THZ1

Biological Activity

In vitro: THZ1 uses a unique mechanism, combining ATP-site and allosteric covalent binding, as a means of attaining potency and selectivity for CDK7. THZ1 irreversibly inhibits RNAPII CTD phosphorylation by covalently targeting a unique cysteine located outside the kinase domain of CDK7. THZ1, but not THZ1-R, completely inhibits the phosphorylation of the established intracellular CDK7 substrate RNAPII CTD at Ser 5 and Ser 7, with concurrent loss of Ser 2 phosphorylation at 250 nM in Jurkat cells. THZ1 exhibits strong antiproliferative effects across a broad range of cancer cell lines from various cancer types. In Jurkat cells, low-dose THZ1 has a profound effect on a small subset of genes, including the key regulator RUNX1, thus contributing to subsequent loss of the greater gene expression program and cell death. THZ1 causes defects in Pol II(polymerase II) phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. In vivo: THZ1 reduces the proliferation of KOPTK1 T-ALL cells in a human xenograft mouse model. THZ1 is well tolerated at 10 mg/kg with no observable body weight loss or behavioural changes, suggesting that it causes no overt toxicity in the animals.

Product Citations

• 

  Mol Cancer Ther. 2021 Oct;20(10):1868-1879.

  Pharmaceutical Interference of the EWS-FLI1–driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma
  THZ1 purchased from AbMole

• 

  Cancer Lett. 2020 Feb 28;471:27-37.

  The covalent CDK7 inhibitor THZ1 enhances temsirolimus-induced cytotoxicity via autophagy suppression in human renal cell carcinoma.
  THZ1 purchased from AbMole

• 

  Cells. 2019 Nov 19;8(11).

  Suppression of Angiogenesis by Targeting Cyclin-Dependent Kinase 7 in Human Umbilical Vein Endothelial Cells and Renal Cell Carcinoma: An In Vitro and In Vivo Study.
  THZ1 purchased from AbMole

Protocol (for reference only)

Cell Experiment
Cell lines	Jurkat, Loucy, KOPTK1 and DND-41 cell lines
Preparation method	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.
Concentrations	0-10 μM
Incubation time	4 h

Animal Experiment
Animal models	Bioluminescent xenografted mouse model
Formulation	10% DMSO in D5W
Dosages	10 mg/kg
Administration	i.v.

Chemical Information

Molecular Weight	566.05
Formula	C31H28ClN7O2
CAS Number	1604810-83-4
Solubility (25°C)	DMSO: ≥ 25 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Nilson KA, et al. Mol Cell. THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing.

[2] Kwiatkowski N, et al. Nature. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor.