THZ1 (CDK7 inhibitor) is a selective covalent inhibitor of CDK7, exhibiting binding affinity to the cysteine residue located at the outer end of the classical kinase domain, thus conferring high selectivity for CDK7, with an IC50 of 3.2 nM.

CDK7:3.2 nM

方法：Loucy 细胞用 THZ1 (0.8-500 nM) 处理 4 h，通过 LanthaScreen Eu Kinase Binding assay 检测结合情况。
结果：THZ1 在体外对 CDK7 具有时间依赖性抑制作用，并具有细胞内 CDK7 的共价结合作用。[1]
方法：NSCLC 细胞系 H1299、A549 和 H292 用 THZ1 (10-10000 nM) 处理 48 h，通过 crystal violet 检测细胞活力。
结果：THZ1 剂量依赖性地抑制了 NSCLC 细胞的迁移增殖。[2]

方法：为检测体内抗肿瘤活性，将 THZ1 (10 mg/kg) 静脉注射给携带 MYCN 扩增的人 NB 异种移植物的 NU/NU 小鼠，每天两次，持续 28 天。
结果：THZ1 抑制人 MYCN 扩增 NB 小鼠模型中的肿瘤生长。[3] 方法：为检测体内抗肿瘤活性，将 THZ1 (10 mg/kg) 腹腔注射给携带 KYSE510 肿瘤的 NSG 小鼠，每天两次，持续 24 天。
结果：THZ1 抑制 KYSE510 异种移植物的生长并抑制 NSG 小鼠模型的肺转移。[4]

For kinase assays following immunoprecipitation of FLAG-CDK7 protein from HCT116 or FLAG-CDK12 from 293A cellular lysates, cells are first treated with THZ1, THZ1-R, or DMSO for 4 hrs at 37°C. Cells are then harvested by lysis in 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, and protease/phosphatase cocktails. Exogenous CDK7 or CDK12 proteins are immunoprecipitated from cellular lysates using FLAG antibody- conjugated agarose beads. Precipitated proteins are washed with lysis buffer 6 times, followed by 2 washes with kinase buffer (40 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) and subjected to in vitro kinase assays at 30°C for 45 minutes using 1 μg of the large subunit of RNAPII (RPB1) as substrate and 25 μM ATP and 10 μCi of 32P ATP. Kinase assays using recombinant CDK7/TFIIH/MAT1 are conducted in the manner as described above using 25 ng of CAK complex per reaction. For kinase assays designed to test time-dependent inactivation of CDK7 kinase activity, CAK complex is pre-incubated with indicated concentrations of THZ1, THZ1-R, or DMSO in kinase buffer without ATP for 4 hrs at 30°C prior to being subjected to kinase assay conditions[1].

Cells are treated with THZ1, THZ1-R or dimethylsulphoxide (DMSO) for 0-6?h to assess the effect of time on the THZ1-mediated inhibition of RNAPII CTD phosphorylation. For subsequent experiments cells are treated with compounds for 4?h as determined by the time-course experiment described earlier, unless otherwise noted. For inhibitor washout experiments, cells are treated with THZ1, THZ1-R or DMSO for 4?h. Medium containing inhibitors is subsequently removed to effectively 'washout' the compound and the cells are allowed to grow in the absence of inhibitor. For each experiment, lysates are probed for RNAPII CTD phosphorylation and other specified proteins.(Only for Reference)