1 3
A P P L I C A T I O N N O T E
PRIO1 AND 2 HITS FURTHER CHARACTERIZATION.
Affinity Screening. Aer lead prioritization as
shown in Figure 4, 62 compounds in Prio1 and
(1.2 % of the library, tiers 1, 2 and 3), and 81
compounds in Prio2 (1.6 % of library, tier 4) were
taken forward for further characterization. Each
compound was tested against MEK1 in affinity
titrations. A 12 point, 1:1 dilution series of the
compounds in technical duplicate was gener-
ated from 10 µM to 5 nM and mixed with 5 nM
MEK1-L021 for Spectral Shi measurement (Fig-
ure 6, Supplementary Figure 1 and Supplemen-
tary Figure 2).
Out of 62 Prio1 hits, more than 50 % (35) of com-
pounds showed at least weak binding (where a
dose response curve was fitted). To further nar-
row down the hit list, a threshold of K
d
< 10 µM
Figure 5.n=2 screening consistency,
where black dashed line is line of unity.
Tier 1-3 hits were prioritized where both
replicates showed consistency, with
both replicates having a minimum RSDE
of > 4. Inconsistent hits are shown in
grey, where at least one of replicate had
a RSDE > 4.
yielded 19 compounds with at least 50 % satu-
ration at highest ligand concentration (Supple-
mentary Figure 1). Two compounds exhibited
sub-100 nM affinity.
From Prio2, 20 % (16) compounds showed at
least weak binding, with 9 of these with a K
d
low-
er than 10 µM (Supplementary Figure 2). Explor-
ing each tier individually (Figure 6C), tier 1 has
the highest number of strong binders, tier 2 and
3 are roughly equal, and affinity distribution dra-
matically drops off for tier 4.
