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E. L. Michelotti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5274–5279
9. Procedure for enzymatic assay: The ATPase activity of
activated p38a was determined using a EnzCheck phos-
phate assay kit (Molecular Probe, OR, USA). The
reactions were carried out at 30 °C in 0.1 M Hepes buffer,
pH 7.6, containing 10 mM MgCl2 and 10% glycerol.
Unless otherwise indicated, the phosphorylated p38a
concentrations (Upstate, NY, USA) were maintained at
100 nM. Test compounds and controls were dissolved in
DMSO and then diluted with reaction buffer such that the
final DMSO concentration in enzymatic reaction was 1%.
Compounds were preincubated with p38a for 15 min and
enzymatic reactions were initiated by the addition of 1 U/
mL nucleoside phosphorylase, 200 lM nucleoside sub-
strate (MESG), and 150 lM ATP. The kinetic analyses
were conducted in a 96-well plate on a Molecular Devices
spectrophotometer (Molecular Devices Corporation, CA,
USA). The IC50 was defined as the concentration of the
test compound that caused a 50% decrease in the maximal
inhibition of p38a activity and was calculated using
GraphPad Prizm software.
stability. It is also possible that the inhibitor reduces the
extent of the disorder by blocking a region of the avail-
able space into which the loop would have been free to
move. In addition, there is an extensive network of
hydrogen bonds facilitated by water molecules which
could play a role in binding and stabilization of the
glycine-rich loop. In particular, a water molecule
simultaneously interacts with Lys53, Glu71, and the
diphenylether oxygen of the Series 2 inhibitors (Fig. 3).
In conclusion, we have identified two new and distinct
classes of p38a inhibitors and characterized their binding
mode by using crystallographic, biochemical, and compu-
tational analysis. The similarities in the binding modes of
these two inhibitor classes, the structure–activity relation-
ships as well as the computational simulations indicate
that the diphenylether motif may be an important phar-
macophore element within the p38a ATP-binding site.
The remaining portions of the two inhibitor classes
reported here have mixed character and adopt confor-
mations that are determined by the intramolecular
conformational preference as well as intermolecular inter-
actions. Although the two chemical classes presented here
share several characteristics with those of previously
reported inhibitors, they also provide new scaffolds for
development of new p38a inhibitor classes.
10. Guarnieri, F. U.S. Patent No. 6,735,530 issued May 11,
2004, 2004.
11. Moore, W. R. Curr. Opin. Drug Discovery Dev. 2005, 8, 1.
12. Procedure for fragment annealing computational simula-
tions: Computations for predicting fragment binding
modes were carried using a modification of previously
reported methods based on simulated annealing of chem-
ical potential as computed using a grand canonical
ensemble.10,11,16,17 Briefly, the coordinates of the pub-
lished crystal structure of apo-p38 (1P38) were prepared
for computations by adding hydrogens and minimizing
rotor states. Binding of the diphenylether fragment to the
p38 molecular surface was analyzed by computational
simulated annealing over the entire surface. The simula-
tion produced an ensemble of equipotent poses (i.e., a
fragment distribution) within the hydrophobic pocket of
the ATP-binding region along with the energy associated
with the fragment distribution.
Acknowledgments
We thank Randy Abramowitz and XiaoChun Yang of
beamline X4A of the National Synchrotron Light Source,
Brookhaven National Laboratory, for assistance with X-
ray data collection, and Arifa Husain, Judith Lalonde,
and Kendal Williams for comments on the manuscript.
13. Procedure for crystallography: p38a protein expression
using E. coli RosettaTM DE3 host strain, purification, and
crystallization has been described previously.18 For the
crystallization, protein/inhibitor complex solution was
mixed with reservoir solution (10–20% PEG 4000, 0.1 M
cacodylic acid, pH 6, and 50 mM n-octyl-b-D-glucoside) at a
3:2 protein/solution volume ratio. Hanging or sitting drops
of the mixture were placed over the reservoir solution and
crystals were grown by vapor diffusion at 20 °C. For
cryoprotection, crystals were gradually transferred to a
solution of 18% ethylene glycol, 25% PEG 4000, and 0.1 M
cacodylic acid, pH 6, and were flash-frozen in liquid
nitrogen. X-ray diffraction data were collected at 100 K,
at the X4A beamline of the National Synchrotron Light
Source (Upton, NY). Analysis of diffraction data using the
HKL program package19 indicated that the crystals are
orthorhombic, of space group P212121 and approximate cell
Supplementary data
Supplementary data associated with this article can be
References and notes
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˚
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