Structure-guided design of potent and selective pyrimidylpyrrole inhibitors of extracellular signal-regulated kinase (ERK) using conformational control

Article abstract of DOI:10.1021/jm900630q

The Ras/Raf/MEK/ERK signal transduction, an oncogenic pathway implicated in a variety of human cancers, is a key target in anticancer drug design. A novel series of pyrimidylpyrrole ERK inhibitors has been identified. Discovery of a conformational change

Full text of DOI:10.1021/jm900630q

SUPPORTING INFORMATION
*
Alex M. Aronov, Qing Tang, Gabriel Martinez-Botella, Guy W. Bemis, Jingrong Cao, Guanjing
Chen, Nigel P. Ewing, Pamella J. Ford, Ursula A. Germann, Jeremy Green, Michael R. Hale,
Marc Jacobs, James W. Janetka, Francois Maltais, William Markland, Mark N. Namchuk,
Suganthini Nanthakumar, Srinivasu Poondru. Judy Straub, Ernst ter Haar, and Xiaoling Xie
Vertex Pharmaceuticals Inc., 130 Waverly St., Cambridge, Massachusetts 02139-4242
Contents of Supporting Information:
1. Description of biochemical and cellular assays.
a. Kinase counterscreening data for compound 11e.
2. PK data for 11e.
3. Crystallographic information.
S-1

1. Description of biochemical and cellular assays.
ERK Inhibition Assay: Compounds were assayed for the inhibition of ERK2 by a
spectophotometric coupled-enzyme assay as described in Fox et al. (Fox, T., Coll, J.T., Xie,
X., et al. Protein Science 1998, 7, 2249-2255). In this assay, a fixed concentration of activated
ERK2 (10 nM) was incubated with various concentrations of the compounds in DMSO
(2.5%) for 10 min. at 30 °C in 0.1 M HEPES buffer, pH = 7.5, containing 10 mM MgCl2, 2.5
mM phosphoenolpyruvate, 200 ꢀM NADH, 150 ꢀg/mL pyruvate kinase, 50 ꢀg/mL lactate
dehydrogenase and 200 ꢀM erktide peptide. The reaction was initiated by the addition of 65
ꢀM ATP. The rate of decrease of absorbance at 340 nM was monitored. The IC50 was
evaluated from the data as a function of inhibitor concentration.
Kinase Counterscreening: % Inhibition values below were measured at Upstate/Millipore (2
ꢀM) and Nanosyn (1 ꢀM).
Table S1. Kinase counterscreening data for 11e.
OH
O
N
Cl
N
N
H
NH
HN
Cl
F
Assay
ERK2
GSK3
AURA
CDK2
FLT3
KI, ꢀM
< 0.002
0.40
0.54
0.85
1.4
ROCK1
JNK3
1.4
1.4
S-2

TAK1
PKA
3
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 5
> 5
> 10
> 10
> 10
> 10
SRC
JAK2
JAK3
SYK
MET
IRAK4
AKT3
KDR
MEK1
P38α
COT
ITK
MEKK3
PLK1
ZAP70
IKKi
NIK
CIT
FAK
IRAK1
MLK2
S-3

NAK
PDK1
SGK1
> 10
> 10
> 10
Additional counterscreening data (% INH at fixed concentration)
ABL1 (%INH @ 1 uM): 3
AKT1 (%INH @ 1 uM): 0
AKT2 (%INH @ 1 uM): 0
AKT3 (%INH @ 1 uM): 0
AMPK (%INH @ 2 uM): 5
BLK (%INH @ 2 uM): 7
BMX (%INH @ 1 uM): 3
BRSK1 (%INH @ 1 uM): 0
BRSK2 (%INH @ 1 uM): 2
BTK (%INH @ 1 uM): 5
CAMKII (%INH @ 2 uM): 1
CAMKIV (%INH @ 2 uM): 0
CDK1 (%INH @ 1 uM): 7
CDK5 (%INH @ 1 uM): 24
CHK1 (%INH @ 2 uM): 40
CK1 (%INH @ 2 uM): 25
CK2 (%INH @ 2 uM): 0
CLK3 (%INH @ 1 uM): 9
CSK (%INH @ 2 uM): 0
CSK (%INH @ 1 uM): 3
DAPK1 (%INH @ 1 uM): 2
DCAMKL2 (%INH @ 1 uM): 1
DYRK1A (%INH @ 1 uM): 3
DYRK2 (%INH @ 1 uM): 3
EGFR (%INH @ 1 uM): 6
EPH-B2 (%INH @ 1 uM): 0
EPH-B4 (%INH @ 1 uM): 0
FER (%INH @ 1 uM): 3
FGFR1 (%INH @ 1 uM): 3
FGFR2 (%INH @ 1 uM): 12
FGFR3 (%INH @ 1 uM): 8
FGFR4 (%INH @ 1 uM): 0.3
FLT-1 (%INH @ 1 uM): 0
FLT-4 (%INH @ 1 uM): 8
FMS (%INH @ 1 uM): 6
FYN (%INH @ 2 uM): 11
S-4

HCK (%INH @ 1 uM): 3
IGF1R (%INH @ 1 uM): 2
IKKa (%INH @ 2 uM): 0
IKKb (%INH @ 2 uM): 8
INSR (%INH @ 1 uM): 4
IRR (%INH @ 1 uM): 0
JNK1 (%INH @ 2 uM): 34
JNK2 (%INH @ 2 uM): 43
KIT (%INH @ 1 uM): 8
LCK (%INH @ 2 uM): 3
LYN (%INH @ 2 uM): 7
MAP4K4 (%INH @ 1 uM): 18
MAPKAPK-2 (%INH @ 1 uM): 0
MAPKAPK-3 (%INH @ 1 uM): 5
MARK1 (%INH @ 1 uM): 12
MER (%INH @ 1 uM): 9
MINK (%INH @ 1 uM): 18
MKK4 (%INH @ 2 uM): 69
MKK6 (%INH @ 2 uM): 9
MKK7 (%INH @ 2 uM): 6
MSK1 (%INH @ 1 uM): 7
MSK2 (%INH @ 1 uM): 6
MST1 (%INH @ 1 uM): 4
NEK1 (%INH @ 1 uM): 6
NEK2 (%INH @ 1 uM): 3
P38β (%INH @ 2 uM): 25
P38δ (%INH @ 2 uM): 54
P38γ (%INH @ 2 uM): 56
P70S6K (%INH @ 2 uM): 18
PAK1 (%INH @ 1 uM): 3
PAK2 (%INH @ 1 uM): 0
PAK3 (%INH @ 1 uM): 3
PAR-1Bα (%INH @ 1 uM): 5
PASK (%INH @ 1 uM): 19
PDGFRα (%INH @ 2 uM): 3
PDK1 (%INH @ 2 uM): 10
PHKγ2 (%INH @ 1 uM): 4
PIM1 (%INH @ 1 uM): 0
PIM2 (%INH @ 1 uM): 3
PKCβ1 (%INH @ 1 uM): 4
PKCη (%INH @ 1 uM): 2
PKCα (%INH @ 2 uM): 12
PKCβ2 (%INH @ 2 uM): 4
PKCγ (%INH @ 2 uM): 11
PKCθ (%INH @ 2 uM): 17
S-5

PRK2 (%INH @ 2 uM): 14
PRKD1 (%INH @ 1 uM): 43
PRKD2 (%INH @ 1 uM): 50
PRKD3 (%INH @ 1 uM): 21
PRKX (%INH @ 1 uM): 3
PYK2 (%INH @ 1 uM): 6
RAFc (%INH @ 2 uM): 3
RET (%INH @ 1 uM): 3
ROCK II (%INH @ 2 uM): 57
RON (%INH @ 1 uM): 1
ROS (%INH @ 1 uM): 3
RSK1 (%INH @ 1 uM): 4
RSK2 (%INH @ 1 uM): 6
RSK3 (%INH @ 1 uM): 7
RSK4 (%INH @ 1 uM): 11
SGK1 (%INH @ 1 uM): 15
SGK2 (%INH @ 1 uM): 41
SGK3 (%INH @ 1 uM): 2
TBK1 (%INH @ 1 uM): 3
TIE2 (%INH @ 1 uM): 0
TSSK2 (%INH @ 1 uM): 3
TYRO3 (%INH @ 1 uM): 0
YES (%INH @ 1 uM): 5
ERK Inhibition Cell Proliferation Assay:
The cell line, Colo205, was obtained from ATCC. Colo205 proliferation was measured by 3H-
thymidine incorporation. The cells were plated at a concentration of 10,000 cells/well in a 96-
well plate using growth media, RPMI 1640 containing 10% FBS. Serially diluted compounds
were added. The cells and compounds were incubated for 48 hours at 37°C incubator. After
48 hours 0.4 µCi of 3H-thymidine (NEN, Cat. #NET-027) was added to each wells for 8 hours
and returned to the 37°C incubator. The cells were harvested using a Tomtec 96-well cell
harvester and the CPM was determined using the Wallac 1205 BETAPLATE liquid
scintillation counter. The IC50 is the 50% inhibition of control (cells with vehicle).
2. PK data for 11e.
Mol.Wt. 499; logP = 5.1; logD = 4.3; solubility (pH 7.4) 10 ꢀM; human plasma PB 99% at 1
ꢀM.
S-6

Rat iv PK (3 mg/kg; DMI): Cl = 24 mL/min/kg; t1/2 = 3.2 h.; Vss = 4.9 L/kg; AUC (0-8h) =
1.6 ꢀg*h/mL; C4h = 123 ng/mL; C8h = 50 ng/mL.
Rat po PK (10 mg/kg; 0.5% MC, 1% SLS): F = 65%; t1/2 = 3 h; C4h = 329 ng/mL; C8h = 267
ng/mL.
Mouse iv PK (5 mg/kg; DMI): Cl = 55 mL/min/kg; t1/2 = 1.6 h.; Vss = 5.6 L/kg; AUC (0-8h)
= 1.6 ꢀg*h/mL; C4h = 79 ng/mL; C8h = 17 ng/mL.
Mouse po PK (33 mg/kg; 0.5% MC, 1% SLS): F = 67%; t1/2 = 4.4 h; C4h = 488 ng/mL; C8h =
122 ng/mL.
3. Crystallographic Information.
Methods: Erk-2 protein was expressed, purified and crystallized as described in Fox et al.
(Fox, T., Coll, J.T., Xie, X., et al. Protein Science 1998, 7, 2249-2255). Briefly, full-length
ERK1 (Met1-Ser360) was expressed as a fusion with an N-terminal (His)6 tag in E. coli. The
protein was purified using a combination of metal-affinity chromatography (Talon resin) and
anion-exchange chromatography (Q sepharose resin). Crystals were grown by vapor diffusion
using a reservoir solution containing 100 mM MES buffer, pH 6.5, 26-28% PEG-MME 2000,
200 mM ammonium sulfate and 20mM 2-mercaptoethanol.
JNK-3 protein was expressed, purified and crystallized as described in Xie et al. (Xie, X., Gu,
Y., Fox, T., et al. Structure 1998, 15, 983-991). Briefly, a truncated JNK-3 construct (Ser40-
Glu402) was expressed in E. coli. The protein was purified using cation-exchange
chromatography (SP Sepharose resin). Crystals were grown by vapor diffusion using a
reservoir solution containing 20-24% PEG-MME 550, 10% ethylene glycol, 100 mM HEPES
buffer, pH 7.5, and 20mM 2-mercaptoethanol.
X-ray data were collected on an Raxis IIC image plate and processed using
DENZO/SCALEPACK. Model building and refinement were performed using Quanta and
CNX respectively (Accelrys).
S-7

X-ray statistics:
protein
ERK2
ERK2
GSK3
compound
9a
2
2
Data collection
Space group
Unit cell parameters
P212121
44.1 x 70.3 x 119.6 Å
P212121
43.9 x 70.8 x 118.5 Å
P212121
82.75 x 86.1 x
178.29 Å
37.3 – 2.3
6.3
Resolution (Å)
Redundancy
20 – 2.5
3.4
20 - 2.2
3.4
Completeness (%)*
95.5 (92.0)
0.043 (0.29)
12.9 (3.8 )
95.2 (75.4)
0.065 (0.28)
19.5 (7.2)
95.8 (78.9)
0.057 (0.26)
22.3 (7.2)
Rmerge
*
<I/σ>*
Refinement
Reflections used
Test reflections
R-factor
12456
1012
0.207
0.248
18070
1421
0.214
0.257
51693
2635
0.179
0.222
Free R-factor
RMS deviations
Bond lengths (Å)
Bond angles (°)
Dihedral angles (°)
0.008
1.05
21.1
0.009
1.16
21.6
0.012
1.22
17.6Í
*Values for the highest resolution shell are shown in parentheses.
Rmerge I(hkl) − I(hkl) / I(hkl)i over i observations of reflection hkl.
=
∑ ∑
∑ ∑
i
hkl
i
hkl
i
R-factor =
F − F
/
F
where Fobs and Fcalc are the observed and calculated
∑
∑
obs
calc
obs
structure factors, respectively. Free R-factor is calculated from a randomly chosen subset of
reflections not used for refinement.
S-8