Antimicrotubule Benzylidene-9(10H)-anthracenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3393
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enenitrilo)tetraacetic acid) by adding 0.25 mM GTP and by
warming the samples to 37 °C. The steady state level, at which
the mass of tubulin in the polymerized state shows no further
increase, was usually observed by turbidity measurements
after an incubation time of 20 min.
The turbidimetric measurement was carried out by spec-
trophotometry at 360 nm, and the temperature in the as-
sembly/disassembly cuvette was regulated with a cryostat/
thermostat combination. The tubulin effectors, dissolved in
DMSO, were added to the cold MTP solution (MTP concentra-
tion 1 mg/mL, temperature 2 to 4 °C) in PIPES buffer (for
composition see above) at pH 6.8. Immediately at the begin-
ning of the measurement, the temperature was raised to 37
°C. After a gap phase, the MTP assembled into microtubule
(MT), and the solution became turbid and achieved finally the
steady state. To prove reversibility of the assembly, stabiliza-
tion by the effectors or to discriminate the turbidity from an
unspecific protein precipitation, the solution was cooled to 2
°C and kept for 5 min at this temperature, resulting in MT
disassembly. The MTP solution became transparent again. If
the drug binds to or interferes with tubulin, the assembly
steady state level is decreased for assembly inhibitors or
increased for MT stabilizers. The IC50 value is defined as the
concentration that causes a 50% deviation from the maximum
tubulin assembly rate.
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3H Colch icin e Com p etition -Bin d in g Assa y.44 3H-Colchi-
cine was diluted, and biotin-labeled tubulin (T333, Cytoskel-
eton, Denver, CO) was reconstituted according to the manu-
facturers protocol. The diluted compounds and the 3H-
colchicine were transferred to a 96-well isoplate (PE-Wallac,
Boston, MA), buffer, and the reconstituted biotin-labeled
tubulin was added. After incubation, streptavidin-coated yt-
trium SPA beads (Amersham Pharmacia Biotech, Piscataway,
NJ ) were added, and the bound radioactivity was determined
using a MicroBeta Trilux Microplate scintillation counter (PE-
Wallac Boston, MA). IC50 values were obtained by nonlinear
regression (GraphPad Prism).
Acqu isition a n d An a lysis of EM Da ta . For EM studies,
tubulin (1 mg/mL) with MAPs was incubated with 9h (0.7 µM)
or colchicine (1 µM) in assay buffer buffer solution containing
20 mM PIPES (1,4-piperazine diethane sulfonic acid, pKa 6.8),
80 mM NaCl, 0.5 mM MgCl2, 1 mM EGTA (ethylenebis-
(oxyethylenenitrilo)tetraacetic acid), and 0.25 mM GTP (gua-
nosin-5′-triphosphate). The assembly was started by incuba-
tion at 37 °C and followed by turbidity measurements at 360
nm. The samples were applied to carbon mica interface, and
the floating carbon films were transferred to a 2% aequous
uranyl acetate solution as a negative stain and mounted on
copper grids. Electron micrographs were obtained using a Zeiss
EM 902 A transmission electron microscope with an accelerat-
ing voltage of 80 kV. The samples were fixed, processed, and
examined under EM at ×50 000.
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Ack n ow led gm en t. We are grateful to Rie Onose,
Antibiotics Laboratory, the Institut for Physical and
Chemical Research (Riken), for having performed a flow
cytometry study with K562 cells. We wish to thank
Angelika Zinner, Katrin Meuser, and Christiane Schet-
tler for excellent technical assistance.
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