Abstract
Novel potential inhibitors of the postsqualene portion of cholesterol synthesis were screened in HepG2 cells. 2-(4-Phenethylpiperazin-1-yl)-1-(pyridine-3-yl)ethanol (LK-980) was identified as a prospective compound and was characterized further in cultures of human primary hepatocytes from seven donors. In vitro kinetic measurements show that the half-life of LK-980 is at least 4.3 h. LK-980 does not induce CYP3A4 mRNA nor enzyme activity. Target prediction was performed by gas chromatography-mass spectrometry, allowing simultaneous separation and quantification of nine late cholesterol intermediates. Experiments indicated that human sterol Δ7-reductase (DHCR7) is the major target of LK-980 (34-fold increase of 7-dehydrocholesterol), whereas human sterol Δ14-reductase (DHCR14), human sterol Δ24-reductase (DHCR24), and human sterol C5-desaturase (SC5DL) represent minor targets. In the absence of purified enzymes, we used the mathematical model of cholesterol synthesis to evaluate whether indeed more than a single enzyme is inhibited. In silico inhibition of only DHCR7 modifies the flux of cholesterol intermediates, resulting in a sterol profile that does not support experimental data. Partial inhibition of the DHCR14, DHCR24, and SC5DL steps, in addition to DHCR7, supports the experimental sterol profile. In conclusion, we provide experimental and computational evidence that LK-980, a novel inhibitor from the late portion of cholesterol synthesis, inhibits primarily DHCR7 and to a lesser extent three other enzymes from this pathway.
Footnotes
- Received August 12, 2010.
- Accepted October 13, 2010.
This work was supported by the European Community [Grant LSHG-CT-2005-512096, STEROLTALK]; the Slovenian-Hungarian Intergovernmental S&T Cooperation Programme [Grant Sl-5/2008]; and the Research Agency of the Republic Slovenia [Grants J1-9438, P1-0104].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.035840.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- HMGCR
- HMG-CoA reductase
- DHCR14
- human sterol Δ14-reductase
- FF-MAS
- follicular fluid meiosis activating sterol (4,4-dimethyl-5a-cholesta-8,14,24-triene-3b-ol)
- T-MAS
- testis meiosis activating sterol(4,4-dimethyl-5a-cholesta-8,24-diene-3b-ol)
- DHCR24
- human sterol Δ24-reductase
- DHCR7
- human sterol Δ7-reductase
- SC5DL
- human sterol C5-desaturase
- LK-980
- 2-(4-phenethylpiperazin-1-yl)-1-(pyridine-3-yl)ethanol
- GC
- gas chromatography
- MS
- mass spectrometry
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- LSD
- least significant difference
- Ay 9944
- trans-1,4-bis(2-chlorobenzaminomethyl) cyclohexane dihydrochloride
- SR 31747
- N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)propen-2-ylamine hydrochloride
- SKF 104976
- 3β-hydroxylanost-8,15-dien-32-carboxylic acid
- CYP51A1
- lanosterol 14α-demethylase
- MER-29
- triparanol
- LK-935
- 2-((3,4-dichlorophenethyl)(propyl)amino)-1-(pyridin-3-yl)ethanol.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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