CDK12715-1038/CycK11-267 complex (used for crystallography, assays)
Human CDK12 (a.a. 715-1038) and CycK (a.a. 11-267) were each cloned into pFB-LIC-Bse, co-expressed in Sf9 cells and purified using Ni-affinity and size exclusion chromatography.
CDK12715-1052/CycK11-267 complex (used for crystallography, assays)
Human CDK12 (a.a. 715-1052) and CycK (a.a. 11-267) were each cloned into pFB-LIC-Bse, co-expressed in Sf9 cells and purified using Ni-affinity and size exclusion chromatography.
Full length CDK12/CycK complex (used for in vitro kinase assays)
6xHis-CDK12/GST-CycK or GST-CDK12/6xHis-CycK were prepared by baculoviral co-expression and purified sequentially using Ni-affinity and glutathione sepharose chromatography (Invitrogen pDEST10 and pDEST20 vectors).
Note, soluble expression of CDK12 was only observed upon co-expression with CycK. Phosphorylated CDK12 was optimally obtained by additional co-expression of yeast CAK.
RNA Pol II CTD (used for in vitro kinase assays)
GST-CTD substrate containing all 52 heptad repeats was cloned into Creator System plasmid V1544 for bacterial expression and glutathione sepharose chromatography purification.
- Core structure of apo CDK12715-1038/CycK11-267 complex (3.15 Å, PDB: 4UN0 supersedes 4CJY)
- Extended structure of CDK12715-1052/CycK11-267 complex with AMP-PNP (3.15 Å, PDB: 4CXA).
- THZ531 inhibitor bound structure of CDK12715-1052/CycK11-267 complex (2.7 Å, PDB: 5ACB).
The structures capture the phosphorylated (pT893) active kinase conformation. Two novel features for a CDK were identified (i) a large β4-β5 loop insertion that contributes to the specific N-lobe interaction with CycK; and (ii) a C-terminal kinase extension (αK helix) observed in alternative conformations that open and close the ATP pocket and allows for covalent inhibitor design. Binding interactions were defined for
AMP-PNP and the covalent inhibitor THZ531, which binds irreversibly to Cys1039 in two conformations of the C-terminal kinase extension. Sequence alignment and modelling shows that CDK12 Cys1039 adopts a distinct position to CDK7 Cys312 (which binds THZ1, (1)) allowing for kinase selectivity.
In vitro assays
In vitro kinase assay: Activity assays were performed using 32P-ATP and GST-CTD as substrates and measured by radiography. The full length (FL) CDK12/CycK complex was 10x more active than the core domains solved by crystallography. KmATP values for the full-length and core complexes were 2 mM and 25 mM, respectively. KmGST-CTD values for the full-length and core complexes were 0.3 mM and 2 mM, respectively. Kinases were present at 13 nM concentration.
CAK assay: Intact mass spec showed that yeast CDK-activating kinase (CAK) could phosphorylate the CDK12 activation loop residue Thr893.
Covalent inhibitor binding assay: THZ series derivatives were screened for covalent CDK12 binding by mass spec. Domain mapping experiments confirmed Cys1039 as the covalent attachment site. CDK12715-1052 could be fully labelled with THZ531, whereas CDK12715-1038 was resistant.
Chemical starting points
Chemical matter with co-structures included AMP-PNP as well as the novel covalent inhibitor THZ531 developed by Nathanael Gray with the following characterisation: (i) KiNativ profiling against 213 targets showed CDK12 and CDK13 as the only proteins labelled >55%; (ii) time dependent IC50 values (30 mins: CDK12, 158 nM; CDK13, 69 nM) with efficient labelling at 50 nM and 50-100 fold selectivity against CDK7 and CDK9; (iii) In cells, 50 nM THZ531 reduced expression of DNA damage response genes (e.g. BRCA1, FANCF, ERCC4), while 200 nM reduced super–enhancer–associated transcription factors (e.g. RUNX1, MYB, TAL1, and GATA3); (iv) anti-proliferativity activity and apoptosis were observed in CML HAP1 cell line and Jurkat T-ALL cells (e.g. IC50 = 50 nM, CellTiterGlo assay).
IMPORTANT: Please note that the existence of small molecules within this TEP indicates that chemical matter can bind to the protein in a functionally relevant pocket. As such these molecules should not be used as tools for functional studies of the protein unless otherwise stated as they are not sufficiently potent or well-characterised to be used in cellular studies. The small molecule ligands are intended to be used as the basis for future chemistry optimisation to increase potency and selectivity and yield a chemical probe or lead series.
New literature since TEP approval
The group of Matthias Meyer have published the crystal structure of the CDK13/CycK complex (PDB 5EFQ).
Chemical screening using the CDK12 inhibitor THZ531 has revealed that Ewing sarcoma cells are especially sensitive to CDK12 inhibition (Cancer Cell (2018), 33, 202-216). A synthetic lethality of CDK12 inhibition with the EWS/FLI is suggested based on tumour cell sensitivity to DNA damage.
CDK12 polyclonal antibodies (anti-rabbit) have been used by our collaborators and were kind gifts of Arno Greenleaf (original description in J Biol Chem 290, 1786-1795) or Jonathon Pines (original description in
J Cell Sci 114, 2591-603).
CRISPR/Cas9 reagents targeting human CDK12 have been published by others in either CDK12-focussed research e.g. Cell Reports (2016) 17, 2367–2381, or in genomic studies e.g. Nat Biotech (2016) 34, 184–191. Latter reagents are available from Addgene (e.g. plasmids #75915, #75916, #75917). Alternatively, shRNA and siRNA reagents are commercially available from companies such as Dharmacon.
- Further optimisation of chemical matter
- Further exploration of CDK12 as a therapeutic target in cancer cells
- Chemistry partner: Nathanael Gray, Harvard University
- Kinase Km assays: Gregg Morin, University of British Columbia