THZ1 Hydrochloride is a selective and potent covalent CDK7 inhibitor with an IC₅₀ of 3.2 nM. THZ1 Hydrochloride also inhibits the closely related kinases CDK12 and CDK13 and downregulates MYC expression^[1][2].

THZ1 inhibits Jurkat cell and Loucy cell with IC₅₀ of 50 nM, and 0.55 nM, respectively. THZ1 demonstrates time-dependent inhibition of CDK7 in vitro and covalent binding of intracellular CDK7. THZ1 (9, 27, 83, 250, 750, and 2500 nM) inhibits CDK12 but at higher concentrations compared to CDK7. THZ1 (1 μM) irreversibly inhibits RNAPII CTD and CAK phosphorylation. THZ1 (2.5 µM) irreversibly inhibits RNAPII CTD phosphorylation by covalently targeting a unique cysteine located outside the kinase domain of CDK7 in Hela S3 cells. THZ1 (250 nM) causes decreased cellular proliferation and an increase in apoptotic index with concomitant reduction in anti-apoptotic proteins, most notably MCL-1 and XIAP in T-ALL cell lines^[1].
All genotypically-distinct human (hSCLC) cell lines exhibit high sensitivity to THZ1, with an IC₅₀ in the range of 5-20 nM^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

THZ1 (10 mg/kg) demonstrates potent killing of primary chronic lymphocytic leukemia (CLL) cells and anti-proliferative activity against primary TALL cells and in vivo against a human T-ALL xenograft^[1].
THZ1 (10 mg/kg, i.v.) inhibits tumor growth in a mouse model of human MYCN-amplified NB and shows no toxicity^[4].
THZ1 (10 mg/kg, i.p.) completely suppresses oesophageal squamous cell carcinoma tumour growth in vivo without loss of body weight or other common toxic effects^[5].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

602.51

C₃₁H₂₉Cl₂N₇O₂

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

In Vitro: 

DMSO : 22.5 mg/mL (37.34 mM; Need ultrasonic and warming)

H₂O : < 0.1 mg/mL (ultrasonic;warming;heat to 60°C) (insoluble)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.17 mg/mL (3.60 mM); Clear solution

  此方案可获得 ≥ 2.17 mg/mL (3.60 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: 2.17 mg/mL (3.60 mM); Suspended solution; Need ultrasonic

  此方案可获得 2.17 mg/mL (3.60 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.17 mg/mL (3.60 mM); Clear solution

  此方案可获得 ≥ 2.17 mg/mL (3.60 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

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

  [2]. Jiang YY, et al. Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma. Gut. 2016 May 10. pii: gutjnl-2016-311818.

  [3]. Christensen CL, et al. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor. Cancer Cell. 2014 Dec 8;26(6):909-22.

  [4]. Chipumuro, et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell. 2014 Nov 20;159(5):1126-39. ?

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. Note: no additional inhibitors are added to the kinase reaction mixture, therefore any inhibition results from the activity of the compounds that are added directly to cells (ie-intracellular inhibition). This suggests that the inhibitors are either covalently tethered to the kinase or have strong non-covalent character. 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].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Jurkat, Loucy, KOPTK1, and DND-41 cell lines are seeded in 384-well microplates at 15% confluency in medium with 5% FBS and penicillin/streptavidin. Cells are treated with THZ1 (2, 10, 50, 250, 1250, and 6250 nM) or DMSO for 72 hrs and cell viability is determined using resazurin^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Thirty-two NOD-SCIDIL2Rcγnull (NSG) 9-week old female mice are divided into treatment groups based on mean BLI as follows: THZ1 10 mg/kg qD, THZ1 10 mg/kg BID, and vehicle (10% DMSO in D5W) BID (n=10 for all groups). Two mice are excluded, one with the highest and one with the lowest BLI. All treatments are administered via IV injection in the lateral tail vein in a volume of 3.3 μL/g (non-blinded). Mice are imaged and weighed every 3-5 days. Mice are treated for four weeks and on the final day mice are imaged, dosed and sacrificed approximately 5-6 hrs post dose. Upon sacrifice, blood is collected via cardiac puncture in EDTA tubes; a portion (300 uL) is processed for plasma. Liver and spleen tissues are collected from each mouse with half of each sample flash frozen and half of each sample fixed. Blood plasma and liver samples are processed for pharmacokinetics analysis of THZ1. Spleen tissues are homogenized and lysed and processed for pharmacodynamics analysis of THZ1 target engagement.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

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

  [2]. Jiang YY, et al. Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma. Gut. 2016 May 10. pii: gutjnl-2016-311818.

  [3]. Christensen CL, et al. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor. Cancer Cell. 2014 Dec 8;26(6):909-22.

  [4]. Chipumuro, et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell. 2014 Nov 20;159(5):1126-39. ?