6
A P P L I C A T I O N N O T E
MEK1 labeling. MEK1 (Crelux Prime Proteins, Munich, Germany) was labelled with Spectral Shi
Optimized Dye (NanoTemper Technologies, Cat #L-021) according to the standard protocol. Briefly,
10 µM of protein was labelled with a 3-fold excess of NHS ester reactive dye in 130 mM NaHCO
3
, 50 mM
NaCl, pH 8.3 with the addition of 10 mM MgCl
2
and 2 mM ATP to protect the active site of the protein,
and incubated at room temperature in the dark for 30 minutes, before excess dye was removed using
a 9 ml desalting column. MEK1-L021 was stored in optimized assay buffer (50 mM phosphate, 150
mM NaCl, 0.005% Tween-20, pH 7.4). Protein was flash frozen at -80 °C in 10 µL aliquots if not used
directly in assays.
Compound Library preparation. The HLL Representative Set of 5120 compounds was bought from
Enamine as 10 µL of 1 mM DMSO solutions in 384 well plates. The compound library was pre-diluted
to 30 µM in 35 µL assay buffer in 384 well U-bottom low volume plates (Corning Cat #4513).
N=2 10 µM Single Point Screen. From the pre-dilution plate, 2.5 µL was dispensed in duplicate
into Dianthus uHTS 1536 well plate (Cat #NT-P002). Following out-of-line compound dispense, the
plates were loaded on a self-built automation platform (see below) for target dispense (5 µL of 5
nM MEK1-L021), mixing, centrifugation, sealing, a 4 hour incubation at 25 °C, before Spectral Shi
measurement.
Raw data were analyzed using an internally developed Python-based analysis soware. Analysis
was performed in a web-based front end developed in Streamlit integrated with open-libraries such
as pandas, numpy, JSON, and internal NanoTemper analysis algorithms. A full description of the
analysis soware is below.
Assay quality statistics were monitored by recording the robust Z' score of each plate and fluorescence
CV of the DMSO reference wells. For hit picking, individual plates were normalized using the robust
Z score method [13], [14] . Compounds were prioritized based on reproducibility and signal strength
relative to controls. Full details of hit prioritization are provided in the results section.
Materials and Methods
