Department of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USA.
Senex Biotechnology, Inc., Columbia, SC 29208, USA.
Cells. 2019 Oct 6;8(10):1208. doi: 10.3390/cells8101208.
Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC values in the WT reporter assay showed near-perfect correlation (R = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition.
基于细胞的 CDK8/19 抑制作用测定方法不容易确定，因为目前还没有已知的细胞功能是这些激酶所特有的。为了解决这个问题，我们利用 CRISPR 敲除技术构建了一个或两个 CDK8 和 CDK19 敲除的 293 细胞系。同时敲除 CDK8 和 CDK19（而非单独敲除）降低了 NFκB（一种 CDK8/19 增强的转录因子）诱导的转录，并且消除了 CDK8/19 抑制剂对这种诱导的作用。我们构建了表达 NFκB 依赖性启动子的荧光素酶的野生型（WT）和双敲除（dKO）细胞系。选择性 CDK8/19 抑制剂而非其他 CDKs 抑制剂能使 TNFα 诱导的 WT 细胞中的荧光素酶表达降低约 80%，但对 dKO 细胞中的荧光素酶诱导没有影响。相比之下，非选择性 CDK 抑制剂 flavopiridol 和 dinaciclib 以及一种 CDK7/12/13 抑制剂 THZ1（而非 CDK4/6 抑制剂 palbociclib）抑制 WT 和 dKO 细胞中的荧光素酶诱导，表明其他 CDKs 在 NFκB 途径中发挥着独特的作用。我们利用该测定方法对一系列具有体外骨合成活性的噻吩吡啶类化合物进行了表征，其中一种被鉴定为选择性 CDK8/19 抑制剂。噻吩吡啶类化合物抑制 WT 细胞中的荧光素酶诱导，但不抑制 dKO 细胞中的荧光素酶诱导，它们在 WT 报告基因测定中的 IC 值与它们在骨合成活性测定中的报道活性具有近乎完美的相关性（R = 0.98），证实了后者的功能是由 CDK8/19 介导的，并验证了我们的测定方法是一种强大而定量的 CDK8/19 抑制作用测定方法。
