CDK7 (IC50 = 3.2 nM)

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]

Inhibitor treatment experiments [1]
Time-course experiments such as those described in Extended Data Fig. 5a were conducted to determine the minimal time required for full inactivation of CDK7. Cells were treated with THZ1, THZ1-R, or DMSO for 0–6 hrs to assess the effect of time on the THZ1 –mediated inhibition of RNAPII CTD phosphorylation. For subsequent experiments cells were treated with compounds for 4 hrs as determined by time-course experiment described above, unless otherwise noted. For inhibitor washout experiments (Fig. 2e, f; Extended Data Fig. 5) cells were treated with THZ1, THZ1-R, or DMSO for 4 hrs. Medium containing inhibitors was subsequently removed to effectively ‘washout’ the compound and the cells were allowed to grow in the absence of inhibitor. For each experiment, lysates were probed for RNAPII CTD phosphorylation and other specified proteins.

High-throughput cell line panel viability assay [1]
Cells were seeded in 384-well microplates at ~15% confluency in medium with 5% FBS and penicillin/streptavidin. Cells were treated with THZ1 or DMSO for 72 hrs and cell viability was determined using resazurin.

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Cell proliferation assays [2]
After virus infection and selection with puromycin, cells were seeded in 12-well plates (at the density of 5 × 103) in 1 ml medium. 14 days later, cells were fixed with 1% formaldehyde for 15 minutes, and stained with crystal violet (0.05%, wt/vol), a chromatin-binding cytochemical stain for 15 minutes. The plates were washed extensively in plenty of deionized water, dried upsidedown on filter paper, and imaged with Epson scanner. For the 3-day cell proliferation assay in 96-well plate, cells were plated at the density of 6000 to 10,000 cells per well and treated with THZ1 or YKL-1–116 of various concentrations on the next day. After 72 hr incubation, CellTiter-Glo reagent was added to cells directly and luminescent signal was read on a plate reader

[1].Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature. 2014;511(7511):616-620.

[2].THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing. Mol Cell. 2015;59(4):576-587.

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]

C31H30CL3N7O2

638.97

637.152

2095433-94-4

129896819

Typically exists as solid at room temperature

6

6

9

43

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.Cl.Cl

AJTGQOACYBCREM-QVLKBJGCSA-N

InChI=1S/C31H28ClN7O2.2ClH/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);2*1H/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;dihydrochloride

THZ1 Dihydrochloride; 2095433-94-4; THZ1 2HCl; (E/Z)-THZ1 (dihydrochloride); N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-4-[[(E)-4-(dimethylamino)but-2-enoyl]amino]benzamide;dihydrochloride; THZ1Dihydrochloride; 1604810-83-4(free base);

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)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)