Atp competitive kinase inhibitors

Thermal stabilization assays were performed as described^{28 (link), 56 (link)}. Purified VRK1_3-364 and VRK2_14-335 were screened against a library of 378 structurally diverse, cell permeable ATP-competitive kinase inhibitors purchased from Selleckchem (Houston, TX, USA; catalog No. L1200). DSF experiments were performed in a 384-well plate format. Each well contained 25 μL of 1 μM kinase in potassium phosphate buffer and the Protein Thermal Shift dye at the recommended concentration of 1:1000 (Applied Biosystems; the composition of the buffer and the dye solutions are not disclosed). Compounds (10 mM) in DMSO were added to 16 μM final concentration to complete a total assay volume of 25.8 μL (3.1% final DMSO). Plates were sealed using optically clear films and transferred to a QuantStudio 6 qPCR instrument (Applied Biosystems). Fluorescence intensity data were acquired in a temperature gradient from 25 to 95 °C at a constant rate of 0.05 °C/sec and protein melting temperatures were calculated based on a Boltzmann function fitting to experimental data, as implemented in the Protein Thermal Shift Software (Applied Biosystems). Protein in 3.1% DMSO was used as a reference.

CAMKK1-KD protein was screened against a library of 378 structurally diverse and cell-permeable ATP-competitive kinase inhibitors available from Selleckchem (Houston, TX, United States; catalog No. L1200). DSF measurements were made in a 384-well plate. Each well contained 20 μL of 1 μM kinase in 100 mM potassium phosphate pH 7.0, 150 mM NaCl, 10% glycerol and the Applied Biosystems Protein Thermal Shift dye at the recommended concentration of 1:1000.
The compounds, previously solubilized in DMSO, were used at 10 µM final concentration and 0.1% DMSO. Plates were sealed using optically clear films and transferred to a QuantStudio 6 qPCR instrument (Applied Biosystems). The fluorescence intensity was measured during a temperature gradient from 25 to 95 °C at a constant rate of 0.05 °C/s and protein melting temperatures were calculated based on a Boltzmann function fitting to experimental data, as implemented in the Protein Thermal Shift Software (Applied Biosystems). Protein in 0.1% DMSO was used as a reference. Compounds that caused a shift in melting temperature of the protein (ΔT_m) of 2 °C or higher compared to the reference were considered positive hits.