CDK7 (Kd = 142 nM)

THZ1-R 的 IC50 为 146 nM，对 CDK7 的亲和力比 THZ1 低。

THZ1 减少人类异种移植小鼠模型中 KOPTK1 T-ALL 细胞的增殖。 THZ1 在 10 mg/kg 时具有良好的耐受性，没有观察到体重减轻或行为变化，这表明它不会对动物造成明显的毒性

抑制剂处理实验[1]
进行了扩展数据图5a中描述的时间过程实验，以确定CDK7完全失活所需的最短时间。用THZ1、THZ1-R或DMSO处理细胞0-6小时，以评估时间对THZ1介导的RNAPII CTD磷酸化抑制的影响。对于后续实验，除非另有说明，否则用上述时间过程实验确定的化合物处理细胞4小时。对于抑制剂洗脱实验（图2e，f；扩展数据图5），用THZ1、THZ1-R或DMSO处理细胞4小时。随后去除含有抑制剂的培养基以有效“洗脱”化合物，并允许细胞在没有抑制剂的情况下生长。对于每个实验，检测裂解物的RNAPII CTD磷酸化和其他特定蛋白质。

高通量细胞系平板活性测定[1]
将细胞接种在384孔微孔板中，在含有5%FBS和青霉素/链霉抗生物素的培养基中以约15%的融合率接种。用THZ1或DMSO处理细胞72小时，并用刃天青测定细胞存活率。

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细胞增殖试验[2]
在病毒感染和嘌呤霉素选择后，将细胞接种在1ml培养基中的12孔板（密度为5×103）中。14天后，细胞用1%甲醛固定15分钟，用结晶紫（0.05%，wt/vol）染色15分钟，这是一种染色质结合细胞化学染色。这些板在大量去离子水中广泛清洗，在滤纸上倒置干燥，并用爱普生扫描仪成像。 对于96孔板中的3天细胞增殖试验，细胞以每孔6000至10000个细胞的密度铺板，并在第二天用不同浓度的THZ1或YKL-116处理。孵育72小时后，将CellTiter-Glo试剂直接加入细胞中，并在平板阅读器上读取发光信号。

[1]. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature. 2014 Jul 31;511(7511):616-20.

THZ1 belongs to the indole class of compounds, with the structure 1H-indole, substituted at position 3 with 5-chloro-2-[3-(4-{[(2E)-4-(dimethylamino)but-2-enoyl]amino}benzamido)anilino]pyrimidin-4-yl]. It is a selective and potent covalent inhibitor of CDK7, exhibiting antiproliferative activity in cancer cell lines. It can function as an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an antitumor drug. THZ1 belongs to the indole, aminopyrimidine, benzamide, organochlorine, enamide, and secondary carboxamide classes. Tumor oncogenes include several transcription factors that utilize universal transcriptional mechanisms to maintain tumorigenesis, but direct pharmacological inhibition of these transcription factors remains challenging to date. However, transcriptional mechanisms involve various enzymatic cofactors, such as cyclin-dependent kinases (CDKs), which can serve as targets for developing novel therapeutic candidates. This article reports the discovery and characterization of a covalent CDK7 inhibitor, THZ1. THZ1 has an unprecedented ability to target distal cysteine residues located outside the classical kinase domain, thus providing an unexpected pathway to achieve CDK7 selectivity. Cancer cell line analysis showed that some cancer cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL), are abnormally sensitive to THZ1. Genome-wide analysis of Jurkat T-ALL cells showed that THZ1 has a disproportionate effect on RUNX1 transcription, suggesting that sensitivity to THZ1 may stem from the vulnerability conferred by the RUNX1 superenhancer and the key role of RUNX1 in the core transcriptional regulatory circuits of these tumor cells. Therefore, pharmacologically modulating CDK7 kinase activity may provide a pathway for identifying and treating tumor types that depend on transcription to maintain oncogenic status. [1]

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High-grade serous ovarian cancer is characterized by extensive copy number variations, with MYC oncogene amplification occurring in nearly half of the tumors. We have confirmed that ovarian cancer cells are highly dependent on MYC to maintain their oncogenic growth, suggesting that MYC is a potential target for treating this refractory malignancy. However, direct targeting of MYC has proven to be very difficult. We screened small molecules that target transcriptional and epigenetic regulation and found that THZ1 (an inhibitor of CDK7, CDK12 and CDK13) can significantly downregulate MYC expression. It is noteworthy that targeting CDK7 alone cannot completely inhibit MYC expression, and it is necessary to combine the inhibition of CDK7, CDK12 and CDK13. In 11 xenograft models derived from ovarian cancer patients who had received extensive pretreatment, administration of THZ1 significantly inhibited tumor growth and suppressed MYC expression. Our study suggests that targeting these transcriptional CDKs with drugs such as THZ1 may be an effective approach for treating MYC-dependent ovarian malignancies. [2]

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Small cell lung cancer (SCLC) is a highly aggressive and deadly disease, so there is an urgent need to find effective drug strategies to target the biological characteristics of SCLC. We used a high-throughput cell screening method to screen multiple compound libraries and found that SCLC is sensitive to transcription-targeting drugs, especially THZ1, a newly discovered covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We found that the expression of super-enhancer-related transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly sensitive to THZ1 treatment. We propose that the downregulation of these transcription factors contributes to the sensitivity of small cell lung cancer (SCLC) to transcription inhibitors, and THZ1 represents a prototype targeted therapy for SCLC. [3]

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MYC oncoproteins are thought to stimulate the growth and proliferation of tumor cells by amplifying gene transcription, a mechanism that has hampered most attempts to use MYC function as a potential cancer therapy. We used a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, and the results showed that the downregulation of the oncoprotein led to a significant inhibition of MYCN-driven overall transcriptional amplification. This response translated into significant tumor regression in a high-risk neuroblastoma mouse model without systemic toxicity. Significant therapeutic selectivity in MYCN-overexpressing cells was associated with preferential downregulation of super-enhancer-related genes, including MYCN and other known oncogenes in neuroblastoma. These results suggest that CDK7 inhibitors may have therapeutic value against cancers driven by MYC family oncoproteins by selectively targeting mechanisms that promote global transcriptional amplification in tumor cells. Objective: Esophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and a major histological subtype of esophageal cancer. Despite increased awareness of OSCC genetic abnormalities through large-scale genomic analyses in recent years, few targetable genomic lesions have been identified, and no molecularly targeted therapies exist. This study aimed to identify potential drug targets in this tumor. Design: A high-throughput small-molecule inhibitor screening method was used to screen for effective anti-OSCC compounds. The mechanism of action of CDK7 inhibitors in OSCC was elucidated using whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). We performed various in vitro and in vivo cell experiments to determine the impact of candidate genes on the malignant phenotype of oral squamous cell carcinoma (OSCC). [4] Results: Through unbiased high-throughput screening of small molecule inhibitors, we identified a highly potent anti-OSCC compound, THZ1, which is a specific CDK7 inhibitor. RNA sequencing showed that low-dose THZ1 treatment selectively inhibited a variety of oncogenic transcripts. Notably, further analysis of the genomic signatures of these THZ1-sensitive transcripts revealed that they were often associated with super-enhancers (SEs). Furthermore, SE analysis alone revealed many OSCC lineage-specific master regulators. Finally, an integrated analysis of THZ1-sensitive and SE-associated transcripts identified several novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2, and YAP1, with PAK4 being a potential drug-targeting kinase. Conclusion: Our integrated approach constructed a catalog of SE-associated master regulators and oncogenic transcripts, which could significantly advance our understanding of OSCC biology and the development of more innovative therapies. [5]

C₃₁H₃₀CLN₇O₂

568.07

567.214

C, 65.54; H, 5.32; Cl, 6.24; N, 17.26; O, 5.63

1621523-07-6

THZ1;1604810-83-4;THZ1 Hydrochloride

119081415

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.720

4.7

115

4

6

10

41

854

0

TUERFPPIPKZNKE-UHFFFAOYSA-N

InChI=1S/C31H30ClN7O2/c1-39(2)16-6-11-28(40)35-21-14-12-20(13-15-21)30(41)36-22-7-5-8-23(17-22)37-31-34-19-26(32)29(38-31)25-18-33-27-10-4-3-9-24(25)27/h3-5,7-10,12-15,17-19,33H,6,11,16H2,1-2H3,(H,35,40)(H,36,41)(H,34,37,38)

N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-4-[4-(dimethylamino)butanoylamino]benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.40 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.40 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。