CDK7 (IC50 = 3.2 nM)

THZ1 抑制 Jurkat 细胞和 Loucy 细胞，IC50 值分别为 50 nM 和 0.55 nM。 CDK12 被 THZ1（9、27、83、250、750 和 2500 nM）抑制，但浓度高于 CDK7。 THZ1 (1 μM) 不可逆地磷酸化 CAK 和 RNAPII CTD。在 Hela S3 细胞中，THZ1 (2.5 μM) 共价靶向 CDK7 激酶结构域外的特定半胱氨酸，以不可逆地阻止 RNAPII CTD 磷酸化。在 T-ALL 细胞系中，THZ1 (250 nM) 会导致抗凋亡蛋白（最明显的是 MCL-1 和 XIAP）下降，以及凋亡指数增加和细胞增殖降低 [1]。所有基因型人类 (hSCLC) 细胞系的 IC50 范围为 5 至 20 nM，均表现出对 THZ1 的高度敏感性 [3]。

THZ1 (10 mg/kg) 可有效杀死原发性慢性淋巴细胞白血病 (CLL) 细胞，并对原发性 TALL 细胞和体内人 T-ALL 异种移植物具有抗增殖活性 [1]。 THZ1（10 mg/kg，静脉注射）可减少肿瘤生长，并且在人 MYCN 扩增的 NB 小鼠模型中没有显示出毒性 [4]。 THZ1（10 mg/kg，腹腔注射）完全抑制动物食管鳞状细胞癌肿瘤的生长，而不会出现体重减轻或其他常见不良症状[5]。

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为了比较THZ1的单药效力与药物的联合作用，我们对THZ1和PARP抑制剂奥拉帕尼（一种FDA批准的治疗复发性卵巢癌症的药物，无论BRCA1/2状态如何）进行了联合研究。我们首先进行了耐受性研究，发现以10mg/kg的剂量每天两次（BID）腹腔注射THZ1具有良好的耐受性，根据体重和动物行为判断，没有明显的毒性迹象（数据未显示）。在疗效研究中，我们首先将腹水衍生的卵巢肿瘤细胞植入小鼠体内，7天后将动物分为四组，分别接受载体对照（10 ml/kg，口服，每日一次）或THZ1（10 mg/kg，IP，每日两次）或奥拉帕尼（100 mg/kg，口服，每天一次），或组合（THZ1+Olaparib）治疗27天，并在5个时间点（0、6、13、20和27天）进行生物发光成像（图5A-B）。与之前对THZ1或奥拉帕尼的研究一致，抑制剂对小鼠体重的影响最小（图5--图补充1）。在所有11个独立的PDX模型中，THZ1的给药对肿瘤细胞生长产生了显著的抑制作用（图5B-C）。值得注意的是，在四种模型（DF-149、172、83和86）中，THZ1诱导了对肿瘤生长的完全抑制（图4C，称为i类）。在六种模型（DF-101、106、118、20、68和216）中，THZ1首先导致肿瘤负担明显减轻，但在以后的时间点重新生长（称为ii类）。只有一个模型（DF-181，称为iii类）没有显示肿瘤消退，而是在THZ1治疗后肿瘤细胞生长较慢。奥拉帕尼的给药并没有显著抑制肿瘤生长，在三种模型（DF-106、68和83）中仅显示出非常温和的效果。然而，THZ1和奥拉帕尼的组合显示出协同作用，在五种模型（DF-106、118、86、181和68）中观察到对肿瘤生长的进一步抑制。此外，我们发现THZ1治疗后，肿瘤中MYC和MCL-1的蛋白质丰度几乎被消除（图5D）。总体而言，THZ1在我们的卵巢肿瘤模型中抑制肿瘤生长的效力是惊人的，因为在之前使用THZ1的研究中很少观察到肿瘤消退。联合研究表明，THZ1与临床PARP抑制剂联合应用可能是治疗卵巢癌症的有前途的未来治疗方法[2]。

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。

Researchers used the ovarian patient-derived xenografts (PDX) models that they established previously (Liu et al., 2017). The primary tumor cells were transduced with luciferase gene to enable the use of non-invasive bioluminescent imaging (BLI) for measurement of tumor growth. Briefly, ovarian cancer cells were taken from consented patients with HGSOC and implanted intraperitoneally into immunocompromised NOD-SCID IL2Rγnull mice. 5 × 106 ascites-derived cells were implanted in each mouse and 7 days post implantation, mice were imaged by BLI and assigned to four groups of treatment with vehicle control via oral gavage (PO) once daily (QD) at the dose of 10 ml/kg; THZ1 intraperitoneally (IP) twice daily (BID) at the dose of 10 mg/kg; Olaparib via PO, QD at the dose of 100 mg/kg; the combination of THZ1 and Olaparib. Tumor growth was assessed every 7 days (0, 6, 13, 20, 27 days) using BLI until day 27. Upon harvesting, tumors or other tissues were snap-frozen in liquid nitrogen for preparation of lysates and immunoblotting.[2]

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Pharmacokinetics study of THZ1 from KOPTK1 T-ALL human xenograft mouse efficacy study samples. [1]
The concentration of THZ1 was determined in plasma and liver samples obtained at the end of the efficacy study. Plasma was generated using standard centrifugation techniques and liver samples were snap frozen and stored at -80OC until analysis. On the day of analysis, plasma and liver samples were thawed on ice, mixed with acetonitrile (1:5 v:v or 1:5 w:v, respectively), sonicated with a probe tip sonicator, and centrifuged through a Millipore Multiscreen Solvinter 0.45 micron low binding PTFE hydrophilic filter plate prior to analysis by LC-MS/MS. Concentrations were determined using an ABSciex 5500 mass spectrometer. THZ1 was detected using a mass transition of 566.4 to 186.1. Plasma drug levels were quantitated using standards made in blank mouse plasma and liver levels against standards made in blank mouse liver homogenate.[1]

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Patient-derived xenograft (PDX) treatment with THZ1. [1]
Patient-derived xenograft cells were treated with THZ1 for 3 hrs in vitro followed by compound washout (3 washes with DPBS). An aliquot of input cells was then counted by flow cytometry using a known quantity of flow cytometry calibration beads (data not shown; Molecular Probes). The remaining cells were plated onto MS5-DL1 feeder cells in the presence of serum-free media7 . 72 hrs later, cultures were harvested by vigorous pipetting with trypsin, filtered through nylon mesh to deplete feeders, and counted by flow cytometry using a known quantity of flow cytometry calibration beads. Based on an estimated average of 1.6-fold expansion with standard deviation of 0.7 and estimated minimum average of 0.4 following treatment with compound (i.e. at least 75% cell death), the test would be 80% powered for a p=0.05 difference with 3 samples.

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KOPTK1 T-ALL human xenograft mouse efficacy study. [1]
Thirty-two NOD-SCIDIL2Rcγ null (NSG) 9-week old female mice were injected intravenously with 2x106 KOPTK-1 cells expressing luciferase. Leukemia burden was established by bioluminescence imaging (BLI) using an IVIS Spectrum system beginning one week following cell injection. At this time, mice were divided into treatment groups based on mean BLI as follows: THZ1 10mg/kg qD, THZ1 10mg/kg BID, and vehicle (10% DMSO in D5W) BID (n=10 for all groups). Two mice were excluded, one with the highest and one with the lowest BLI. All treatments were administered via IV injection in the lateral tail vein in a volume of 3.3µL/g (non-blinded). Mice were imaged and weighed every 3-5 days. Mice were treated for four weeks and on the final day mice were imaged, dosed and sacrificed approximately 5-6 hrs post dose. Upon sacrifice, blood was collected via cardiac puncture in EDTA tubes; a portion (~300 uL) was processed for plasma. Liver and spleen tissues were collected from each mouse with half of each sample flash frozen and half of each sample fixed. Blood plasma and liver samples were processed for pharmacokinetics analysis of THZ1. Spleen tissues were homogenized and lysed and processed for pharmacodynamics analysis of THZ1 target engagement.

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Formulated in 10% DMSO in D5W; 10 mg/kg; i.v. injection

Bioluminescent xenografted mouse model

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

[2]. Targeting MYC dependency in ovarian cancer through inhibition of CDK7 and CDK12/13. Elife. 2018 Nov 13;7. pii: e39030.

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

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

[5]. Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma. Gut. 2016 May 10. pii: gutjnl-2016-311818.

THZ1 is a member of the class of indoles that is 1H-indole substituted by a 5-chloro-2-[3-(4-{[(2E)-4-(dimethylamino)but-2-enoyl]amino}benzamido)anilino]pyrimidin-4-yl group at position 3. It is a selective and potent covalent inhibitor of CDK7 that exhibits anti-proliferative effects in cancer cell lines. It has a role as an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an antineoplastic agent. It is a member of indoles, an aminopyrimidine, a member of benzamides, an organochlorine compound, an enamide and a secondary carboxamide.

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Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.[1]

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High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.[2]

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Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy.[3]

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The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins. Objectives: Oesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour. Design: High-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes. [4]

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Results: The unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase. Conclusions: Our integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.[5]

C31H28CLN7O2

566.05

565.199

C, 65.78; H, 4.99; Cl, 6.26; N, 17.32; O, 5.65

1604810-83-4

bio-THZ1;1604811-14-4;THZ1-R;1621523-07-6;THZ1 Hydrochloride

73602827

Off-white to yellow solid powder

1.4±0.1 g/cm3

1.735

5.08

115

4

6

9

41

896

0

CN(C)C/C=C/C(=O)NC1=CC=C(C=C1)C(=O)NC2=CC=CC(=C2)NC3=NC=C(C(=N3)C4=CNC5=CC=CC=C54)Cl

OBJNFLYHUXWUPF-IZZDOVSWSA-N

InChI=1S/C31H28ClN7O2/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/h3-15,17-19,33H,16H2,1-2H3,(H,35,40)(H,36,41)(H,34,37,38)/b11-6+

N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-4-[[(E)-4-(dimethylamino)but-2-enoyl]amino]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: 5 mg/mL (8.83 mM) in 10% DMSO + 90% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*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 (4.42 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 3 中的溶解度: ≥ 2.5 mg/mL (4.42 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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配方 4 中的溶解度: 2.08 mg/mL (3.67 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将100μL 20.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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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。