| Assay Method Information | |
| | Thermal Shift Assay |
| Description: | Recombinant kinase domains of STK3 and STK4 at 2.0 μM in 10 mM HEPES pH 7.5 and 500 mM NaCl were mixed with 10 UM of the test inhibitors. Temperature-dependent protein unfolding profiles were measured using a Real-Time PCR Mx3005p machine (Stratagene: San Diego, CA, USA). Data evaluation and melting temperature calculation were performed using Protein Thermal Shift Software v1.2. To further evaluate binding of these compounds to STK3/4, differential scanning fluorimetry (protein thermal shift using ThermoFluor technology) assay was used to benchmark the capacity to stabilize the kinase domain. The melting temperature (Tm) of STK3 or STK4 was measured in the presence of vehicle (DMSO) or 6, 11 or 23. A Tm shift of >7.4° C. was obtained for all three potent compounds tested, and comparable results were obtained for compounds 5, 7, 15, and 16. The thermal shift results corresponded closely to the potencies measured using the ADP-Glo kinase assay. |
| Affinity data for this assay | |
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