THZ531

THZ531 is a high quality irreversible cysteine-reactive covalent acrylamide inhibitor of CDK12 and CD13 with high selectivity over other human protein kinases. Several lines of evidence, including mass spectrometry and a co-crystal structure demonstrate covalent bond formation with the target cysteine and the target binding mode as well as requirement for the target cysteine for inhibitor activity. Kinase profiling data indicate high selectivity for the intended target and an inactive analog provides a selectivity control.

The probe is well characterised, and shows selective activity at CDK12/13 in Jurkat cells, as seen in Kinativ profiling. In particular, good selectivity over other CDK family is observed.  However, the compound does not show good selectivity in Ambit kinase binding profiling showing comparable binding at JNK family, GSK3, and ~10 other targets within 10 fold of the CDK13 activity.  10 fold selectivity over CDK7 is observed Kinativ profiling suggests that in the context of Jurkat cells, this binding affinity translates to reasonable selectivity for CDK12/13 (these two are equiactive), but care is required in interpreting findings with this molecule, particularly in different cell lines where different expression and activity levels of different kinases may be observed. At the time of writing this represents the best available tool for CDK12/13, but combining this with additional tools is recommended, as is the use of C to S mutants of CDK12 (as carried out by Zhang et al) to tease out the contribution of CDK12.