Targeting cellular squalene synthase, an enzyme essential for cholesterol biosynthesis, is a potential antiviral strategy against hepatitis C virus

Journal of Virology

Volumn 89, Issue 4, 2015, Pages 2220-2232

  Saito, Kyoko ^a   Shirasago, Yoshitaka ^a,b   Suzuki, Tetsuro ^c   Aizaki, Hideki ^a   Hanada, Kentaro ^a   Wakita, Takaji ^a   Nishijima, Masahiro ^d   Fukasawa, Masayoshi ^a  

^a NATIONAL INSTITUTE OF INFECTIOUS DISEASES   (Japan)

^b TOKYO UNIVERSITY OF SCIENCE   (Japan)

^c HAMAMATSU UNIVERSITY SCHOOL OF MEDICINE   (Japan)

^d SHOWA PHARMACEUTICAL UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

CHOLESTEROL; CHOLESTEROL ESTER; NONSTRUCTURAL PROTEIN 4B; RNA; SINGLE STRANDED RNA; SMALL INTERFERING RNA; SQUALENE SYNTHASE; VIRUS RNA; ANTIVIRUS AGENT; ENZYME INHIBITOR; QUINUCLIDINE DERIVATIVE; YM 53601;

ARTICLE; CELL VIABILITY; CHOLESTEROL ESTERIFICATION; CHOLESTEROL METABOLISM; CHOLESTEROL SYNTHESIS; CONTROLLED STUDY; HEPATITIS C VIRUS; HUMAN; IN VITRO STUDY; NONHUMAN; PROGENY; PROTEIN ANALYSIS; PROTEIN TARGETING; REPLICON; RNA REPLICATION; VIRUS ENTRY; VIRUS REPLICATION; ANTAGONISTS AND INHIBITORS; BIOSYNTHESIS; CELL LINE; DRUG EFFECTS; ENZYMOLOGY; HEPACIVIRUS; LIVER CELL; PHYSIOLOGY; VIROLOGY;

HEPATITIS C VIRUS;

ANTIVIRAL AGENTS; CELL LINE; CHOLESTEROL; ENZYME INHIBITORS; FARNESYL-DIPHOSPHATE FARNESYLTRANSFERASE; HEPACIVIRUS; HEPATOCYTES; HUMANS; QUINUCLIDINES; VIRUS REPLICATION;

EID: 84921691270     PISSN: 0022538X     EISSN: 10985514     Source Type: Journal    
DOI: 10.1128/JVI.03385-14     Document Type: Article

References (76)

1. World Health Organization. 2014. Hepatitis C fact sheet no. 164. World Health Organization, Geneva, Switzerland. http://www.who.int/mediacentre/factsheets/fs164/en/.
2. Ghany MG, Strader DB, Thomas DL, Seeff LB. 2009. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 49:1335-1374. http://dx.doi.org/10.1002/hep.22759.
3. Alexopoulou A, Papatheodoridis GV. 2012. Current progress in the treatment of chronic hepatitis C. World J Gastroenterol 18:6060-6069. http://dx.doi.org/10.3748/wjg.v18.i42.6060.
4. Doyle JS, Aspinall E, Liew D, Thompson AJ, Hellard ME. 2013. Current and emerging antiviral treatments for hepatitisCinfection. Br J Clin Pharmacol 75:931-943. http://dx.doi.org/10.1111/j.1365-2125.2012.04419.x.
5. Lawitz E, Sulkowski MS, Ghalib R, Rodriguez-Torres M, Younossi ZM, Corregidor A, DeJesus E, Pearlman B, Rabinovitz M, Gitlin N, Lim JK, Pockros PJ, Scott JD, Fevery B, Lambrecht T, Ouwerkerk-Mahadevan S, Callewaert K, Symonds WT, Picchio G, Lindsay KL, Beumont M, Jacobson IM. 2014. Simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis C virus genotype 1 in nonresponders to pegylated interferon and ribavirin and treatment-naive patients: the COSMOS randomised study. Lancet 384:1756-1765. http://dx.doi.org/10.1016/S0140-6736(14)61036-9.
6. Schneider MD, Sarrazin C. 2014. Antiviral therapy of hepatitis C in 2014: do we need resistance testing? Antiviral Res 105:64-71. http://dx.doi.org/10.1016/j.antiviral.2014.02.011.
7. Fraser CS, Doudna JA. 2007. Structural and mechanistic insights into hepatitis C viral translation initiation. Nat Rev Microbiol 5:29-38. http://dx.doi.org/10.1038/nrmicro1558.
8. Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM. 1993. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67:1385-1395.
9. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H. 1993. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67: 3835-3844.
10. Hijikata M, Mizushima H, Tanji Y, Komoda Y, Hirowatari Y, Akagi T, Kato N, Kimura K, Shimotohno K. 1993. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci USA 90:10773-10777. http://dx.doi.org/10.1073/pnas.90.22.10773.
11. Moradpour D, Penin F, Rice CM. 2007. Replication of hepatitis C virus. Nat Rev Microbiol 5:453-463. http://dx.doi.org/10.1038/nrmicro1645.
12. Moradpour D, Gosert R, Egger D, Penin F, Blum HE, Bienz K. 2003. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 60:103-109. http://dx.doi.org/10.1016/j.antiviral.2003.08.017.
13. Felmlee DJ, Hafirassou ML, Lefevre M, Baumert TF, Schuster C. 2013. Hepatitis C virus, cholesterol and lipoproteins-impact for the viral life cycle and pathogenesis of liver disease. Viruses 5:1292-1324. http://dx.doi.org/10.3390/v5051292.
14. Kapadia SB, Barth H, Baumert T, McKeating JA, Chisari FV. 2007. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol 81: 374-383. http://dx.doi.org/10.1128/JVI.01134-06.
15. Aizaki H, Lee KJ, Sung VM, Ishiko H, Lai MM. 2004. Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324:450-461. http://dx.doi.org/10.1016/j.virol.2004.03.034.
16. Shi ST, Lee KJ, Aizaki H, Hwang SB, Lai MM. 2003. Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77:4160-4168. http://dx.doi.org/10.1128/JVI.77.7.4160-4168.2003.
17. Gao L, Aizaki H, He JW, Lai MM. 2004. Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J Virol 78: 3480-3488. http://dx.doi.org/10.1128/JVI.78.7.3480-3488.2004.
18. Lingwood D, Simons K. 2010. Lipid rafts as a membrane-organizing principle. Science 327:46-50. http://dx.doi.org/10.1126/science.1174621.
19. Aizaki H, Morikawa K, Fukasawa M, Hara H, Inoue Y, Tani H, Saito K, Nishijima M, Hanada K, Matsuura Y, Lai MM, Miyamura T, Wakita T, Suzuki T. 2008. Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection. J Virol 82:5715-5724. http://dx.doi.org/10.1128/JVI.02530-07.
20. Matto M, Rice CM, Aroeti B, Glenn JS. 2004. Hepatitis C virus core protein associates with detergent-resistant membranes distinct from classical plasma membrane rafts. J Virol 78:12047-12053. http://dx.doi.org/10.1128/JVI.78.21.12047-12053.2004.
21. Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T, Zayas M, Bartenschlager R, Wakita T, Hijikata M, Shimotohno K. 2007. The lipid droplet is an important organelle for hepatitisCvirus production. Nat Cell Biol 9:1089-1097. http://dx.doi.org/10.1038/ncb1631.
22. Bartenschlager R, Penin F, Lohmann V, Andre P. 2011. Assembly of infectious hepatitis C virus particles. Trends Microbiol 19:95-103. http://dx.doi.org/10.1016/j.tim.2010.11.005.
23. Hishiki T, Shimizu Y, Tobita R, Sugiyama K, Ogawa K, Funami K, Ohsaki Y, Fujimoto T, Takaku H, Wakita T, Baumert TF, Miyanari Y, Shimotohno K. 2010. Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms. J Virol 84:12048-12057. http://dx.doi.org/10.1128/JVI.01063-10.
24. Merz A, Long G, Hiet MS, Brugger B, Chlanda P, Andre P, Wieland F, Krijnse-Locker J, Bartenschlager R. 2011. Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome. J Biol Chem 286:3018-3032. http://dx.doi.org/10.1074/jbc.M110.175018.
25. Goldstein JL, Brown MS. 1990. Regulation of the mevalonate pathway. Nature 343:425-430. http://dx.doi.org/10.1038/343425a0.
26. Schachter M. 2005. Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam Clin Pharmacol 19:117-125. http://dx.doi.org/10.1111/j.1472-8206.2004.00299.x.
27. Ye J, Wang C, Sumpter R, Jr, Brown MS, Goldstein JL, Gale M, Jr. 2003. Disruption of hepatitisCvirusRNAreplication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci USA 100:15865-15870. http://dx.doi.org/10.1073/pnas.2237238100.
28. Kapadia SB, Chisari FV. 2005. Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci USA 102:2561-2566. http://dx.doi.org/10.1073/pnas.0409834102.
29. Ikeda M, Abe K, Yamada M, Dansako H, Naka K, Kato N. 2006. Different anti-HCV profiles of statins and their potential for combination therapy with interferon. Hepatology 44:117-125. http://dx.doi.org/10.1002/hep.21232.
30. Brautbar A, Ballantyne CM. 2011. Pharmacological strategies for lowering LDL cholesterol: statins and beyond. Nat Rev Cardiol 8:253-265. http://dx.doi.org/10.1038/nrcardio.2011.2.
31. Owens CM, Mawhinney C, Grenier JM, Altmeyer R, Lee MS, Borisy AA, Lehar J, Johansen LM. 2010. Chemical combinations elucidate pathway interactions and regulation relevant to hepatitis C replication. Mol Syst Biol 6:375.
32. Rodgers MA, Villareal VA, Schaefer EA, Peng LF, Corey KE, Chung RT, Yang PL. 2012. Lipid metabolite profiling identifies desmosterol metabolism as a new antiviral target for hepatitis C virus. J Am Chem Soc 134: 6896-6899. http://dx.doi.org/10.1021/ja207391q.
33. Takano T, Tsukiyama-Kohara K, Hayashi M, Hirata Y, Satoh M, Tokunaga Y, Tateno C, Hayashi Y, Hishima T, Funata N, Sudoh M, Kohara M. 2011. Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes. J Hepatol 55: 512-521. http://dx.doi.org/10.1016/j.jhep.2010.12.011.
34. Tansey TR, Shechter I. 2000. Structure and regulation of mammalian squalene synthase. Biochim Biophys Acta 1529:49-62. http://dx.doi.org/10.1016/S1388-1981(00)00137-2.
35. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R, Liang TJ. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791-796. http://dx.doi.org/10.1038/nm1268.
36. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wieland SF, Uprichard SL, Wakita T, Chisari FV. 2005. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 102:9294-9299. http://dx.doi.org/10.1073/pnas.0503596102.
37. Shirasago Y, Sekizuka T, Saito K, Suzuki T, Wakita T, Hanada K, Kuroda M, Abe R, Fukasawa M. 25 November 2014. Isolation and characterization of a Huh.7.5.1-derived cell clone highly permissive to hepatitis C virus. Jpn J Infect Dis http://dx.doi.org/10.7883/yoken.JJID.2014.231.
38. Ugawa T, Kakuta H, Moritani H, Matsuda K, Ishihara T, Yamaguchi M, Naganuma S, Iizumi Y, Shikama H. 2000. YM-53601, a novel squalene synthase inhibitor, reduces plasma cholesterol and triglyceride levels in several animal species. Br J Pharmacol 131:63-70. http://dx.doi.org/10.1038/sj.bjp.0703545.
39. Bergstrom JD, Kurtz MM, Rew DJ, Amend AM, Karkas JD, Bostedor RG, Bansal VS, Dufresne C, VanMiddlesworth FL, Hensens OD, Liesch JM, Zink DL, Wilson KE, Onishi J, Milligan JA, Bills G, Kaplan L, Nallin Omstead M, Jenkins RG, Huang L, Meinz MS, Quinn L, Burg RW, Kong YL, Mochales S, Mojena M, Martin I, Pelaez F, Diez MT, Alberts AW. 1993. Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase. Proc Natl Acad Sci USA 90:80-84. http://dx.doi.org/10.1073/pnas.90.1.80.
40. Ross AC, Go KJ, Heider JG, Rothblat GH. 1984. Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035. J Biol Chem 259:815-819.
41. Hanada K, Nishijima M, Kiso M, Hasegawa A, Fujita S, Ogawa T, Akamatsu Y. 1992. Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids. J Biol Chem 267: 23527-23533.
42. Kato T, Matsumura T, Heller T, Saito S, Sapp RK, Murthy K, Wakita T, Liang TJ. 2007. Production of infectious hepatitis C virus of various genotypes in cell cultures. J Virol 81:4405-4411. http://dx.doi.org/10.1128/JVI.02334-06.
43. Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917. http://dx.doi.org/10.1139/o59-099.
44. Mizoguchi T, Edano T, Koshi T. 2004. A method of direct measurement for the enzymatic determination of cholesteryl esters. J Lipid Res 45:396-401. http://dx.doi.org/10.1194/jlr.D300024-JLR200.
45. Kato T, Date T, Miyamoto M, Sugiyama M, Tanaka Y, Orito E, Ohno T, Sugihara K, Hasegawa I, Fujiwara K, Ito K, Ozasa A, Mizokami M, Wakita T. 2005. Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system. J Clin Microbiol 43:5679-5684. http://dx.doi.org/10.1128/JCM.43.11.5679-5684.2005.
46. Krieger N, Lohmann V, Bartenschlager R. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614-4624. http://dx.doi.org/10.1128/JVI.75.10.4614-4624.2001.
47. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, Wakita T. 2003. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808-1817. http://dx.doi.org/10.1053/j.gastro.2003.09.023.
48. Inoue Y, Murakami K, Hmwe SS, Aizaki H, Suzuki T. 2007. Transcriptomic comparison of human hepatoma Huh-7 cell clones with different hepatitis C virus replication efficiencies. Jpn J Infect Dis 60:173-178. http://www0.nih.go.jp/JJID/60/173.pdf.
49. Yamaji T, Nishikawa K, Hanada K. 2010. Transmembrane BAX inhibitor motif containing (TMBIM) family proteins perturbs a trans-Golgi network enzyme, Gb3 synthase, and reduces Gb3 biosynthesis. J Biol Chem 285:35505-35518. http://dx.doi.org/10.1074/jbc.M110.154229.
50. Niwa H, Yamamura K, Miyazaki J. 1991. Efficient selection for highexpression transfectants with a novel eukaryotic vector. Gene 108:193-199. http://dx.doi.org/10.1016/0378-1119(91)90434-D.
51. Yamaji T, Mitsuki M, Teranishi T, Hashimoto Y. 2005. Characterization of inhibitory signaling motifs of the natural killer cell receptor Siglec-7: attenuated recruitment of phosphatases by the receptor is attributed to two amino acids in the motifs. Glycobiology 15:667-676. http://dx.doi.org/10.1093/glycob/cwi048.
52. Bartosch B, Dubuisson J, Cosset FL. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633-642. http://dx.doi.org/10.1084/jem.20021756.
53. Murakami Y, Fukasawa M, Kaneko Y, Suzuki T, Wakita T, Fukazawa H. 2013. Selective estrogen receptor modulators inhibit hepatitis C virus infection at multiple steps of the virus life cycle. Microbes Infect 15:45-55. http://dx.doi.org/10.1016/j.micinf.2012.10.003.
54. Wakita T, Wands JR. 1994. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence. J Biol Chem 269:14205-14210.
55. Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, Moradpour D, Bienz K. 2002. Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76:5974-5984. http://dx.doi.org/10.1128/JVI.76.12.5974-5984.2002.
56. Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, Moradpour D. 2003. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77: 5487-5492. http://dx.doi.org/10.1128/JVI.77.9.5487-5492.2003.
57. Konan KV, Giddings TH, Jr, Ikeda M, Li K, Lemon SM, Kirkegaard K. 2003. Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J Virol 77: 7843-7855. http://dx.doi.org/10.1128/JVI.77.14.7843-7855.2003.
58. Lundin M, Monne M, Widell A, Von Heijne G, Persson MA. 2003. Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol 77:5428-5438. http://dx.doi.org/10.1128/JVI.77.9.5428-5438.2003.
59. Gretton SN, Taylor AI, McLauchlan J. 2005. Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol 86:1415-1421. http://dx.doi.org/10.1099/vir.0.80768-0.
60. Manna D, Aligo J, Xu C, Park WS, Koc H, Heo WD, Konan KV. 2010. Endocytic Rab proteins are required for hepatitis C virus replication complex formation. Virology 398:21-37. http://dx.doi.org/10.1016/j.virol.2009.11.034.
61. Han Q, Aligo J, Manna D, Belton K, Chintapalli SV, Hong Y, Patterson RL, van Rossum DB, Konan KV. 2011. Conserved GXXXG-and S/T-like motifs in the transmembrane domains of NS4B protein are required for hepatitis C virus replication. J Virol 85:6464-6479. http://dx.doi.org/10.1128/JVI.02298-10.
62. Park EM, Nguyen LN, Lim YS, Hwang SB. 2014. Farnesyl-diphosphate farnesyltransferase 1 regulates hepatitis C virus propagation. FEBS Lett 588:1813-1820. http://dx.doi.org/10.1016/j.febslet.2014.03.043.
63. Ishihara T, Kakuta H, Moritani H, Ugawa T, Sakamoto S, Tsukamoto S-I, Yanagisawa I. 2003. Syntheses of 3-ethylidenequinuclidine derivatives as squalene synthase inhibitors. Part 2: Enzyme inhibition and effects on plasma lipid levels. Bioorg Med Chem 11:3735-3745. http://dx.doi.org/10.1016/S0968-0896(03)00336-5.
64. Sakamoto H, Okamoto K, Aoki M, Kato H, Katsume A, Ohta A, Tsukuda T, Shimma N, Aoki Y, Arisawa M, Kohara M, Sudoh M. 2005. Host sphingolipid biosynthesis as a target for hepatitis C virus therapy. Nat Chem Biol 1:333-337. http://dx.doi.org/10.1038/nchembio742.
65. Weng L, Hirata Y, Arai M, Kohara M, Wakita T, Watashi K, Shimotohno K, He Y, Zhong J, Toyoda T. 2010. Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner. J Virol 84:11761-11770. http://dx.doi.org/10.1128/JVI.00638-10.
66. Voisset C, Lavie M, Helle F, Op De Beeck A, Bilheu A, Bertrand-Michel J, Terce F, Cocquerel L, Wychowski C, Vu-Dac N, Dubuisson J. 2008. Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry. Cell Microbiol 10:606-617. http://dx.doi.org/10.1111/j.1462-5822.2007.01070.x.
67. Brusselmans K, Timmermans L, Van de Sande T, Van Veldhoven PP, Guan G, Shechter I, Claessens F, Verhoeven G, Swinnen JV. 2007. Squalene synthase, a determinant of raft-associated cholesterol and modulator of cancer cell proliferation. J Biol Chem 282:18777-18785. http://dx.doi.org/10.1074/jbc.M611763200.
68. Paul D, Hoppe S, Saher G, Krijnse-Locker J, Bartenschlager R. 2013. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol 87:10612-10627. http://dx.doi.org/10.1128/JVI.01370-13.
69. Lange Y. 1991. Disposition of intracellular cholesterol in human fibroblasts. J Lipid Res 32:329-339.
70. Ikonen E. 2008. Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9:125-138. http://dx.doi.org/10.1038/nrm2336.
71. Wolk B, Buchele B, Moradpour D, Rice CM. 2008. A dynamic view of hepatitis C virus replication complexes. J Virol 82:10519-10531. http://dx.doi.org/10.1128/JVI.00640-08.
72. Targett-Adams P, Boulant S, McLauchlan J. 2008. Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol 82:2182-2195. http://dx.doi.org/10.1128/JVI.01565-07.
73. Huang H, Sun F, Owen DM, Li W, Chen Y, Gale M, Jr, Ye J. 2007. Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. Proc Natl Acad Sci USA 104:5848-5853. http://dx.doi.org/10.1073/pnas.0700760104.
74. Zheng YH, Plemenitas A, Fielding CJ, Peterlin BM. 2003. Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions. Proc Natl Acad Sci USA 100:8460-8465. http://dx.doi.org/10.1073/pnas.1437453100.
75. Charlton-Menys V, Durrington PN. 2008. Human cholesterol metabolism and therapeutic molecules. Exp Physiol 93:27-42. http://dx.doi.org/10.1113/expphysiol.2007.035147.
76. Trapani L, Segatto M, Ascenzi P, Pallottini V. 2011. Potential role of nonstatin cholesterol lowering agents. IUBMB Life 63:964-971. http://dx.doi.org/10.1002/iub.522.