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19-Azasqualene-2,3-epoxide and its N-oxide: Metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae

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Lipids

Abstract

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7±2 and 25±5 μM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [14C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 μM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and >100 μM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in “ultrasonically stimulated” cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.

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Abbreviations

IC50 :

concentration of inhibitor that reduced the enzymatic conversion of 2,3-oxidosqualene to lanosterol by 50%

NMR:

nuclear magnetic resonance

OS:

2,3-oxidosqualene

OSC:

2,3-oxidosqualene-lanosterol cyclase

TLC:

thin-layer chromatography

References

  1. Abe, I., Rohmer, M., and Prestwich, G.D. (1993) Enzymatic Cyclization of Squalene and Oxidosqualene to Sterols and Triterpenes, Chem. Rev., 93, 2189–2206.

    Article  CAS  Google Scholar 

  2. Van Tamelen, E.E. (1982) Bioorganic Characterization and Mechanism of the 2,3-Oxidosqualene→Lanosterol Conversion, J. Am. Chem. Soc. 104, 6480–6481.

    Article  Google Scholar 

  3. Nes, W.D., Koike, K., Jia, Z., Sakamoto, Y., Satou, T., Nikaido, T., and Griffin, J.F. (1998) Cyclosterol Analysis by 1H and 13C NMR, Crystallographic Observations, and Molecular Mechanics Calculations, J. Am. Chem. Soc. 120, 5970–5980.

    Article  CAS  Google Scholar 

  4. Moore, W.R., and Schatzman, G.L. (1992) Purification of 2,3-Oxidosqualene Cyclase from Rat Liver, J. Biol. Chem. 267, 22003–22006.

    PubMed  CAS  Google Scholar 

  5. Abe, I., Ebizuka, Y., Seo, S., and Sankawa, U. (1989) Purification of Squalene-2,3-epoxide Cyclase from Cell Suspension Cultures of Rabdosia japonica Hara, FEBS Lett. 249, 100–104.

    Article  CAS  Google Scholar 

  6. Abe, I., Sankawa, U., and Ebizuka, Y. (1992) Purification and Properties of Squalene-2,3-epoxide Cyclase from Pea Seedlings, Chem. Pharm. Bull. 40, 1755–1760.

    CAS  Google Scholar 

  7. Abe, I., and Prestwich, G.D. (1995) Molecular Cloning, Characterization, and Functional Expression of Rat Oxidosqualene Cyclase cDNA, Proc. Natl. Acad. Sci. USA 92, 9274–9278.

    Article  PubMed  CAS  Google Scholar 

  8. Corey, E.J., Matsuda, S.P.T., and Bartel, B. (1994) Molecular Cloning, Characterization, and Overexpression of ERG7, the Saccharomyces cerevisiae Gene Encoding Lanosterol Synthase, Proc. Natl. Acad. Sci. USA 91, 2211–2215.

    Article  PubMed  CAS  Google Scholar 

  9. Buntel, C.J., and Griffin, J.H. (1992) Nucleotide and Deduced Amino Acid Sequences of the Oxidosqualene Cyclase from Candida albicans, J. Am. Chem. Soc. 114, 9711–9713.

    Article  CAS  Google Scholar 

  10. Corey, E.J., Matsuda, S.P.T., and Bartel, B. (1993) Isolation on an Arabidopsis thaliana Gene Encoding Cycloartenol Synthase by Functional Expression in a Yeast Mutant Lacking Lanosterol Synthase by the Use of a Chromatographic Screen, Proc. Natl. Acad. Sci. USA 90, 11628–11632.

    Article  PubMed  CAS  Google Scholar 

  11. Xiao, X., and Prestwich, G.D. (1991) 29-Methylidene-2,3-oxidosqualene: A Potent Mechanism-Based Inactivator of Oxidosqualene Cyclase, J. Am. Chem. Soc. 113, 9673–9674.

    Article  CAS  Google Scholar 

  12. Abe, I., Liu, W., Oehlschlager, A.C., and Prestwich, G.D. (1996) Mechanism-Based Active Site Modification of Oxidosqualene Cyclase by Tritium-Labeled 18-Thia-2,3-oxidosqualene, J. Am. Chem. Soc. 118, 9180–9181.

    Article  CAS  Google Scholar 

  13. Corey, E.J., Cheng, H., Baker, H., Matsuda, S.P.T., Li, D., and Song, X. (1997) Studies of the Substrate Binding Segments and Catalytic Action of Lanosterol Synthase. Affinity Labeling with Carbocations Derived from Mechanism-Based Analogs of 2,3-Oxidosqualene and Site-Directed Mutagenesis Probes, J. Am. Chem. Soc. 119, 1289–1296.

    Article  CAS  Google Scholar 

  14. Ceruti, M., Rocco, F., Viola, F., Balliano, G., Milla, P., Arpicco, S., and Cattel, L. (1998) 29-Methylidene-2,3-oxidosqualene Derivatives as Stereospectific Mechanism-Based Inhibitors of Liver and Yeast Oxidosqualene Cyclase, J. Med. Chem. 41, 540–554.

    Article  PubMed  CAS  Google Scholar 

  15. Eisele, B., Budzinski, R., Muller, P., Maier, R., and Mark, M. (1997) Effects of a Novel 2,3-Oxidosqualene Cyclase Inhibitor on Cholesterol Biosynthesis and Lipid Metabolism in vivo, J. Lipid Res. 38, 564–575.

    PubMed  CAS  Google Scholar 

  16. Zheng, Y.E., Dodd, D.S., Oehlschlager, A.C., and Hartman, P.G. (1995) Synthesis of Vinyl Sulfide Analogs of 2,3-Oxidosqualene and Their Inhibition of 2,3-Oxidosqualene Lanosterol Cyclase, Tetrahedron 51, 5255–5276.

    Article  CAS  Google Scholar 

  17. Cattel, L., Ceruti, M., Balliano, G., Viola, F., Grosa, G., Rocco, F., and Brusa, P. (1995) 2,3-Oxidosqualene Cyclase: from Azasqualenes to New Site-Directed Inhibitors, Lipids 30, 235–246.

    PubMed  CAS  Google Scholar 

  18. Balliano, G., Viola, F., Ceruti, M., and Cattel, L. (1988) Inhibition of Sterol Biosynthesis in Saccharomyces cerevisiae by N,N-Diethylazasqualene and Derivatives, Biochim. Biophys. Acta 959, 9–19.

    PubMed  CAS  Google Scholar 

  19. Ceruti, M., Balliano, G., Viola, F., Cattel, L., Gerst, N., and Schuber, F. (1987) Synthesis and Biologial Activity of Azasqualenes, Bis-azasqualenes and Derivatives, Eur. J. Med. Chem. 22, 199–208.

    Article  CAS  Google Scholar 

  20. Viola, F., Brusa, P., Balliano, G., Ceruti, M., Boutaud, O., Schuber, F., and Cattel, L. (1995) Inhibition of 2,3-Oxidosqualene Cyclase and Sterol Biosynthesis by 10-and 19-Azasqualenes Derivatives, Biochem. Pharmacol. 50, 787–796.

    Article  PubMed  CAS  Google Scholar 

  21. Bradford, M.M. (1976) 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.

    Article  PubMed  CAS  Google Scholar 

  22. Bujons, J., Guajardo, R., and Kyler, K.S. (1988) Enantioselective Enzymatic Sterol Synthesis by Ultrasonically Stimulated Baker’s Yeast, J. Am. Chem. Soc. 110, 604–606.

    Article  CAS  Google Scholar 

  23. Peterson, G.L. (1977) A Simplification of the Protein Assay Method of Lowry et al. Which Is More Generally Applicable, Anal. Biochem. 83, 346–356.

    Article  PubMed  CAS  Google Scholar 

  24. Rickwood, D., Dujon, B., and Darley-Usmar, V.M. (1988) Yeast Mitochondria, in Yeast, A Practical Approach (Campbell, I., and Duffus, J.H., eds.), pp. 185–247, IRL Press, Oxford.

    Google Scholar 

  25. Lineweaver, H., and Burk, D. (1934) The Determination of Enzyme Dissociation Constants, J. Am. Chem. Soc. 56, 658–666.

    Article  CAS  Google Scholar 

  26. Lindsey, S., and Harwood, H.J., Jr. (1995) Inhibition of Mammalian Squalene Synthetase Activity by Zaragozic Acid A Is a Result of Competitive Inhibition Followed by Mechanism-Based Irreversible Inactivation, J. Biol. Chem. 270, 9083–9096.

    Article  PubMed  CAS  Google Scholar 

  27. Ceruti, M., Rocco, F., Viola, F., Balliano, G., Dosio, F., and Cattel, L. (1993) Synthesis and Biological Activity of 19-Azasqualene 2,3-Epoxide as Inhibitor of 2,3-Oxidosqualene Cyclase, Eur. J. Med. Chem. 28, 675–682.

    Article  CAS  Google Scholar 

  28. Ceruti, M., Grosa, G., Rocco, F., Dosio, F., and Cattel, L. (1994) A Convenient Synthesis of [3-3H]Squalene and [3-3H]-2,3-Oxidosqualene, J. Labelled Compd. Radiopharm. 34, 577–585.

    Article  CAS  Google Scholar 

  29. Balliano, G., Milla, P., Ceruti, M., Carrano, L., Viola, F., Brusa, P., and Cattel, L. (1994) Inhibition of Sterol Biosynthesis in Saccharomyces cerevisiae and Candida albicans by 22,23-Epoxy-2-aza-2,3-dihydrosqualene and the Corresponding N-Oxide, Antimicrob. Agents Chemother. 38, 1904–1908.

    PubMed  CAS  Google Scholar 

  30. Nes, W.D., and Parish, E.J. (1988) Metabolism of 2(RS),3-Epiminosqualene to 24(RS),25-Epiminolanosterol by Gibberella fujikuroi, Lipids 13, 375–376.

    Google Scholar 

  31. Cattel, L., Ceruti, M., Balliano, G., Viola, F., Grosa, G., and Schuber, F. (1989) Drug Design Based on Biosynthetic Studies: Synthesis, Biological Activity, and Kinetics of New Inhibitors of 2,3-Oxidosqualene Cyclase and Squalene Epoxidase, Steroids 53, 363–391.

    Article  PubMed  CAS  Google Scholar 

  32. Cattel, L., and Ceruti, M. (1997) Inhibitors of 2,3-Oxidosqualene Cyclase as Tools for Studying the Mechanism and Function of the Enzyme, in Biochemistry and Function of Sterols (Parish, E.J., and Nes, W.D., eds.), pp. 1–21, CRC Press, Boca Raton.

    Google Scholar 

  33. Kitamura, S., and Tatsumi, K. (1984) Reduction of Tertiary Amine N-Oxides by Liver Preparations: Function of Aldehyde Oxidase as a Major N-Oxide Reductase, Biochem. Biophys. Res. Commun. 121, 749–754.

    Article  PubMed  CAS  Google Scholar 

  34. Balliano, G., Viola, F., Ceruti, M., and Cattel, L. (1992) Characterization and Partial Purification of Squalene-2,3-oxide Cyclase from Saccharomyces cerevisiae, Arch. Biochem. Biophys. 293, 122–129.

    Article  PubMed  CAS  Google Scholar 

  35. Leber, R., Zinser, E., Zellnig, G., Paltauf, F., and Daum, G. (1994) Characterization of Lipid Particles of the Yeast, Saccharomyces cerevisiae, Yeast 10, 1421–1428.

    Article  PubMed  CAS  Google Scholar 

  36. Zinser, E., Sperka-Gottlieb, C.D.M., Fasch, E.-V., Kohlwein, S.D., Paltauf, F., and Daum, G. (1991) Phospholipid Synthesis and Lipid Composition of Subcellular Membranes in the Unicellular Eukaryote Saccharomyces cerevisiae, J. Bacteriol. 173, 2026–2034.

    PubMed  CAS  Google Scholar 

  37. Zinser, E., Paltauf, F., and Daum, G. (1993) Sterol Composition of Yeast Organelle Membranes and Subcellular Distribution of Enzymes Involved in Sterol Metabolism, J. Bacteriol. 175, 2853–2858.

    PubMed  CAS  Google Scholar 

  38. Leber, R., Landl, K., Zinser, E., Ahorn, H., Spök, A., Kohlwein, S.D., Turnowsky, F., and Daum G. (1998) Dual Localization of Squalene Epoxidase, Erg 1p, in Yeast Reflects a Relationship Between the Endoplasmic Reticulum and Lipid Particles, Mol. Biol. Cell. 9, 375–386.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Via Pietro Giuria, 9, 10125, Torino, Italy

    Paola Milla, Franca Viola, Maurizio Ceruti, Flavio Rocco, Luigi Cattel & Gianni Balliano

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  1. Paola Milla
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  2. Franca Viola
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  3. Maurizio Ceruti
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  4. Flavio Rocco
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  5. Luigi Cattel
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  6. Gianni Balliano
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Correspondence to Gianni Balliano.

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Milla, P., Viola, F., Ceruti, M. et al. 19-Azasqualene-2,3-epoxide and its N-oxide: Metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae . Lipids 34, 681–688 (1999). https://doi.org/10.1007/s11745-999-0413-5

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  • DOI: https://doi.org/10.1007/s11745-999-0413-5

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