Application Notes

7 A P P L I C A T I O N N O T E Dose-Response and Affinity Determination. Selected hits were subjected to dose-response screening using a 12-point, 1:1 dilution series in duplicate, starting at 10 µM, with 5 nM labelled MEK1 in assay buffer. Hit picking from pre-dilution plates, and subsequent dilutions series in Dianthus uHTS 1536 well plates were performed using an Integra Assist+, programmed with ViaLab (v. 3.3.1). 5 µL of 7.5 nM MEK1-L021 (final concentration 5 nM) was added to the wells. Binding affinities (K d ) were estimated from fitted binding curves as described in [1]. Hit Confirmation via Thermal Shi Assay. Thermal shi assays to monitor melting temperature shis were performed in Andromeda X, monitoring protein unfolding as a function of temperature. 2.5 µL of 30 µM hits were mixed with 5 µL of 7.5 nM MEK1-L021 to a final concentration of 10 µM compound, 5 nM MEK1-L021 in duplicate. Samples were loaded in High Sensitivity Capillaries and were loaded onto the temperature unit of the Andromeda X. With an excitation of 100 %, spectral shi of the samples was monitored while performing a temperature ramp from 25–95 °C at 4 °C/min (An.X Control v. 1.0). Melting scans were loaded in to An.X Analysis (v. 1.0) and duplicate measurements merged. Data was fitted to a 2-state fit, and T m reported. ADP Glow Assays (Promega Corporation, Madison WI, USA) were performed in 10 mM TRIS, pH 7.5, 8 mM MgCl 2 . 54 nM MEK1 protein was preincubated with either 10 µM U0126 control compound, 10 µM Ligand or DMSO reference for 10 minutes at room temperature, before addition of 100 µM ATP and 250 µg / µL CHKtide and incubation at 25 °C for 60 minutes. The reaction was quenched with an equal volume of ADP Glow detection reagent (5 µL) for 40 minutes, prior to the addition of 10 µL Kinase Detection Reagent. Assays were incubated in the dark for 30 minutes before luminescence measurement on a CLARIOStar Plate Reader (BMG LabTech, Ortenberg, Germany), with an integration time of 0.5 s, gain 3600 and 12 mm focal height. Samples were blanked to an enzyme free sample blank. RFU was converted to ATPase Activity Inhibition comparing samples to DMSO reference using Equation 2 where S is the Sample Luminescence, and x is the mean of the negative DMSO control signal.

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