Xu Hua, Gopalsamy Ariamala, Hett Erik C, Salter Shores, Aulabaugh Ann, Kyne Robert E, Pierce Betsy, Jones Lyn H
Worldwide Medicinal Chemistry, Pfizer, 610 Main St., Cambridge, Massachusetts 02139, USA.
Org Biomol Chem. 2016 Jul 14;14(26):6179-83. doi: 10.1039/c6ob01078d. Epub 2016 May 24.
Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies, the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case, the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity.
在生理相关环境中证明药物与靶点的结合是成功进行治疗靶点验证的关键支柱。我们开发了两种正交技术，即细胞热迁移分析（CETSA）和共价化学探针报告方法（利用磺酰氟酪氨酸标记及后续的点击化学），以测量活细胞中小分子抑制剂对mRNA去帽清除酶DcpS的占有率。使用等温剂量反应指纹图谱（ITDRFCETSA）测定的酶亲和力与使用化学探针报告分析测定的占据50%酶所需的浓度（OC50）非常相似。在这种情况下，由于可逆抑制剂的长偏移动力学（使用荧光染料标记探针测定），化学探针方法效果良好。这项工作表明，由于其简单性，CETSA可能成为确定细胞内靶点结合的首选分析方法。
