1 4
A P P L I C A T I O N N O T E
Thermal Shi Assay. We performed Spectral Shi Differential Scanning Fluorimetry (SpS.DSF) mea-
surements using an Andromeda X instrument to determine the melting inflection point of MEK1 in
the presence of either 10 µM hit compounds, the U0126 positive control, or negative control DMSO
(Figure 7A). 67 compounds from Prio 1 and Prio 2 induced a significant shi in temperature inflection
point (IP) compared to DMSO (pink area Figure 7B).
Plotting IP against calculated compound affinity (Figure 7C) highlights that there is no correlation
between temperature stabilization of a protein-ligand complex and the affinity for compound.
Figure 6. A. Example dose response cur ves for ligands with a variety of affinity. Assays were performed in duplicate
(except U0126 performed in quadruplicate). Dashed black lines are theoretical and described by the Kd Fit equation.
B. Histogram of calculated affinities for the Tier 1-4 hits, insert, percent active (i.e compounds with at least weak
binding) from each tier. C. Breakdown of activities within each tier of compounds, where weak binders showed at
least some activity, and strong binders with a Kd < 10 µM.
