Dart Melanie L, Machleidt Thomas, Jost Emily, Schwinn Marie K, Robers Matthew B, Shi Ce, Kirkland Thomas A, Killoran Michael P, Wilkinson Jennifer M, Hartnett James R, Zimmerman Kristopher, Wood Keith V
Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States.
Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States.
ACS Med Chem Lett. 2018 Apr 16;9(6):546-551. doi: 10.1021/acsmedchemlett.8b00081. eCollection 2018 Jun 14.
Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.
蛋白质热迁移分析（TSA）提供了一种通过配体诱导的热稳定性来表征靶点结合的方法。尽管这些分析在药物发现项目中被广泛用于筛选文库和验证命中化合物，但它们可能会带来繁琐的操作要求，例如需要纯化的蛋白质或选择性抗体。将目标蛋白与小型荧光素酶（NanoLuc）连接，可使热变性与发光输出相耦合，为评估在诸如透化细胞等成分复杂的环境中的靶点结合提供了一种快速且灵敏的方法。NanoLuc的固有热稳定性高于哺乳动物蛋白，我们的结果表明，连接的荧光素酶不会改变目标蛋白的热变性。我们已成功将NanoLuc荧光素酶热迁移分析（NaLTSA）应用于几个临床相关的蛋白质家族，包括激酶、溴结构域和组蛋白脱乙酰酶。我们还证明了这种分析方法适用于文库筛选和化合物分析。
