1 5
A P P L I C A T I O N N O T E
ATPase Activity Inhibition Assays. Prio1 compounds only were tested with MEK1 and the model
substrate CHKtide for inhibition of ATPase activity using the ADP Glo assay. 40 Prio 1 compounds
showed significant inhibition of ATPase activity of MEK1 at 10 µM (Figure 8A and B). 22 compounds
indicated binding without inhibiting MEK1 ATPase activity (Figure 8B), while 17 also inhibited ATPase
activity. From this we can hypothesize that with the Spectral Shi binding assay we are able to find
binders which are not antagonists, i.e. binding at an allosteric site on MEK1.
Figure 7. Spectral Shift-based differential scanning fluorometr y to monitor thermal stability of MEK1. A. normalized
averaged temperature ramps of ratio 670 nm / 650 nm signal against temperature, grey traces are the prioritized hits;
with controls in color as indicated in legend. B. Inflection point distribution shown for each Hit Tier categor y. C.
Inflection point as determined by 2-state fit vs affinity K
d
measurement from Dianthus uHTS DRC assay. Pink shaded
area indicate the area of insignificance as determined by the inflection point for reference DMSO.
