U. Grädler, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127551
Fig. 1. Published ERK1/2 inhibitors (1–4).
and 14 µM) against ERK3 in line with inactivity in the DX binding assay
(Kd > 1 µM, Table 1). Compounds 5–9 demonstrated MST binding in
the Kd range of 120 nM up to 5.6 µM and in correlation with DX binding
data. ERK3 co-crystallization of inhibitors 7–9 failed, but we solved the
inhibitor binding, we conducted MST binding experiments with dif-
ferent wildtype and S189E mutant ERK3 proteins either produced from
E. coli or insect cells (Table 1). Inhibitors 5–9 demonstrated similar Kds
against different ERK3 proteins indicating no major impact on binding
mode by Ser189 phosphorylation. Based on the ERK3•7 structure, we
selected further analogs from our corporate compound library for
testing in a more functional biochemical ERK3 assay. We established an
ADP-Glo assay applying different ERK3 proteins (1-365, 1-471 and full-
length) together with MK5 kinase dead mutant (K51A) as substrate.31
Inhibitors 6–21 showed similar biochemical potencies using different
proteins in line with DX and MST binding data (Table 2). Compounds
13–16 in Supplement Scheme 2–5).
structure with 7 by soaking into ERK3 (9–327, L290V) apo crystals
(Table S2).
The ERK3‧7 crystal structure revealed a dimer in the
asymmetric unit (ASU) with clear electron density for the inhibitor in
the ATP site of chain A, while electron density for 7 was lacking in
chain B (Fig. S1). Inhibitor 7 comprises two classical and one non-
classical H-bonds of the aminopyrimidine ring towards Met111 and
Glu109 of the hinge region (Fig. 2). The triazolo[4,5-d]pyrimidin-5-
and π–π-interactions with Phe159. The 4-aminocyclohexyl ring of 7 is
sandwiched in vdW contacts between Leu26 and Thr113, whereas the
amino group is solvent exposed. The methoxyphenyl ring of 7 adopts
vdW interactions to Val34 of the GC loop and is in crystal contacts with
Glu163 and Asp164 of chain B (Fig. 2). We have no experimental evi-
dence, if the ERK3 dimer found in the ASU of the crystal structure is
also present in solution to draw any further conclusions on the potential
relevance of this structural arrangement (Fig. S2). The activation loop
in chain A is well defined except for residues 179–186 revealing Asp171
and Phe172 in DFG-in conformation. The orientations of the R/C-
(active CIDI: αC-helix-in/DFG-in) in the ERK3•7 structure.28–30
Synthesis of compounds 5–12 and 17–21: a) R1-NH2, NEt3 (1 eq.),
THF, 0–23 °C, o/n, b) H2, sponge-Ni, THF, RT, o/n, c) Fuming HCl,
Sodium Nitrite (2 eq.), 0 °C, 1 h, d) R2-NH2, methoxyethanol, 80 °C, 3 h.
All four stereoisomers of 1,3-diaminocyclopentyl derivatives 9–12
were synthesized and revealed the R,R-enantiomer 9 as most potent
analogue (IC50 range 157–274 nM). Core variations of Nitrogen and
Carbon altering in 1,3-diaminocyclopentyl derivatives 13–16 indicated
significant potency drops. Significant impact on ERK3 potency was
found for 17 and 18 with 1-pyridin-4-yl-pyrrolidin-3-yl-amine sub-
stituents as S-enantiomer (17: IC50s 1.8–4.5 µM) or R-enantiomer (18:
IC50s 73–93 nM). Analogs 19–21 bearing methylpiperidin-pyrazole
substituents in the amino position and variable moieties in the triazolo
position were also very potent (e.g. 20: IC50s 30–55 nM). ERK3 crystal
structures with 18 and 21 were solved from soaking into apo crystals
obtained from optimized crystallization conditions comprising a tet-
ramer in the ASU (Table S2). Inhibitors 18 and 21 were clearly defined
by electron densities in all ATP sites of the tetramer (Fig. S4) with
identical binding modes in comparison to 7 (Fig. 3).
The S-E-G motif is well defined by electron density and phosphor-
ylation of Ser189 can be ruled out for steric reasons (Fig. S3). Quali-
tative MS peptide mapping analysis of ERK3 wildtype protein from E.
coli showed Ser189 phosphorylation, which was not detectable by
Western blot analysis with pSer189 antibody indicating Ser189 phos-
phorylation levels below limit of detection (Fig. S6-S8). In contrast,
Ser189 phosphorylation was confirmed by Western Blot using the same
antibody in ERK3 full-length and ERK3 (9–327) wildtype proteins
produced from insect cells in line with previous reports (Table S1).13 To
further investigate the potential impact of Ser189 phosphorylation on
electron density revealing slightly shifted positions of the 4-pyridine
ring from superimposition of all chains of the tetramer. The same
2