17-Nov-2022

Seminar Dr. Basil Greber (Team Leader, The Institute of Cancer Research, London)

November 17th 2022 / 10.00 / HPL D32

Harnessing the power of cryo-EM for development of cancer therapeutics: High-resolution structures of the human CDK-activating kinase bound to inhibitors

The human CDK-activating kinase (CAK) is a trimeric protein complex that acts as a master regulator of cell growth and division. Multiple drug discovery programmes aiming to find potent CAK inhibitors are underway, and several inhibitors have entered clinical trials. In order to facilitate discovery of next-generation therapeutics, we sought to make the CAK amenable to structure-based drug design. To this end, we initially determined structures of the human CAK bound to nucleotide analogues and two inhibitors using a 200 kV-electron microscope, achieving up to 2.5 Å resolution for the 85 kDa CDK-cyclin core module of the complex.

To uncover the structural basis of inhibitor selectivity, which requires the rapid acquisition of high-quality datasets for large numbers of different inhibitors, we established a screening workflow using a 200 kV Glacios 2 instrument equipped with a Selectris X energy filter and a Falcon 4i direct electron detector. This workflow enables 3D reconstruction of the CAK – which is a small, asymmetric, ligand-bound complex – at 3.5-4.5 Å within one hour from the start of data collection, and at approx. 3 Å within four hours from the start of data collection. Finally, we obtained more than a dozen high-resolution structures of the 85 kDa CDK-cyclin-module of the human CAK bound to inhibitors and nucleotides at up to 1.8 Å resolution using a Titan Krios G4 microscope with a cold-FEG. These structures provide detailed insight into inhibitor binding and suggest a mechanism for target selectivity that we are currently testing.

References
Greber et al. (2020), PNAS 117 (37): 22849-57
Greber et al. (2021), Biophys J. 120 (4): 677-686