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A gas chromatography–mass spectrometry-based whole-cell screening assay for target identification in distal cholesterol biosynthesis

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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Fig. 1: Main cholesterol biosynthesis pathways.
Fig. 2: Separation of Δ7- and Δ8-sterol isomers.
Fig. 3
Fig. 4: Quantitation of 2-13C-acetate incorporation.
Fig. 5: Anticipated results.

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Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Nes, W. D. Biosynthesis of cholesterol and other sterols. Chem. Rev. 111, 6423–6451 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Byskov, A. G. et al. Chemical structure of sterols that activate oocyte meiosis. Nature 374, 559–562 (1995).

    CAS  PubMed  Google Scholar 

  3. Roux, C., Horvath, C. & Dupuis, R. Teratogenic action and embryo lethality of AY 9944R: prevention by a hypercholesterolemia-provoking diet. Teratology 19, 35–38 (1979).

    CAS  PubMed  Google Scholar 

  4. Kirby, T. J. Cataracts produced by triparanol (MER-29). Trans. Am. Ophthalmol. Soc. 65, 494–543 (1967).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Zerenturk, E. J., Sharpe, L. J., Ikonen, E. & Brown, A. J. Desmosterol and DHCR24: unexpected new directions for a terminal step in cholesterol synthesis. Prog. Lipid Res. 52, 666–680 (2013).

    CAS  PubMed  Google Scholar 

  6. Hubler, Z. et al. Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination. Nature 560, 372–376 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Canfrán-Duque, A. et al. Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro. J. Lipid Res. 54, 310–324 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Sánchez-Wandelmer, J. et al. Haloperidol disrupts lipid rafts and impairs insulin signaling in SH-SY5Y cells. Neuroscience 167, 143–153 (2010).

    PubMed  Google Scholar 

  9. Boland, M. R. & Tatonetti, N. P. Investigation of 7-dehydrocholesterol reductase pathway to elucidate off-target prenatal effects of pharmaceuticals: a systematic review. Pharmacogenomics J. 16, 411–429 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Simonen, P. et al. Desmosterol accumulation in users of amiodarone. J. Intern. Med. 283, 93–101 (2017).

    PubMed  Google Scholar 

  11. Kedjouar, B. et al. Molecular characterization of the microsomal tamoxifen binding site. J. Biol. Chem. 279, 34048–34061 (2004).

    CAS  PubMed  Google Scholar 

  12. Giavini, E. & Menegola, E. Are azole fungicides a teratogenic risk for human conceptus? Toxicol. Lett. 198, 106–111 (2010).

    CAS  PubMed  Google Scholar 

  13. Pilmis, B. et al. Antifungal drugs during pregnancy: an updated review. J. Antimicrob. Chemother. 70, 14–22 (2014).

    PubMed  Google Scholar 

  14. Brown, A. J., Ikonen, E. & Olkkonen, V. M. Cholesterol precursors: more than mere markers of biosynthesis. Curr. Opin. Lipidol. 25, 133–139 (2014).

    CAS  PubMed  Google Scholar 

  15. Sharpe, L. J. & Brown, A. J. Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA Reductase (HMGCR). J. Biol. Chem. 288, 18707–18715 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Rozman, D. & Monostory, K. Perspectives of the non-statin hypolipidemic agents. Pharmacol. Ther. 127, 19–40 (2010).

    CAS  PubMed  Google Scholar 

  17. Kim, H.-Y. H. et al. Inhibitors of 7-dehydrocholesterol reductase: screening of a collection of pharmacologically active compounds in Neuro2a cells. Chem. Res. Toxicol. 29, 892–900 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Giera, M., Plössl, F. & Bracher, F. Fast and easy in vitro screening assay for cholesterol biosynthesis inhibitors in the post-squalene pathway. Steroids 72, 633–642 (2007).

    CAS  PubMed  Google Scholar 

  19. Giera, M., Renard, D., Plössl, F. & Bracher, F. Lathosterol side chain amides—a new class of human lathosterol oxidase inhibitors. Steroids 73, 299–308 (2008).

    CAS  PubMed  Google Scholar 

  20. Kloos, D.-P. et al. Comprehensive gas chromatography–electron ionisation mass spectrometric analysis of fatty acids and sterols using sequential one-pot silylation: quantification and isotopologue analysis. Rapid Commun. Mass Spectrom. 28, 1507–1514 (2014).

    CAS  PubMed  Google Scholar 

  21. Müller, C., Binder, U., Bracher, F. & Giera, M. Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography–mass spectrometry. Nat. Protoc. 12, 947–963 (2017).

    PubMed  Google Scholar 

  22. Horling, A., Muller, C., Barthel, R., Bracher, F. & Imming, P. A new class of selective and potent 7-dehydrocholesterol reductase inhibitors. J. Med. Chem. 55, 7614–7622 (2012).

    CAS  PubMed  Google Scholar 

  23. Müller, C. et al. Fungal sterol C22-desaturase is not an antimycotic target as shown by selective inhibitors and testing on clinical isolates. Steroids 101, 1–6 (2015).

    PubMed  Google Scholar 

  24. Mailänder-Sánchez, D. et al. Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion. PLoS ONE 12, e0184438 (2017).

    PubMed  PubMed Central  Google Scholar 

  25. van der Kant, R. et al. Cholesterol metabolism is a druggable axis that independently regulates tau and amyloid-beta in iPSC-derived Alzheimer’s disease neurons. Cell Stem Cell 24, 363–375.e369 (2019).

    PubMed  PubMed Central  Google Scholar 

  26. Müller, C. et al. New chemotype of selective and potent inhibitors of human delta 24-dehydrocholesterol reductase. Eur. J. Med. Chem. 140, 305–320 (2017).

    PubMed  Google Scholar 

  27. Krojer, M., Müller, C. & Bracher, F. Steroidomimetic aminomethyl spiroacetals as novel inhibitors of the enzyme Δ8,7-sterol isomerase in cholesterol biosynthesis. Arch. Pharm. (Weinheim) 347, 108–122 (2014).

    CAS  Google Scholar 

  28. Keller, M. et al. Arylpiperidines as a new class of oxidosqualene cyclase inhibitors. Eur. J. Med. Chem. 109, 13–22 (2016).

    CAS  PubMed  Google Scholar 

  29. König, M., Müller, C. & Bracher, F. Stereoselective synthesis of a new class of potent and selective inhibitors of human Δ8,7-sterol isomerase. Bioorg. Med. Chem. 21, 1925–1943 (2013).

    PubMed  Google Scholar 

  30. Fanter, L., Müller, C., Schepmann, D., Bracher, F. & Wünsch, B. Chiral-pool synthesis of 1,2,4-trisubstituted 1,4-diazepanes as novel σ1 receptor ligands. Bioorg. Med. Chem. 25, 4778–4799 (2017).

    CAS  PubMed  Google Scholar 

  31. Knappmann, I. et al. Lipase-catalyzed kinetic resolution as key step in the synthesis of enantiomerically pure σ ligands with 2-benzopyran structure. Bioorg. Med. Chem. 25, 3384–3395 (2017).

    CAS  PubMed  Google Scholar 

  32. Renard, D., Perruchon, J., Giera, M., Müller, J. & Bracher, F. Side chain azasteroids and thiasteroids as sterol methyltransferase inhibitors in ergosterol biosynthesis. Bioorg. Med. Chem. 17, 8123–8137 (2009).

    CAS  PubMed  Google Scholar 

  33. Fernández, C., Martín, M., Gómez-Coronado, D. & Lasunción, M. A. Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression. J. Lipid Res. 46, 920–929 (2005).

    PubMed  Google Scholar 

  34. Giera, M., Müller, C. & Bracher, F. Analysis and experimental inhibition of distal cholesterol biosynthesis. Chromatographia 78, 343–358 (2015).

    CAS  Google Scholar 

  35. Shackleton, C., Pozo, O. J. & Marcos, J. GC/MS in recent years has defined the normal and clinically disordered steroidome: will it soon be surpassed by LC/tandem MS in this role? J. Endocr. Soc. 2, 974–996 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Müller, C., Staudacher, V., Krauss, J., Giera, M. & Bracher, F. A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects on total ergosterol biosynthesis. Steroids 78, 483–493 (2013).

    PubMed  Google Scholar 

  37. Honda, A. et al. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS. J. Lipid Res. 49, 2063–2073 (2008).

    CAS  PubMed  Google Scholar 

  38. Folch, J., Lees, M. & Stanley, G. H. S. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).

    CAS  PubMed  Google Scholar 

  39. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).

    CAS  Google Scholar 

  40. Sánchez-Wandelmer, J. et al. Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes. Biochim. Biophys. Acta 1788, 1731–1739 (2009).

    PubMed  Google Scholar 

  41. Korade, Ž. et al. Effect of psychotropic drug treatment on sterol metabolism. Schizophr. Res. 187, 74–81 (2017).

    PubMed  PubMed Central  Google Scholar 

  42. Korade, Z. et al. Vulnerability of DHCR7+/− mutation carriers to aripiprazole and trazodone exposure. J. Lipid Res. 58, 2139–2146 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Chua, N. K., Coates, H. W. & Brown, A. J. Cholesterol, cancer, and rebooting a treatment for athlete’s foot. Sci. Transl. Med. 10, eaat3741 (2018).

  44. Brown, A. J. & Gelissen, I. C. Cholesterol and desmosterol dancing to the beat of a different drug. J. Intern. Med. 283, 102–105 (2018).

    CAS  PubMed  Google Scholar 

  45. Grimm, C. et al. High susceptibility to fatty liver disease in two-pore channel 2-deficient mice. Nat. Commun. 5, 4699 (2014).

    CAS  PubMed  Google Scholar 

  46. Mutemberezi, V., Guillemot-Legris, O. & Muccioli, G. G. Oxysterols: from cholesterol metabolites to key mediators. Prog. Lipid Res. 64, 152–169 (2016).

    CAS  PubMed  Google Scholar 

  47. Lamberson, C. R. et al. Propagation rate constants for the peroxidation of sterols on the biosynthetic pathway to cholesterol. Chem. Phys. Lipids 207, 51–58 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Plössl, F., Giera, M. & Bracher, F. Multiresidue analytical method using dispersive solid-phase extraction and gas chromatography/ion trap mass spectrometry to determine pharmaceuticals in whole blood. J. Chromatogr. A 1135, 19–26 (2006).

    PubMed  Google Scholar 

  49. Kloos, D. et al. Analysis of biologically-active, endogenous carboxylic acids based on chromatography-mass spectrometry. Trends Anal. Chem. 61, 17–28 (2014).

    CAS  Google Scholar 

  50. Goad, L. J. & Akihisa, T. Analysis of Sterols 115–143 (Springer, Netherlands, 1997).

  51. Gerst, N., Ruan, B., Pang, J., Wilson, W. K. & Schroepfer, G. J. An updated look at the analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry. J. Lipid Res. 38, 1685–1701 (1997).

    CAS  PubMed  Google Scholar 

  52. Griffiths, W. J. & Sjövall, J. Bile acids: analysis in biological fluids and tissues. J. Lipid Res. 51, 23–41 (2010).

    PubMed  PubMed Central  Google Scholar 

  53. Griffiths, W. J. et al. Cholesterolomics: an update. Anal. Biochem. 524, 56–67 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Bowers, L. D. & Sanaullah Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry. J. Chromatogr. B Biomed. Appl. 687, 61–68 (1996).

    CAS  PubMed  Google Scholar 

  55. Hill, M. et al. Steroid metabolome in plasma from the umbilical artery, umbilical vein, maternal cubital vein and in amniotic fluid in normal and preterm labor. J. Steroid Biochem. Mol. Biol. 121, 594–610 (2010).

    CAS  PubMed  Google Scholar 

  56. Häkkinen, M. R. et al. Analysis by LC–MS/MS of endogenous steroids from human serum, plasma, endometrium and endometriotic tissue. J. Pharm. Biomed. Anal. 152, 165–172 (2018).

    PubMed  Google Scholar 

  57. Griffiths, W. J. & Wang, Y. Analysis of oxysterol metabolomes. Biochim. Biophys. Acta 1811, 784–799 (2011).

    CAS  PubMed  Google Scholar 

  58. Herron, J. et al. Identification of environmental quaternary ammonium compounds as direct inhibitors of cholesterol biosynthesis. Toxicol. Sci. 151, 261–270 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Saraiva, D., Semedo, R., Castilho, Md. C., Silva, J. M. & Ramos, F. Selection of the derivatization reagent—the case of human blood cholesterol, its precursors and phytosterols GC–MS analyses. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. B 879, 3806–3811 (2011).

    CAS  Google Scholar 

  60. Wassif, C. A. et al. HEM dysplasia and ichthyosis are likely laminopathies and not due to 3β-hydroxysterol Δ14-reductase deficiency. Hum. Mol. Genet. 16, 1176–1187 (2007).

    CAS  PubMed  Google Scholar 

  61. Brunetti-Pierri, N. et al. Lathosterolosis, a novel multiple-malformation/mental retardation syndrome due to deficiency of 3β-hydroxysteroid-Δ5-desaturase. Am. J. Hum. Genet. 71, 952–958 (2002).

    PubMed  PubMed Central  Google Scholar 

  62. Has, C. et al. Gas chromatography–mass spectrometry and molecular genetic studies in families with the Conradi–Hünermann–Happle syndrome. J. Invest. Dermatol. 118, 851–858 (2002).

    CAS  PubMed  Google Scholar 

  63. Kelley, R. I. Diagnosis of Smith–Lemli–Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts. Clin. Chim. Acta 236, 45–58 (1995).

    CAS  PubMed  Google Scholar 

  64. Blassberg, R., Macrae, J. I., Briscoe, J. & Jacob, J. Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome. Hum. Mol. Genet. 25, 693–705 (2016).

    CAS  PubMed  Google Scholar 

  65. Korade, Z. et al. The effect of small molecules on sterol homeostasis: measuring 7-dehydrocholesterol in Dhcr7-deficient Neuro2a cells and human fibroblasts. J. Med. Chem. 59, 1102–1115 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Dias, C. et al. Desmosterolosis: an illustration of diagnostic ambiguity of cholesterol synthesis disorders. Orphanet J. Rare Dis. 9, 94 (2014).

    PubMed  PubMed Central  Google Scholar 

  67. Thompson, E. et al. Lamin B receptor-related disorder is associated with a spectrum of skeletal dysplasia phenotypes. Bone 120, 354–363 (2019).

    CAS  PubMed  Google Scholar 

  68. He, M., Smith, L. D., Chang, R., Li, X. & Vockley, J. The role of sterol-C4-methyl oxidase in epidermal biology. Biochim. Biophys. Acta 1841, 331–335 (2014).

    CAS  PubMed  Google Scholar 

  69. Acimovic, J. et al. Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 2081–2086 (2009).

    CAS  PubMed  Google Scholar 

  70. McDonald, J. G., Smith, D. D., Stiles, A. R. & Russell, D. W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53, 1399–1409 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Herron, J., Hines, K. M. & Xu, L. Assessment of altered cholesterol homeostasis by xenobiotics using ultra-high performance liquid chromatography–tandem mass spectrometry. Curr. Protoc. Toxicol. 78, e65 (2018).

    PubMed  Google Scholar 

  72. Griffiths, W. J. & Wang, Y. Sterolomics: state of the art, developments, limitations and challenges. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1862, 771–773 (2017).

    CAS  PubMed  Google Scholar 

  73. Liere, P. & Schumacher, M. Mass spectrometric analysis of steroids: all that glitters is not gold. Expert Rev. Endocrinol. Metab. 10, 463–465 (2015).

    CAS  PubMed  Google Scholar 

  74. Krone, N. et al. Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS). J. Steroid Biochem. Mol. Biol. 121, 496–504 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Nicholson, J. D. Derivative formation in the quantitative gas-chromatographic analysis of pharmaceuticals. Part II. A review. Analyst 103, 193–222 (1978).

    CAS  Google Scholar 

  76. Cerqueira, N. M. F. S. A. et al. Cholesterol biosynthesis: a mechanistic overview. Biochemistry 55, 5483–5506 (2016).

    CAS  PubMed  Google Scholar 

  77. Prabhu, A. V., Luu, W. & Brown, A. J. in Cholesterol Homeostasis: Methods and Protocols (eds Gelissen, I. C. & Brown, A. J.) 211–219 (Springer, New York, 2017).

  78. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    CAS  Google Scholar 

  79. Kohler, I., Verhoeven, A., Derks, R. J. & Giera, M. Analytical pitfalls and challenges in clinical metabolomics. Bioanalysis 8, 1509–1532 (2016).

    CAS  PubMed  Google Scholar 

  80. Nes, W. R. in Methods in Enzymology, Vol. 111 (eds Law, J. H. & Rilling, H. C.) 3–37 (Academic Press, 1985).

  81. Zhou, W. et al. Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1863, 1164–1178 (2018).

    CAS  PubMed  Google Scholar 

  82. Chugh, A., Ray, A. & Gupta, J. B. Squalene epoxidase as hypocholesterolemic drug target revisited. Prog. Lipid Res. 42, 37–50 (2003).

    CAS  PubMed  Google Scholar 

  83. Matzno, S. et al. Inhibition of cholesterol biosynthesis by squalene epoxidase inhibitor avoids apoptotic cell death in L6 myoblasts. J. Lipid Res. 38, 1639–1648 (1997).

    CAS  PubMed  Google Scholar 

  84. Mark, M., Muller, P., Maier, R. & Eisele, B. Effects of a novel 2,3-oxidosqualene cyclase inhibitor on the regulation of cholesterol biosynthesis in HepG2 cells. J. Lipid Res. 37, 148–158 (1996).

    CAS  PubMed  Google Scholar 

  85. Chuang, J.-C. et al. Sustained and selective suppression of intestinal cholesterol synthesis by Ro 48-8071, an inhibitor of 2,3-oxidosqualene:lanosterol cyclase, in the BALB/c mouse. Biochem. Pharmacol. 88, 351–363 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Pfeifer, T. et al. Synthetic LXR agonist suppresses endogenous cholesterol biosynthesis and efficiently lowers plasma cholesterol. Curr. Pharm. Biotechnol. 12, 285–292 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Lepesheva, G. I. & Waterman, M. R. Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms. Biochim. Biophys. Acta 1770, 467–477 (2007).

    CAS  PubMed  Google Scholar 

  88. Beynen, A. C., Buechler, K. F. & J. Van Der Molen, A. Inhibition of lipogenesis in isolated hepatocytes by 3-amino-1,2,4-triazole. Toxicology 22, 171–178 (1981).

    CAS  PubMed  Google Scholar 

  89. Shefer, S. et al. Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. J. Lipid Res. 39, 2471–2476 (1998).

    CAS  PubMed  Google Scholar 

  90. Porter, F. D. & Herman, G. E. Malformation syndromes caused by disorders of cholesterol synthesis. J. Lipid Res. 52, 6–34 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Kanungo, S., Soares, N., He, M. & Steiner, R. D. Sterol metabolism disorders and neurodevelopment—an update. Dev. Disabil. Res. Rev. 17, 197–210 (2013).

    PubMed  Google Scholar 

  92. Rohanizadegan, M. & Sacharow, S. Desmosterolosis presenting with multiple congenital anomalies. Eur. J. Med. Genet. 61, 152–156 (2018).

    PubMed  Google Scholar 

  93. Tomková, M., Marohnic, C., Baxová, A. & Martasek, P. Antley-Bixler syndrome or POR deficiency? Cas Lek Cesk 147, 261–265 (2008).

    PubMed  Google Scholar 

  94. Ho, A. C. C. et al. Lathosterolosis: a disorder of cholesterol biosynthesis resembling smith-lemli-opitz syndrome. JIMD Rep. 12, 129–134 (2013).

    PubMed  PubMed Central  Google Scholar 

  95. DeBarber, A. E., Eroglu, Y., Merkens, L. S., Pappu, A. S. & Steiner, R. D. Smith–Lemli–Opitz syndrome. Expert Rev. Mol. Med. 13, e24 (2011).

    PubMed  PubMed Central  Google Scholar 

  96. Brooks, C. J. W., Horning, E. C. & Young, J. S. Characterization of sterols by gas chromatography–mass spectrometry of the trimethylsilyl ethers. Lipids 3, 391–402 (1968).

    CAS  PubMed  Google Scholar 

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Acknowledgements

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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Correspondence to Martin Giera.

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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

Supplementary Information

Supplementary Figures 1–6 and Supplementary Tables 1–4 and 8

Reporting Summary

Supplementary Table 5

Supplementary Table 5

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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