3102 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Brief Articles
(3) Karin, M.; Yamamoto, Y.; Wang, Q. M. The IKK NF-κB system: a
treasure trove for drug development. Nat. ReV. Drug DiscoVery 2004,
3, 17–26.
(4) Bonizzi, G.; Karin, M. The two NF-kB activation pathways and their
role in innate and adaptive immunity. Trends Immunol. 2004, 25, 280–
288.
(5) Prajapati, S.; Tu, Z.; Yamamoto, Y.; Gaynor, R. B. IKKR regulates
the mitotic phase of the cell cycle by modulating Aurora A phospho-
rylation. Cell Cycle 2006, 5, 2371–2380.
(6) Blazkova, H.; von Schubert, C.; Mikule, K.; Schwab, R.; Angliker,
N.; Schmuckli-Maurer, J.; Fernandez, P. C.; Doxsey, S.; Dobbelaere,
D. A. The IKK inhibitor BMS-345541 affects multiple mitotic cell
cycle transitions. Cell Cycle 2007, 6, 2531–2540.
1-737) was expressed in baculovirus as a C-terminal GST-tagged
fusion protein. GST-IκBR substrate in a volume of 3 µL (25 nM
final)/ATP (1 µM final) diluted in assay buffer (50 mM HEPES,
10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01%
w/v BSA) was added to wells containing various concentrations
of compound or DMSO vehicle (less than 5% final). The reaction
mixture was initiated by the addition 3 µL of IKK-ꢀ (typically 0.5
nM final) in a total volume of 6 µL. The reaction was incubated
for 15 min at room temperature, then terminated by the addition of
stop reagent (3 µL) containing 50 mM EDTA and detection reagents
in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v
BSA). Detection reagents comprise antiphosphoserine-IκBR-32/36
monoclonal antibody 12C2 (Cell Signalling Technology, Beverly,
MA) labeled with W-1024 europium chelate (Perkin-Elmer, Bea-
consfield, U.K.) and an allophycocyanin-labeled anti-GST antibody
(Prozyme, San Leandro, CA). The mixture (9 µL total volume)
was further incubated for at least 30 min at room temperature. The
degree of phosphorylation of GST-IκBR was measured using a
suitable time-resolved fluorimeter as a ratio of specific 665 nm
energy transfer signal to reference europium 620 nm signal. IKK-R
kinase inhibitory activity was determined in an analogous fashion,
using 6-his-tagged full length IKK-R. Typical assay variability, as
defined by measuring the pIC50 reproducibility of standard com-
pounds [IKK-R, eight standard compounds with mean pIC50 values
ranging from 5.4 to 7.2; IKK-ꢀ, seven standard compounds with
mean pIC50 values ranging from 5.1 to 8.0], all run greater than
100 times in total, showed an average standard deviation around
the measured pIC50 of (0.2 log units for both assays.
(7) Irelan, J. T.; Murphy, T. J.; DeJesus, P. D.; Teo, H.; Xu, D.; Gomez-
Ferreria, M. A.; Zhou, Y.; Miraglia, L. J.; Rines, D. R.; Verma, I. M.;
Sharp, D. J.; Tergaonkar, V. A role for IκB kinase 2 in bipolar spindle
assembly. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16940–16945.
(8) Coish, P. D. G.; Wickens, P. L.; Lowinger, T. B. Small molecule
inhibitors of IKK kinase activity. Expert Opin. Ther. Pat. 2006, 16,
1–12.
(9) Christopher, J. A.; Avitabile, B. G.; Bamborough, P.; Champigny,
A. C.; Cutler, G. J.; Dyos, S. L.; Grace, K. G.; Kerns, J. K.; Kitson,
J. D.; Mellor, G. W.; Morey, J. V.; Morse, M. A.; O’Malley, C. F.;
Patel, C. B.; Probst, N.; Rumsey, W.; Smith, C. A.; Wilson, M. J.
The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-R
and IKK-ꢀ kinases. Bioorg. Med. Chem. Lett. 2007, 17, 3972–3977.
(10) Reagent selection, filtering, and array enumeration were carried out
using proprietary software based on the Daylight toolkit (www.
daylight.com). The starting point was a list of available aryl bromides
or iodides from the corporate collection or from external suppliers.
These were filtered to remove certain defined functionalities that are
potentially unstable or undesirable. Substructure searches were used
to exclude compounds containing more than one aryl halide or any
boronic acid, sulfonyl halide, aldehyde, acid chloride, ꢀ-keto ester,
thiol, anhydride, isothiocyanate, or epoxide. Compounds containing
over 25 heavy atoms were also excluded. Approximately 8000 aryl
halides remained.
(11) The 3D aryl halide database was built and searched and the
com). Fast conformer generation was used with a maximum of 50
conformers per molecule. The standard H-bond acceptor function was
modified to include only strong, neutral, planar H-bonding groups,
and excluded volume spheres were placed with reference to the
homology model. Docking studies into the homology model (see ref
9) were performed using version 3.0 of Gold (Cambridge Crystal-
scores and binding modes was carried out visually, using a combination
of software including the Spotfire Decision Site (spotfire.tibco.com)
interfaced with Accelrys Viewer Pro using proprietary code.
(12) Hass, H. B.; Bender, M. L. Reaction of benzyl halides with the sodium
salt of 2-nitropropane. A general synthesis of substituted benzalde-
hydes. J. Am. Chem. Soc. 1949, 71, 1767–1769.
Acknowledgment. We thank Duncan B. Judd and Bob Blade
for assistance in the scale-up of 14, Bill Leavens for HRMS
analysis, and members of the Screening and Compound Profiling
Departments based in Harlow, Essex, U.K., and Research
Triangle Park, NC, for the generation of kinase inhibition data.
Supporting Information Available: Synthetic details and
characterization data for 14, 15, 40-42, and 23-39; characterization
data for 6, 7, 16, 18-21; LCMS traces for 17, 20, and 21;
experimental details for the PBMC and human whole blood assays.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Parang, K.; Sun, G. Design strategies for protein kinase inhibitors.
Curr. Opin. Drug DiscoVery DeV. 2004, 7, 617–629.
(2) Ha¨cker, H; Karin, M. Regulation and function of IKK and IKK-related
kinases. Sci. STKE 2006, 357, re13.
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