Inhibitory assays

A panel of 10 kinases consisting of FAK, Flt3, JAK2, JAK3, CSF-1R, SRC, VEGFR3, Aurora A, LIMK1, and ROCK2 was initially used to evaluate kinase inhibition profiles. 15 μL assay reactions were performed in Greiner brand white 384-well, low-volume plates. All reactions contained assay buffer (10 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% (v/v) Tween-20, 50 μM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol), a N-terminally biotinylated peptide substrate (concentration 111 nM), and ATP at a concentration equaling the KM (ATP) of the kinase. The compounds were added in a volume of 100 nL from an 11-point-dilution series prepared in DMSO, positive and negative control reactions receiving the same volume of DMSO without compound. The kinases were added at predetermined concentrations, generally ranging between 0.2 and 8 nM, with the enzyme being omitted from negative control reactions. The reactions were incubated for 90 min at 30 ℃ and stopped by adding 5 μL of Stop buffer (10 mM HEPES pH 7.4, 25 mM NaCl, 100 mM EDTA, 0.01% (v/v) Tween-20) containing streptavidin-coated donor and antiphosphotyrosine (P-Tyr-100) acceptor beads. Plates were incubated for 4–6 h before being read on a PerkinElmer EnVision plate reader in HTS Alphascreen mode.

Cell lines

MDA-MB-231 Cells

Preparation method

The solubility of this compound in DMSO is >21.9 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

1 μM

Applications

CTX-0294885 exhibited inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

CTX-0294885 is a a novel bisanilino pyrimidine; exhibits inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent.Target: Kinase capture reagentUse of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ～10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies.

References:
[1]. Zhang L, et al. Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling. J Proteome Res. 2013 Jul 5;12(7):3104-16.