title: Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2-aza-2,3-dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor.
abstract: The effect of 2-aza-2,3-dihydrosqualene, a new compound designed to inhibit the 2,3-oxidosqualene-lanosterol cyclase [A. Duriatti et al., Biochem. Pharmac. 34, 2765 (1985)] was studied as inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment with the drug of cells which were grown for 2 days in a delipidated medium resulted in a marked decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 = 0.3 microM was calculated when the cells were preincubated for a period of 4 hr with 2-aza-2,3-dihydrosqualene. This inhibition was correlated with an intracellular accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene, indicating that the cyclase was indeed an intracellular target of the drug. A precursor-product relationship of the accumulated [14C]squalene oxide(s) and the [14C]sterols was demonstrated in chase experiments in the absence of drug. Sterols more polar than cholesterol were also detected in treated fibroblasts and in the cells which underwent chase experiments; they were mainly composed of 24,25-epoxycholesterol. The C27-[14C]sterols of [14C]acetate pulse labeled cells consisted in a mixture of desmosterol and cholesterol; treatment of the cells with 2-aza-2,3-dihydrosqualene resulted in a decreased conversion of desmosterol into cholesterol indicating that the delta 24-sterol reductase might be another target of the drug. 2-Aza-2,3-dihydrosqualene at 1 microM affected normal growth of 3T3 fibroblasts, this effect could be prevented by addition of exogeneous cholesterol (50 microM). Growth arrest of the treated cells was correlated with a decrease in cellular sterol content to less than 40% of controls. About 30% of the C27-sterol fraction, of the treated cells, was desmosterol. Our work demonstrates that 2-aza-2,3-dihydrosqualene is a valuable new inhibitor of cholesterol biosynthesis in mammalian cells.
mesh_terms: D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002784:Cholesterol / Q000096:biosynthesis*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000037:antagonists & inhibitors*; D009589:Nitrogen Oxides / Q000494:pharmacology; D013185:Squalene / Q000031:analogs & derivatives* / Q000494:pharmacology; D013261:Sterols / Q000096:biosynthesis
chemical_list: D002250:Carbon Radioisotopes; D009589:Nitrogen Oxides; D013261:Sterols; C050287:2-aza-2,3-dihydrosqualene N-oxide; D013185:Squalene; C039926:2-aza-2-dihydrosqualene; D002784:Cholesterol; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase