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

Abstract

Cyclin-dependent kinases (CDKs) constitute a family of 20 serine/threonine protein kinases that play pivotal roles in the regulation of numerous important molecular and cellular processes. CDKs have long been considered promising therapeutic targets in a variety of pathologies, and the recent therapeutic success of CDK4/6 inhibitors in breast cancers has renewed interest in their therapeutic potential. Small-molecule inhibitors have been identified for every human CDK, except for CDK10. The only recent discovery of an activating cyclin (CycM) for CDK10 enabled us to identify its first phosphorylation substrates and gain insights into its biological functions. Yet, our knowledge of this kinase remains incomplete, despite it being the only member of its family that causes severe human developmental syndromes, when mutated either on the cyclin or the CDK moiety. CDK10 small-molecule inhibitors would be useful in exploring the functions of this kinase and gauging its potential as a therapeutic target for some cancers. Here, we report the identification of an optimized peptide phosphorylation substrate of CDK10/CycM and the development of the first homogeneous, miniaturized CDK10/CycM in vitro kinase assay. We reveal the ability of known CDK inhibitors, among which clinically tested SNS-032, riviciclib, flavopiridol, dinaciclib, AZD4573 and AT7519, to potently inhibit CDK10/CycM. We also show that NVP-2, a strong, remarkably selective CDK9 inhibitor is an equally potent CDK10/CycM inhibitor. Finally, we validate this kinase assay for applications in high-throughput screening campaigns to discover new, original CDK10 inhibitors.

CDK10 in Gastrointestinal Cancers: Dual Roles as a Tumor Suppressor and Oncogene

“…Discovery of a small molecule inhibitor for CDK10 would facilitate further exploration of its biological functions and affirm its candidacy as a therapeutic target, specifically for CRC. Recently, Robert et al described a novel CDK10/Cyclin M in vitro activity assay (81). This luminescence-based assay uses a synthetic peptide phosphorylation substrate for the CDK10/Cyclin M complex.…”

“…Recently, Robert et al. described a novel CDK10/Cyclin M in vitro activity assay ( 81 ). This luminescence-based assay uses a synthetic peptide phosphorylation substrate for the CDK10/Cyclin M complex.…”

“…It has been shown to inhibit CDK1, CDK2, CDK4, CDK6, CDK9 and CDK10. While flavopiridol can inhibit CDK10, the IC50 for flavopiridol inhibition against CDK10/Cyclin M is less potent than flavopiridol inhibition of other CDKs ( 81 ). In vitro studies in gastrointestinal cells demonstrated that flavopiridol was effective in inducing apoptosis through blockage of cell cycle progression at G 1 ( 82 , 83 ).…”

“…Additional CDK inhibitors such as dinaciclib, SDS-032, AZD4573, AT7519 and riviciclib were all tested on CDK10/Cyclin M, however none of these potently inhibited CDK10 kinase activity ( 81 ). Ibrahim et al synthesized novel flavopiridol analogs and assessed their inhibitory activity on CDK2, CDK5 and CDK9 ( 86 ).…”

Targeting CDK9 for Anti-Cancer Therapeutics

“…to attain their kinase activity. Presently, this family comprises of 21 members, further classified into three sub-types, broadly based on their regulatory functions: (1) regulators of the cell-cycle-CDKs 1, 2, 4 and 6; (2) regulators of transcription- CDKs 7,8,9,12,13 and 19; (3) regulators of diverse or as yet undefined functions- CDKs 5,10,11,14,15,16,17,18 and 20 [1][2][3]. At present, 29 cyclins have been identified in humans.…”