Virtual Screening for 5-Lipoxygenase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2645
Three-Dimensional Pharmacophore Modeling. Three-dimen-
sional conformations and an initial rigid alignment were obtained
from MOE (v.2005, Chemical Computing Group Inc., Montreal,
Canada). The final alignment was adjusted manually. An extended
version of SQUID25 was used for visualization of pharmacophore
features (Y. Tanrikulu et al., unpublished). Potential pharmacophore
points (PPPs) are visualized as ellipsoids representing Gaussian
feature densities. Features were clustered into PPPs by use of the
following cluster radii: lipophilic PPPs, 2 Å; hydrogen-bond donor
PPPs, 2 Å; and hydrogen-bond acceptor PPPs, 2 Å. Visualization
was performed with PyMOL (The PyMOL Molecular Graphics
System, DeLano Scientific, San Carlos, CA).
after 10 min at 37 °C by addition of 1 mL of ice-cold methanol,
and the formed metabolites were analyzed by HPLC as described
for intact cells (vide supra). Each compound was tested at least
three times, and the mean and standard error of the mean were
determined.
Acknowledgment. Hugo Kubinyi and Hans Peter Albrecht
are thanked for valuable discussion and support. This research
was supported by the Beilstein-Institut zur F o¨ rderung der
Chemischen Wissenschaften, Frankfurt am Main.
Supporting Information Available: Details on virtual screen-
ing results and compound synthesis. This material is available free
of charge via the Internet at http://pubs.acs.org.
Assay Systems: (A) Materials. Arachidonic acid and calcium
ionophore A23187 were from Sigma (Deisenhofen, Germany).
HPLC solvents were from Merck (Darmstadt, Germany).
(
B) Cell Preparation. Human PMNL were freshly isolated from
References
leukocyte concentrates obtained at St. Markus Hospital (Frankfurt,
Germany). In brief, venous blood was taken from healthy adult
donors and leukocyte concentrates were prepared by centrifugation
at 4000g for 20 min at 20 °C. PMNL were immediately isolated
by dextran sedimentation, centrifugation on Nycoprep cushions
PAA Laboratories, Linz, Austria), and hypotonic lysis of eryth-
rocytes as described previously. Cells were finally resuspended
in phosphate-buffered saline, pH 7.4 (PBS), containing 1 mg/mL
(
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(C) Expression and Purification of 5-LO Protein in Escheri-
chia coli. E. coli MV1190 was transformed with pT3-5LO plasmid,
and recombinant 5-LO protein was expressed at 27 °C as
described.27 For purification of 5-LO protein, E. coli cells were
lysed by incubation in 50 mM triethanolamine hydrochloride, pH
929-937.
(
(
(
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8.0, 5 mM ethylenediaminetetraacetic acid (EDTA), soybean trypsin
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inhibitor (60 µg/mL), 1 mM phenylmethanesulfonyl fluoride
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PMSF), and lysozyme (500 µg/mL); homogenized by sonication
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including 5-LO were precipitated with 50% saturated ammonium
sulfate during stirring on ice for 60 min. The precipitate was
collected by centrifugation at 16000g for 25 min and the pellet
was resuspended in 20 mL of PBS containing 1 mM EDTA and 1
mM PMSF. The resuspended 16000g precipitate from E. coli was
centrifuged at 100000g for 70 min at 4 °C, and the 100000g
supernatant (S100) was applied to an ATP-agarose column. The
column was eluted as described previously.28 Purified 5-LO was
immediately used for 5-LO activity assays. Each compound was
tested at least three times, and the mean and standard error of the
mean were determined.
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(D) Determination of 5-LO Product Formation in Intact Cells.
6
For assays of intact cells, 5 × 10 freshly isolated PMNL were
resuspended in 1 mL of PGC buffer. After preincubation with the
test compounds for 15 min at room temperature, 5-LO product
formation was started by addition of 2.5 µM calcium ionophore
A23187, plus 20 µM exogenous arachidonic acid. After 10 min at
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µL of 1 N HCl, 200 ng of prostaglandin B , and 500 µL of PBS
1
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by HPLC as described.28 5-LO product formation is expressed as
6
nanograms of 5-LO products per 10 cells, which includes LTB
4
35, 2600-2609.
and its all-trans isomers, 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-
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4 4 4
, D , and E ) were not detected, and oxidation
(
(
products of LTB were not determined. Each compound was tested
4
at least three times, and the mean and standard error of the mean
were determined.
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Systems. For determination of the activity of partially purified 5-LO
from E. coli, 5-LO (corresponding to 0.5 µg of 5-LO protein) was
added to 1 mL of a 5-LO reaction mix (PBS, pH 7.4, 1 mM EDTA,
and 1 mM ATP). Samples were preincubated with the test
compounds. After 5-10 min at 4 °C, samples were prewarmed for
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30 s at 37 °C, and 2 mM CaCl
2
and 20 µM arachidonic acid were
added to start 5-LO product formation. The reaction was stopped