SCREENING FOR INHIBITORS OF THE HMG-COA REDUCTASE PROMOTER IN HEPG2 CELLS - IDENTIFICATION OF 4 NON-OXYSTEROL INHIBITORS

The 4.9-kb segment of the Chinese hamster hydroxymethylglutaryl-CoA re ductase 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 ge ne, lacZ of E. coli. The new construct pRed3lacZ was transfected into human hepatoma cells (HepG2), and a single clone, G52, was isolated. P romoter activity was monitored by measuring beta-galactosidase activit y in cell lysates in microtiter wells using a fluorogenic substrate, 4 -methyl-umbelliferyl beta-D-galactoside. The amount of beta-galactosid ase activity in cell lysates was directly proportional to the initial cell inoculum up to 30,000 cells per well. Fidelity of the reductase p romoter in G52 was confirmed by (1) suppression of beta-galactosidase synthesis (62.7%) by a sterol mixture consisting of 1.6 x 10(-5) M 25- hydroxycholesterol and 3.1 x 10(-6) M cholesterol, and (2) increased s ynthesis (51.3%) of beta-galactosidase in the presence of 10(-7) M mev inolin, a competitive inhibitor of HMG-CoA reductase activity. Using t his cell line, we examined 5,400 compounds and found four compounds, U -9888, U-20685, U-51862, and U-71690, that inhibit the reductase promo ter with IC50 values of 13.3, 12.0, 12.3, and 14.3 mu M, respectively. In wild-type HepG2 cells, these compounds reduced the synthesis of HM G-CoA reductase by 37, 48, 32, and 22%, respectively. Significantly, n one of these compounds inhibit the binding of LDL to its receptor, sug gesting an important separation of these two coordinately regulated ac tivities. 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 reducta se expression regulators. (C) 1997 Wiley-Liss, Inc.