Screening for inhibitors of the HMG-CoA reductase promoter in HepG2 cells: Identification of four non-oxysterol inhibitors
✍ Scribed by Yoshihiko Yagi; Debra J. Bevis; Karen L. Hart; Gerard F. Hess; Dac M. Dinh; Barbara J. Keiser; Scott D. Larsen; Charles H. Spilman
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 106 KB
- Volume
- 40
- Category
- Article
- ISSN
- 0272-4391
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✦ Synopsis
The 4.9-kb segment of the Chinese hamster hydroxymethylglutaryl-CoA reductase promoter found in pRedCAT-3 consists of 1.4-kb of the promoter and a 3.5-kb intron. We placed this segment upstream of a reporter gene, lacZ of E. coli. The new construct pRed3lacZ was transfected into human hepatoma cells (HepG2), and a single clone, G52, was isolated. Promoter activity was monitored by measuring βgalactosidase activity in cell lysates in microtiter wells using a fluorogenic substrate, 4-methyl-umbelliferyl β-D-galactoside. The amount of β-galactosidase activity in cell lysates was directly proportional to the initial cell inoculum up to 30,000 cells per well. Fidelity of the reductase promoter in G52 was confirmed by (1) suppression of β-galactosidase synthesis (62.7%) by a sterol mixture consisting of 1.6 × 10 -5 M 25hydroxycholesterol and 3.1 × 10 -6 M cholesterol, and (2) increased synthesis (51.3%) of β-galactosidase in the presence of 10 -7 M mevinolin, a competitive inhibitor of HMG-CoA reductase activity. Using this cell line, we examined 5,400 compounds and found four compounds, U-9888, U-20685, U-51862, and U-71690, that inhibit the reductase promoter with IC50 values of 13.3, 12.0, 12.3, and 14.3 µM, respectively. In wild-type HepG2 cells, these compounds reduced the synthesis of HMG-CoA reductase by 37, 48, 32, and 22%, respectively. Significantly, none of these compounds inhibit the binding of LDL to its receptor, suggesting an important separation of these two coordinately regulated activities. Furthermore, all four of these inhibitors lie outside of the class of compounds typically defined as classic oxysterols, and thus represent new potential templates for the discovery of HMG-CoA reductase expression regulators.