These businesses are developing novel therapies for cancer. 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 around 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 Hoechst 33258 analog 5 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 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, initial CDK10 inhibitors. kinase assay. We also unveil Hoechst 33258 analog 5 the ability of known CDK inhibitors, some of which tested in clinical trials, to potently inhibit CDK10/CycM kinase assays with recombinant GST-CDK10/Strep2-CycM around the peptide substrate TNFSF13B library in the presence of ATP[-32P]. We carried out these reactions in 10 mM MgCl2, 25mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM DTT, and 50 g/mL heparin at 30C for 90 min. The peptides, which are biotinylated at their C-termini, were blotted onto streptavidin-conjugated membranes and imaged with a Typhoon FLA 7000 phosphorimager. Detailed information around the protocol is provided elsewhere (Turk et al., 2006). We quantified the spot densities from the blot array and we normalized by each row. We used these values to score the amino acid sequence surrounding each identified phospho-site and we applied them to predict highest scoring substrate peptides. Protein Kinase Assays CDK10/CycM We performed the kinase reaction assays in white opaque, flat-bottom 384-well microplates (Optiplate, Perkin Elmer) in a total volume of 6 L, adding kinase reaction buffer (final concentrations: 25 mM Tris-HCl pH7.5, 10 mM MgCl2, 1 mM EGTA, 1mM DTT, 50 g/mL Heparin, 3 g/mL BSA), DMSO 1% (or molecules diluted in 1% DMSO), recombinant purified GST-CDK10/Strep2-CycM (50 nM) and peptide substrate (150 M) (except when indicated otherwise in figure legends), and ATP 10 M (except for the Km, ATP determination assay). We incubated the plates 30 min at 30C and we measured the protein kinase activity using the ADP-Glo kinase assay (Promega). We added 6 L of ADP-Glo reagent and we incubated the plates 50 min at room temperature. We then added 12 L of kinase detection reagent and Hoechst 33258 analog 5 we incubated the plates 60C90 min at room temperature. We mildly agitated the plates during all incubation actions. We measured the luminescence using an Envision plate reader (Perkin Elmer). All measurements were performed in triplicates except for the measurements of the IC50 values, which were performed in duplicates. For the validation of the screening assay in a 384-well plate, we used columns 1 and 24 for a no-substrate control and we filled columns 2C23 in an interleaved format of high (DMSO), low (NVP-2) and no (vacant wells) signals, leaving the first and last two rows vacant. We filled the plate using a Janus Expanded automated liquid handling system (Perkin Elmer). CDK9/CycT1 We followed a similar procedure, using 80 M of CDK7/9tide peptide (YSPTSPSYSPTSPSYSPTSPSKKKK) as a substrate and 17 nM of enzyme. Results Identification of Peptide Phosphorylation Substrates We set out to develop a non-radioactive CDK10/CycM kinase assay amenable to high-throughput screening campaigns. Based on prior successful experiences with other kinases, we opted for a luminescent assay that quantifies ADP Hoechst 33258 analog 5 produced by a kinase reaction with.