 8.
Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30 %), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (e.g. small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94 %, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput.
差示扫描荧光法（DSF）是一种用于体外评估蛋白质稳定性的生物物理分析方法。在DSF实验中，利用诸如Sypro Orange等溶剂化显色染料荧光增强来检测蛋白质在加热过程中的解折叠。然而，Sypro Orange仅与少数蛋白质（<30%）兼容，限制了该方法的应用范围。我们最近报道蛋白质适应性DSF（paDSF）可部分解决此问题，其中先针对约300种化学性质各异的染料（称为Aurora文库）对蛋白质进行预筛选。虽然这种方法显著增加了适合DSF分析的靶标数量，但对于某些蛋白质仍无法产生蛋白质 - 染料对。在此，我们报道将染料文库扩展至Aurora 2.0，其总共包含517种结构各异的分子和多种新化学类型。为评估性能，针对一组约100种蛋白质对这些染料进行筛选，部分蛋白质被挑选出来以代表最具挑战性的靶标（例如小尺寸蛋白质）。通过这项工作，Aurora 2.0取得了高达94%的成功率，包括为原始文库中未匹配的一些靶标产生了染料。这些发现支持了这样一种观点，即更大、化学性质更多样的文库能提高在更广泛蛋白质范围内检测熔解转变的可能性。我们认为Aurora 2.0使paDSF成为一种在高通量研究蛋白质稳定性、配体结合及其他生物物理性质方面日益强大的方法。
