Abstract 2821: Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes

Abstract

Members of the family of cyclin dependent kinases (CDKs) have been recognized as pivotal regulators of cell cycle progression for more than 20 years. Concordant to their central role in the control of cell division they have been in the focus of research of proliferation associated diseases ever since, most prominently amongst these cancer. Although initial results obtained from first and second generation, low specificity CDK inhibitors (e.g. Flavopyridol, Roscovitine, Dinaciclib, AT7519, R547) have been sobering the recent approval of the first CDK-inhibitor Palbociclib for the treatment of certain forms of breast cancer clearly demonstrates the suitability of cell cycle CDKs as targets in oncology. Furthermore, in addition to cell cycle CDKs a second group of CDKs have been shown to have important roles in the regulation of gene transcription, and several of the “transcriptional” CDKs have become interesting targets in oncology. Recent results underline the notion that for being effective in the treatment of cancer, CDK inhibition requires very high specificity towards the respective target CDK(s). For example CDK1 knockdown or CDK9 inhibition have been shown to be synthetically lethal in combination with MYC overexpression. Selectivity of compounds within the family of CDKs could so far only be tested using a quite limited number of CDK-Cyclin complexes expressed in human cells. To date there are 20 CDK genes and at least 17 different Cyclin genes described, many of which give rise to different variants, e.g. there are 3 D-type cyclins, two A- and E-type cyclins etc.. Experimental data indicates that at least 50-60 different, biologically relevant CDK-Cyclin complexes may exist, but only a limited number of these are available for biochemical testing of drug candidates so far. We have recombinantly expressed and purified 28 different CDK-Cyclin complexes, covering a significant part of the CDK family, and established in-vitro kinase-activity assays for these recombinant enzymes. The resulting CDK panel represents the most comprehensive array for biochemical testing of this enzyme group currently available. We characterized the specificity of several CDK inhibitors that have been or are currently in preclinical or clinical development with this CDK collection. Results will be presented showing the specificity of these inhibitors not only for CDKs but also for CDKs complexed to different Cyclins. In several cases we could detect signifcant differences in the inhibition of the same CDK complexed to different Cyclins, e.g. a 10fold difference was seen for CDK6 complexes with Cyclin D1-3. A >100 fold difference was detected for CDK3 complexed to either Cyclin E1 or Cyclin C. This screening panel allows generating data on compound selectivity early in development, diminishing the risk of designing a compound with suboptimal target specificity. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Christian Beisenherz-Huss, Diane Kraemer, Carolin Heidemann-Dinger, Constance Ketterer, Chris Eckert, Michael H.G. Kubbutat. Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2821.

MiR-219-5p suppresses cell proliferation and cell cycle progression in esophageal squamous cell carcinoma by targeting CCNA2

“…Under complex pathological conditions, the entry of cancer cells and their progeny into uncontrolled cell growth are driven by a limited number of “mitosis events” [26]. Cyclin family members have long been considered as critical regulators of cell cycle progression [27]. CCNA2 is a ubiquitously expressed member of the cyclin family [28, 29].…”

Development of a CDK10/CycM in vitro Kinase Screening Assay and Identification of First Small-Molecule Inhibitors

“…Strikingly, among the CDK family, only two CDKs currently appear in the class of proteins for which no small-molecule inhibitor is available: CDK20 and CDK10 1 . However, a sub-nanomolar inhibitor that targets a few CDKs including CDK20 has recently been reported (Mueller et al, 2016).…”

AlphaFold accelerates artificial intelligence powered drug discovery: efficient discovery of a novel CDK20 small molecule inhibitor

“…AAPK-25 was also reported as a CDK20 inhibitor with a Kd value of 8020 nM 46 . Moreover, Mueller et al described several potential CDK20 inhibitors in their recent poster, including Palbociclib, Flavopiridol, Dinaciclib, and Roscovitine with IC50 values ranging from 1260 nM to 8680 nM 47 . Besides, they also identified another potent CDK20 inhibitor MER-128 with an IC50 value of 2 nM, however, the structure of this molecule was not disclosed 47 .…”

“…Moreover, Mueller et al described several potential CDK20 inhibitors in their recent poster, including Palbociclib, Flavopiridol, Dinaciclib, and Roscovitine with IC50 values ranging from 1260 nM to 8680 nM 47 . Besides, they also identified another potent CDK20 inhibitor MER-128 with an IC50 value of 2 nM, however, the structure of this molecule was not disclosed 47 . Figure 3 describes our generative procedures for the identification of CDK20 inhibitors starting from structure extracting to hit generation through the SBDD approach by utilizing Chemistry42 27, 48-55 .…”

“… 47 Besides, they also identified another potent CDK20 inhibitor MER-128 with an IC 50 value of 2 nM; however, the structure of this molecule was not disclosed. 47 Fig. 3 describes our generative procedures for the identification of CDK20 inhibitors starting from structure extraction to hit generation through the SBDD approach by utilizing Chemistry42.…”