Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by 15 alpha-fluorolanost-7-en-3 beta-ol. A mechanism-based inhibitor of cholesterol biosynthesis.
The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15 alpha-fluorolanost-7-en-3 beta-ol, are described. The 15 alpha-fluorosterol is shown to be a competitive inhibitor of the lanosterol 14 alpha-methyl demethylase (Ki = 315 microM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15 alpha-fluorosterol is converted to the corresponding 15 alpha-fluoro-3 beta-hydroxylanost-7-en-32-aldehyde by hepatic microsomal lanosterol 14 alpha-methyl demethylase but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 15 alpha-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14 alpha-methyl demethylase-deficient mutant cell line, AR45, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14 alpha-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15 alpha-fluorosterol-treated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in HMG-CoA reductase protein is due to inhibition of enzyme synthesis, suggesting that the 15 alpha-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the 15 alpha-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms.
Trzaskos
J M
JM
Du Pont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880-0400.
Magolda
R L
RL
Favata
M F
MF
Fischer
R T
RT
Johnson
P R
PR
Chen
H W
HW
Ko
S S
SS
Leonard
D A
DA
Gaylor
J L
JL
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology*; D001711:Biotransformation; D015152:Blotting, Northern; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007202:Indicators and Reagents; D007810:Lanosterol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D016296:Mutagenesis; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
D000924:Anticholesteremic Agents; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007202:Indicators and Reagents; C083839:15-fluorolanost-7-en-3-ol; D007810:Lanosterol; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D006903:Hydroxymethylglutaryl CoA Reductases; D058886:Sterol 14-Demethylase