Cyclin K
October 2023
Cyclin K
Cyclin K is a regulatory protein that forms complexes with cyclin-dependent kinases (CDKs) and plays a crucial role in regulating the cell cycle and transcription, making it an attractive target for cancer research. However, cyclin K has long been considered an “undruggable” protein target since it does not possess an obvious site for the development of drugs with traditional drug discovery approaches.
Several compounds have been recently observed to target the complex CDK12-cyclin K, inducing cyclin K degradation and acting as molecular glue degraders. These are a class of emerging therapeutics with a different mechanism of action than “classic” inhibitors, opening new drug discovery opportunities for proteins that have always been considered undruggable.
Molecular glue degraders are small molecule compounds that mediate the interaction between the protein target and the cell’s protein degradation machinery. This association allows the machinery to mark the protein target for degradation (ubiquitination), ultimately triggering its depletion in the cell (ubiquitin-proteosome system).
Read our blog post on a molecular glue use case to learn more.
Despite the clinical success of some molecular glue degraders, we still have limited knowledge of how to design and rationally develop this class of drugs, thus hindering a broader application of this methodology in drug discovery.
In a recent paper, scientists investigated the structure-activity relationship of a large set of small molecules that promoted cyclin K degradation. They observed that even compounds with high chemical diversity - including known kinase inhibitors - showed molecular glue activity for cyclin K. Their findings paved the way for rational design and optimization of novel molecular glue compounds.
The scientists observed that the compounds reported in the literature to degrade cyclin K lacked any obvious chemical structure similarity. Therefore, they decided to investigate the structural basis for the molecular glue activity of chemically diverse ligands.
The researchers started examining the binding mode of the cyclin K degrader CR8. From the crystal structure of the complex of CDK12-cyclin K with CR8 and a protein of the cellular degradation machinery, DDB1 (DDB1-CR8-CDK12-cyclin K), they observed that CR8 bridges CDK12 and DDB1 by binding to the ATP-binding pocket of CDK12 and the surface of DDB1 (Image 1). In this way, CR8 brings the protein degradation cellular machinery in proximity with cyclin K, which is then ubiquitinated and proteolytically degraded.
Image 1. Crystal structure of the DDB1-CR8-CDK12-cyclin K complex: cyclin K in aquamarine, CDK12 in yellow, DDB1 in grey, and ligand CR8 in pink (PDB: 6TD3). In the zoomed panel, focus on the binding mode of CR8 at the interface between CDK12 and DDB1, with the phenylpyridine moiety interacting with the Arg928 on DDB1. The purine scaffold of CR8 occupies the ATP binding pocket and interacts with the hinge region. Pictures produced with the 3decision® software.
The researchers investigated which portion of the CR8 was key for inducing the degradation of cyclin K. They extensively modified the substituents on the purine-scaffold of CR8 (Image 2A), varying their chemical nature and size. They found that the “gluing moiety” (the phenylpyridine in CR8 - Image 1, zoom) was the most impactful on the degrader activity and that the interaction with DDB1 Arg928 was key for stabilizing the complex formation. Interestingly, they also observed that several chemically diverse gluing moieties could be accommodated in this interfacial cavity in disparate ways (Image 2B), translating into an excellent molecular glue activity. The best degraders carried an aromatic ring as gluing moiety, which preserved the interaction with the DDB1 Arg928.
Image 2. A) Chemical structure of CR8. In yellow is highlighted the purine scaffold, in red the gluing moiety (the phenylpyridine substituent). B) Overlay of the crystal structures of the DDB1-ligand-CDK12-cyclin K complex, where ligand is respectively: CR8 in pink (PDB: 6TD3), roscovitine in blue (PDB: 8BU9), DS06 in white (PDB: 8BUJ), DS08 in orange (PDB: 8BUK), DS11 in green (PDB: 8BUL). For clarity, the cartoon representation is not displayed. On the right, the chemical structure of the gluing moiety for each of the compounds. Pictures produced with the 3decision® software.
They next explored the binding mode of compounds with chemically different scaffolds. Using virtual screening and mining the literature;, they identified over 40 compounds with putative cyclin K degradation activity - the most known CDK inhibitors. They solved crystal structures of 28 glue-induced ternary complexes and observed that the overall architecture of the complexes was always very similar (Image 3A). The identified molecular glue degraders had variable sizes (molecular weights: 317 - 722 Da) and polarity (clogP: 0.4 - 5.1), indicating an impressive chemical diversity. What they all shared was an acceptor-donor motif interacting with the CDK12 hinge region and a gluing moiety bearing an aromatic system extending from the hydrogen bond donor (Image 3B).
Image 3. A) Overlay of the crystal structures of the DDB1-ligand-CDK12-cyclin K complex, where ligand is respectively: CR8 in pink (PDB: 6TD3), SR-4835 in blue (PDB: 8BU5), 21195 in white (PDB: 8BU7), 919278 in orange (PDB: 8BUA). On the right, the chemical structures of each of the compounds; the gluing moiety is highlighted in red. The picture is produced using the 3decision® highlight mode, which only shows the different portions of the superposed proteins to easily spot the differences among the binding sites. B) Common chemical motifs for cyclin K molecular glue degraders. In green is highlighted the acceptor-donor motif interacting with the CDK12 hinge region; in red is the aromatic gluing moiety, bound to the hydrogen bond donor.
In this study, the researchers could rationally design more potent and selective compounds than CR8 driven by structural considerations and find many new starting scaffolds for further optimization. Also, the impressive number of new crystal structures determined in this study contributes to our general understanding of the structural basis of molecular glue degraders.
Reference
Kozicka, Z., Suchyta, D.J., Focht, V. et al. Design principles for cyclin K molecular glue degraders. Nat Chem Biol (2023). https://doi.org/10.1038/s41589-023-01409-z