Binding assays

Radioactive kinase activity measurements were performed at a concentration of 0.2 μM CDK–cyclin complex. Typically, 35 μl reaction volumes of 0.2 μM active kinase were equilibrated in kinase buffer (40 mM Hepes (pH 7.6)), 34 mM NaCl, 34 mM KCl, 10 mM MgCl2, 5% glycerol, and 1× PhosSTOP. Cold ATP to a final concentration of 200 μM and 3 μCi [γ-32P]ATP and 50 μM of a substrate peptide containing five phosphorylation sites were added and the reaction mixture incubated for 30 min at 30 ℃ at 350 rpm in a thermomixer. Reactions were stopped by adding EDTA to a final concentration of 50 mM. Aliquots of 15 μl each were spotted onto paper squares using the Optitran BA-S85 reinforced membrane. Paper squares were washed three times for 5 min with 0.75% (v/v) phosphoric acid, with at least 5 ml washing solution per paper. Radioactivity was counted in a Beckman Scintillation Counter (Beckman Coulter) for 1 min. Compounds THZ531 was added at varying concentrations to 0.2 μM CDK–cyclin complex and incubated for varying times from 1 to 540 min at 30 ℃ and 350 rpm before ATP and substrate were added to the reaction mix.

Cell lines

Jurkat cells

Preparation method

This compound is soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

50-1200 nM, 6 h

Applications

THZ531 treatment led to a dramatic and irreversible decrease in Jurkat cell proliferation with an IC50 of 50 nM. Treatment with escalating doses of THZ531 displayed a dose- and time-dependent increase in the number of cells exhibiting sub-G1 content. Higher doses of THZ531 led to a pronounced annexin V signal, with 30–40% annexin-V-stained cells by 72 h. THZ531 selectively reduced Ser2 phosphorylation levels without appreciable effect on CTD pSer5 or pSer7 levels. Treatment with 50 nM THZ531 resulted in the loss of expression of a small subset of genes.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

THZ531 is a first-in-class CDK12 and CDK13 covalent inhibitor with IC50 values of 158 nM and 69 nM, respectively [1].

Complexes containing CDK12 and CDK13 regulate transcriptional elongation as well as processes, including mRNA splicing and 3’-end RNA processing. Loss of CDK12 and CDK13, or of their cofactor cyclin K, impedes both Pol II processivity and RNA processing [1].

THZ531 is an irreversible CDK12 and CDK13 covalent inhibitor. THZ531 potently inhibited CDK12 and CDK13 with IC50 values of 158 nM and 69 nM, whereas inhibition of CDK7 and CDK9 was more than 50-fold weaker, with IC50s of 8.5 μM and 10.5 μM, respectively. In Jurkat cells, THZ531 irreversibly reduced cell proliferation with IC50 of 50 nM. THZ531 induced apoptosis in a dose- and time-dependent way. THZ531 also reduced Pol II C-terminal domain (CTD) Ser2 phosphorylation, a mark of active transcriptional elongation, resulted in the loss of key super-enhancer-associated transcription factor genes and DNA damage response (DDR) genes expression [1].

Reference:

1.Zhang T, Kwiatkowski N, Olson CM, et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol. 2016 Oct;12(10):876-84.