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499
(IC50) activity. An analog of this compound was successfully soaked
into a crystal of the EPHA4 kinase domain, confirming the pre-
dicted binding mode of this scaffold (pdb:2XYU). Selectivity
profiling against 124 protein kinases revealed that this compound
seems to be a moderately selective kinase inhibitor.
The potential applicability of this scaffold was recently
confirmed with the disclosure of a patent and publication by
a group of researchers from Merck [24,25]. The reported IC50 values
of the two compounds are consistent with the data presented in
this work and underscores the potential of our scaffold as a possible
new drug for the treatment of cancer and neuronal injuries by
inhibition of the EPHA4 receptor tyrosine kinase.
mixtures (20
bottom plates (Greiner, Stonehouse, UK) and contained 100 nM
His-tagged EPHA4-KD, 33 M ATP (determined as Km for this
kinase preparation), 200 g/mL E4:Y1 (Sigma, St. Louis, MO) as
ml) were prepared in black polypropylene 384 well flat
m
m
substrate and increasing concentrations of inhibitors. Assay buffer
(pH 7.4) contains 20 mM NaCl, 15 mM Hepes, 20 mM MgCl2, 1 mM
EGTA and 0.1% Tween20. After 75 min at 30 ꢂC, reagents A and B
from the kit were added and the fluorescence intensity was
measured with a Tecan infinite F200 Reader (Tecan, Grödig, Austria)
after 60 min incubation at 30 ꢂC, with an Ex of 535 nm and Em of
590 nm and 25 reads per well. Standard curves, ranging from 30 nM
to 75 mM ADP (dynamic range of the kit), were used as an internal
assay control. Background was measured in reactions containing no
substrate and subtracted from those containing E4:Y1. Dasatinib
was used as a positive control. For confirmed hits, the effects of the
inhibitors on the enzymes in the ADP detection step were deter-
mined in assay buffer and the reagents A and B only.
9. Experimental Section
9.1. Synthesis. General Remarks
Chemicals and reagents were obtained from commercial
suppliers and were used without further purification. Yields given
are isolated yields. Flash column chromatography was carried out
on a Biotage Isolera One flash chromatography system, using pre-
packed Biotage SNAP Cartidge KP-Sil columns with the UV detector
operating at 254 nm. All 1H NMR and 13C NMR spectra were
9.4. Crystallography
The crystal structure of apo-EPHA4 kinase domain and the
kinase domain bound to dasatinib have been elucidated. The
methods and results will be reported elsewhere (manuscript in
preparation). Briefly, purified apo kinase domain was used to grow
crystals at 20 ꢂC using the sitting-drop vapour-diffusion method.
The purified EPHA4 kinase domain was mixed with an equal
volume of a reservoir solution containing 15e21% polyethylene
glycol 10 K, 100 mM ammonium acetate and 100 mM bistris pH 5.5.
Rod-like crystals appeared after 1e3 days. Crystals of apo protein
were soaked in drops of compound 73 at 30 mM. After 6 h incu-
bation at 20 ꢂC, crystals were flash-frozen using 20% glycerol plus
the reservoir solution. Diffraction data was collected at 100 K at the
ESRF. Datasets at 2.12 Å resolution were merged and processed
using XDS [26] and Pointless [27] from the CCP4 [28] suite and the
structure was solved by molecular replacement using AMoRe [29].
The apo-EPHA4 kinase domain was used as a search model. The
structure was subsequently refined using PHENIX [30] and COOT
[31] was used for model building.
recorded on
a Bruker-250, Bruker-400 or Bruker-500 NMR.
Analytical HPLC-MS analyses were conducted using a Shimadzu LC-
20AD liquid chromatograph pump system with a Shimadzu SPD-
M20A UVevis detector with the MS detection performed with
a Shimadzu LCMS-2010 EV liquid chromatograph mass spectrom-
eter. The analyses were performed using the following conditions;
Xbridge (C18) 5
mm column (100 mm ꢀ 4.6 mm) with solvent A
(water with 0.1% formic acid) and solvent B (acetonitrile with 0.1%
formic acid), flow rate of 1.0 mL/min, start 5% B, linear gradient to
90% B in 5 min, then 5 min at 90% B, then 5 min at 5% A, total run
time of 15 min. Compound purities were calculated as the
percentage peak area of the analyzed compound by UV detection at
254 nm. The purity of synthesized compounds 3 and 61e74 was
confirmed to be ꢅ95%. HRMS analyses were conducted using an
Agilent 1200 series pump system with the MS detection performed
with a Bruker MicroTof Q mass spectrometer. Synthetic procedures
for the synthesis of 18e74 are presented in the Supporting
Information.
Appendix. Supplementary material
Detailed synthesis procedures. Spectral and chemical charac-
terization of compounds 33e74. Table with kinase selectivity
profiling results. Supplementary material associated with this
9.2. In silico screening
The pharmacophore features were assigned by the MOE-2007
Pharmacophore Query Editor, after selecting the atoms of interest
on both the ligands and protein. The aromatic feature was gener-
ated by manually selecting the atoms of the adenine ring of
AMPPNP and the thiazole ring of dasatinib. Default radii (1Å) were
used for the size of the features, except for features F4 and F8 (see
Fig. 2) for which a radius of 1.2Å was used. The actual set consists of
2,824,291 non-redundant compounds (both fragments and
ligands) collected from 31 vendors. MACCS Structural Keys (Bit
packed) were used for calculating fingerprints of hits. Jarvis-Patrick
clustering method was used as the fingerprint clustering algorithm.
GOLDScore was used as scoring function for GOLD docking. Typical
scores were between 12.1181 and 41.7002 and for the selected
compounds between 21.2187 and 39.0420.
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