Abstract
Distal cholesterol biosynthesis (CB) has recently taken center stage as a promising drug target in several diseases previously not linked to this biochemical pathway, including cardiovascular disease, cancer, multiple sclerosis and Alzheimer’s disease. Most enzymes involved in this pathway are hard to isolate, warranting dedicated analytical tools for biochemical screening. We describe the use of gas chromatography–electron ionization mass spectrometry (GC–MS) in a whole-cell screening assay aimed at monitoring interactions with all enzymes of distal CB in a single experiment. Following cell culture and lipid extraction, the trimethylsilyl ethers of sterols are analyzed by GC–MS. Analytical data for 23 relevant sterols (intermediates) are provided, allowing their unambiguous identification. Sterol pattern analysis reveals the target enzyme on the basis of characteristic marker sterols, whereas quantification of 2-13C-acetate incorporation correlates with the inhibitory activity of drug candidates. The protocol can be used by both experienced scientists and newcomers to the field, allowing detection and quantification of small molecule–enzyme interactions in distal CB. The entire protocol can be carried out within two working days.
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Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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We thank Niki Zervoudi for the artwork of Fig. 3.
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C.M., J.J. and M.G. carried out the experiments. C.M., F.B. and M.G. designed the protocol. All authors contributed to writing the protocol.
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Key references using this protocol
Van der Kant, R. et al. Cell Stem Cell 24, 363–375.e9 (2019): https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30603-9
Müller, C. et al. Eur. J. Med. Chem. 140, 305–320 (2017): https://www.sciencedirect.com/science/article/pii/S0223523417306141
Keller, M. et al. Eur. J. Med. Chem. 109, 13–22 (2016): https://www.sciencedirect.com/science/article/pii/S0223523415304086
Horling, A., Müller, C., Barthel, R., Bracher, F. & Imming, P. J. Med. Chem. 55, 7614–7622 (2012): https://pubs.acs.org/doi/10.1021/jm3006096]
Sánchez-Wandelmer, J. et al. Neuroscience 167, 143–153 (2010): https://www.sciencedirect.com/science/article/abs/pii/S0306452210001430
Giera, M., Renard, D., Plössl, F. & Bracher, F. Steroids 73, 299–308 (2008): https://www.sciencedirect.com/science/article/pii/S0039128X07002139
Key data used in this protocol
Kloos, D.-P. et al. Rapid Commun. Mass Spectrom. 28, 1507–1514 (2014): https://onlinelibrary.wiley.com/doi/abs/10.1002/rcm.6923
Giera, M., Plössl, F. & Bracher, F. Steroids 72, 633–642 (2007): https://www.sciencedirect.com/science/article/pii/S0039128X07000736
Integrated supplementary information
Supplementary Fig. 1 Comparison of labeled and unlabeled 7-Dehydrocholesterol.
A) Labeled 7-Dehydrocholesterol after incubation of HL60 cells with AY9944 and 2-13C-acetate. The red lines denote the isotopes +4 to +11 of the two most prominent peaks in the spectrum, used for quantification. B) unlabeled control.
Supplementary Fig. 2 Comparison of labeled and unlabeled cholesta-8,14-dien-3β-ol.
A) labeled Cholesta-8,14-dien-3β-ol after incubation of HL60 cells with AY9944 and 2-13C-acetate. The red lines denote the isotopes +4 to +11 of the two most prominent peaks in the spectrum, used for quantification. B) unlabeled control.
Supplementary Fig. 3 Comparison of labeled and unlabeled zymostenol.
A) Labeled Zymostenol after incubation of HL60 cells with AY9944 and 2-13C-acetate. The red lines denote the isotopes +4 to +11 of the two most prominent peaks in the spectrum, used for quantification. B) unlabeled control.
Supplementary Fig. 4 IC50 comparison for AY 9944.
IC50 curves constructed using precursor sterols versus 2-13C acetate accumulation in total cholesterol (right side).
Supplementary Fig. 5 IC50 comparison for tamoxifen.
IC50 curves constructed using precursor sterols versus 2-13C acetate accumulation in total cholesterol (right side).
Supplementary Fig. 6 IC50 comparison for SH42.
IC50 curves constructed using precursor sterols versus 2-13C acetate accumulation in total cholesterol (right side).
Supplementary information
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Supplementary Table 6
Supplementary Table 6
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Müller, C., Junker, J., Bracher, F. et al. A gas chromatography–mass spectrometry-based whole-cell screening assay for target identification in distal cholesterol biosynthesis. Nat Protoc 14, 2546–2570 (2019). https://doi.org/10.1038/s41596-019-0193-z
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DOI: https://doi.org/10.1038/s41596-019-0193-z
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