4280 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Campiani et al.
conducted in conformity with the institutional guidelines that
are in compliance with national (D:L: n. 116, G. U., suppl.
40, Feb.18, 1992) and international laws and policies (EEC
Council Directive 86/609, OJ L 358, 1, Dec.12, 1987; Guide
for the Care and Use of Laboratory Animals, U.S. National
Research Council, 1996). Before they were killed by decapita-
tion (unanesthetized), the rats hed been housed in groups of
five in plastic cages, kept under standard conditions (room
temperature, 21 ( 1 °C; relative humidity, 55 ( 10%; 12/12 h
light-dark cycle), and given tap water and food pellets ad
libitum. After they were decapitated, the brains were rapidly
removed from the skulls and the cortex was dissected out,
frozen on dry ice, and stored at -80 °C until used. Cortices
were homogenized in about 50 volumes of ice cold phosphate-
buffered saline, 50 mM, pH 7.4, using an Ultra Turrax TP-
1810 (2 × 20 s) and centrifuged at 50 000g for 10 min. The
pellet was then washed three times more by resuspensions in
fresh buffer and centrifugations as before. The last pellet was
resuspended just before the binding assay.
For binding assay, 10 mg of original wet tissue weight was
incubated with 1 nM [3H]PK11195 (sa 85.8 Ci/mmol; NEN) in
1 mL (final volume) for 120 min at 4 °C in the presence of
8-12 increasing concentrations of drugs. Nonspecific binding
was determined by using 1 µM clonazepam.
Incubation was stopped by rapid filtration under vacuum
through GF/B fiber filters, which were then washed with 12
mL of ice-cold buffer and counted in 4 mL of Ultima Gold MV
(Packard) in a liquid scintillation spectrometer WALLAC 1409,
with a counting efficiency of about 56%. IC50 values were
determined by nonlinear fitting of binding inhibition curves,
using the Allfit program running on an IBM AT personal
computer. The Ki values were derived from the IC50 values.11
Each point was the mean of triplicate samples.
exceed 1% (v/v), a concentration that on its own had no effect
on steroid production. Following a 2 h incubation with the
compounds, the incubation medium was acidified by the
addition of perchloric acid (107 mM), which lysed the cells.
The incubation plate was then frozen and thawed, and the
contents were neutralized by the addition of K3P04. The
amount of progesterone produced by the cells was quantified
by radioimmunoassay (RIA) using an antibody from ICN
Biochemical Inc. (California), under the conditions described
by the manufacturer. Analysis of the RIA data was performed
using the Apple Macintosh ASSAYZAP program obtained from
Biosoft.
Ack n ow led gm en t. We thank MURST-Rome, Italy
(PRIN 99), for financial support.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails (chemistry and pharmacology, Tables 1 and 2). This
information is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Anholt, R. R. H.; De Souza, E. B.; Oster-Granite, M. L.; Snyder,
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(2) De Souza, E. B.; Anholt, R. R. H.; Murphy, K. M. M.; Snyder, S.
H.; Kuhar, M. J . Peripheral-Type Benzodiazepine Receptors in
Endocrine Organs: Autoradiographic Localisation in Rat Pitu-
itary, Adrenal and Testis. Endocrinology 1985, 116, 567-573.
(3) Papadopoulos, V. Peripheral-Type Benzodiazepine/Diazepam
Binding Inhibitor Receptor: Biological Role in Steroidogenic Cell
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(4) Anholt, R. R. H.; Pedersen, P. L.; De Souza, E. B.; Snyder, S. H.
The Peripheral-Type Benzodiazepine Receptor: Localisation to
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Ra t Testicu la r Mitoch on d r ia l P r ep a r a tion a n d [3H]-
P K11195 a n d [3H]Ro5-4864 Bin d in g. For binding studies,
mitochondria were prepared from the testes of Wistar rats
(200-250 g) killed by cervical dislocation. Testes were removed
and homogenized in buffer A (50 mM Tris/Cl, pH 7.4, contain-
ing 0.25 M sucrose and 1 mM ethylenediaminetetraacetic acid
(EDTA) with a Teflon pestle in a glass homogenizer. The
homogenate was centrifuged at 600g for 10 min, and the
resulting supernatant was then centrifuged at 10 000g for 10
min. The mitochondrial pellet was then suspended in buffer
A and centrifuged at 10 000g for a further 10 min. The
resulting washed mitochondrial pellet was resuspended in 50
(5) (a) Fiorini, I.; Nacci, V.; Ciani, S. M.; Garofalo, A.; Campiani,
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T. Novel Ligands Specific for Mitochondrial Benzodiazepine
Receptors: 6-Arylpyrrolo[2,1-d][1,5]benzothiazepine Derivatives.
Synthesis, Structure-Activity Relationships, and Molecular
Modelling Studies. J . Med. Chem. 1994, 37, 1427-1438. (b)
Greco, G.; Novellino, E.; Fiorini, I.; Nacci, V.; Campiani, G.;
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benzothiazepines Binding Selectively to the Mitochondrial Ben-
zodiazepine Receptor. J . Med. Chem. 1994, 37, 4100-4108. (c)
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G. A Concerted Study Using Binding Measurements, X-ray
Structural Data, and Molecular Modeling on the Stereochemical
Features Responsible for the Affinity of 6-Arylpyrrolo[2,1-d][1,5]-
benzothiazepines Toward Mitochondrial Benzodiazepine Recep-
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M.; Manzoni, C.; Mennini, T. New Pyrrolobenzothiazepine
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mM Tris/Cl, pH 7.4, at
a concentration of 2 mg/mL as
determined by the method of Markwell et al.,12 for use in
binding assays. All procedures were performed at 4 °C.
Testicular mitochondria (20 µg of protein) were incubated with
either [3H]PK11195 (2 nM) or [3H]Ro5-4864 (2 nM) (NEN) in
50 mM Tris/Cl, pH 7.4, with a range of concentrations of the
tested compounds (0.1 nM-1 µM) dissolved in this buffer
containing 0.1% ethanol, in a total volume of 0.5 mL at 4 °C.
Total and nonspecific binding in each case were determined
in the absence and presence, respectively, of unlabeled PK11195
and Ro5-4864 (1 µM). All samples were incubated in triplicate
for 60 min. Assays were terminated by filtration through
Whatman glass fiber filters (GF/B, 2.5 cm) using a Brandel
cell harvester. Radioactivity trapped on the filters was deter-
mined by liquid scintillation counting. The IC50 values and
subsequent Ki values for each compound were generated by
the use of the computer programs EBDA and LIGAND.
Ster oid Biosyn th esis in MA10 Leyd ig Cells. The MA10
Leydig cells were gratefully obtained from Mario Ascoli,
Department of Pharmacology, University of Iowa College of
Medicine, Iowa. They were cultured in Waymouth’s medium
supplemented with horse serum (15% v/v) and gentamycin (50
mg/mL). Cultures were maintained in a humidified atmo-
sphere at 5% CO2/95% air at 37 °C. MA10 Leydig cells were
seeded in 24 well plates at a density of 1 × 105 cells per well
in a final volume of 0.5 mL. The test compounds, PK11195
and Ro5-4864, were then added to the cells at
a final
concentration of 10 µM. The final concentration of ethanol was
constant for all of the wells in each experiment and did not