641
r e f e r e n c e s
and 4-methylcholesta-7-en-3-ol (18) accumulated, as previ-
ously described for in vivo experiments [28], both substances
have been identified due to their mass spectral data, which
was especially possible because of the previous description
of their accumulation under enzyme inhibition with amino-
triazole [28]. At a concentration of 0.1 M AY 9944, the only
accumulating sterol was 7-dehydrocholesterol (9), which is in
accordance with the previously published results [22]. DR 258
cholesta-5,7,24-trien-3-ol (12), which corresponds to the pre-
viously described results for ergosterol, having the same target
enzyme [10]. 12 had been identified due to its mass spectral
data according to ref. [29].
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The IC50 values of clotrimazole and NB 598 could be deter-
mined without the need to use radioactive labeled substances
through the mass spectrometric determination of labeled
cholesterol. The labeling of the target molecule cholesterol
was achieved by the addition of sodium 2-13C-acetate. The
obtained IC50 value for clotrimazole (1.28 × 10−7 M) fits quite
[26], which was determined using human fibroblasts and 14C-
acetate. To show the comparability of our assay to assays using
14C-acetate and HepG2 cells, we determined the IC50 value
of NB 598 which was 1.92 nM (Table 2, Fig. 9). The IC50 val-
ues of NB 598 previously reported are 7.2 nM [24] and 0.75 nM
[25]. The former value was determined using a HepG2 cell
homogenate and [3H]squalene, and the latter value was deter-
mined using HepG2 cells and 14C-acetate. The value obtained
with our assay fits quite well the previously determined val-
ues, suggesting that our presented assay can equally be used.
In general, the IC50 values of NB 598 and clotrimazole previ-
ously described and determined with our assay fit quite well.
In conclusion, inhibition of all of the enzymes involved in
late cholesterol biosynthesis except sterol-C5-desaturase (no
selective inhibitor had been available) can be analyzed qual-
itatively and quantitatively in a single assay. With the assay
described here, fast (more than 50 samples a day) identifica-
tion of late cholesterol biosynthesis inhibitors is possible. The
presented chromatograms can be used as qualitative stan-
dards for the different enzyme inhibitions. New substances
showing the same sterol patterns as the described inhibitors
can be stated as having the same target enzyme.
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The quantitative test procedure allows determining an
overall IC50 value for cholesterol biosynthesis, regardless of
the type of enzyme(s) that was inhibited. Due to the use of
13C-acetate, radioactive substances can be refused owing to
safety and costs.
Taken together, we have worked out a fast and easy in
vitro screening assay for cholesterol biosynthesis inhibitors in
post-squalene pathway that should be useful for the first iden-
tification and characterization of new selective cholesterol
biosynthesis inhibitors; nevertheless, single enzyme assays
should always be used for further substance characterization.
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Acknowledgements
We thank Boehringer Ingelheim Pharma GmbH & Co. KG for
providing BIBX 79 and Dr. D. Renard for preparing and provid-
ing DR 258.