A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

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Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC₅₀ values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Vyšlo v časopise: A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry. PLoS Pathog 6(9): e32767. doi:10.1371/journal.ppat.1001086
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001086

Souhrn

Zdroje

1. ShepardCW

FinelliL

AlterMJ

2005 Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5 558 567

2. LauerGM

WalkerBD

2001 Hepatitis C virus infection. N Engl J Med 345 41 52

3. SimonettiRG

CammaC

FiorelloF

CottoneM

RapicettaM

1992 Hepatitis C virus infection as a risk factor for hepatocellular carcinoma in patients with cirrhosis. A case-control study. Ann Intern Med 116 97 102

4. SeeffLB

1997 Natural history of hepatitis C. Hepatology 26 21S 28S

5. CharltonM

2001 Hepatitis C infection in liver transplantation. Am J Transplant 1 197 203

6. ZeuzemS

2008 Interferon-based therapy for chronic hepatitis C: current and future perspectives. Nat Clin Pract Gastroenterol Hepatol 5 610 622

7. FeldJJ

HoofnagleJH

2005 Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436 967 972

8. MannsMP

WedemeyerH

CornbergM

2006 Treating viral hepatitis C: efficacy, side effects, and complications. Gut 55 1350 1359

9. SarrazinC

ZeuzemS

2010 Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology 138 447 462

10. RongL

DahariH

RibeiroRM

PerelsonAS

2010 Rapid emergence of protease inhibitor resistance in hepatitis C virus. Sci Transl Med 2 30ra32

11. BarthH

SchaferC

AdahMI

ZhangF

LinhardtRJ

2003 Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J Biol Chem 278 41003 41012

12. BasuA

KandaT

BeyeneA

SaitoK

MeyerK

2007 Sulfated homologues of heparin inhibit hepatitis C virus entry into mammalian cells. J Virol 81 3933 3941

13. GermiR

CranceJM

GarinD

GuimetJ

Lortat-JacobH

2002 Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption. J Med Virol 68 206 215

14. MolinaS

CastetV

Fournier-WirthC

Pichard-GarciaL

AvnerR

2007 The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus. J Hepatol 46 411 419

15. MonazahianM

BohmeI

BonkS

KochA

ScholzC

1999 Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol 57 223 229

16. OwenDM

HuangH

YeJ

GaleMJr

2009 Apolipoprotein E on hepatitis C virion facilitates infection through interaction with low-density lipoprotein receptor. Virology 394 99 108

17. MolinaS

CastetV

Pichard-GarciaL

WychowskiC

MeursE

2008 Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent. J Virol 82 569 574

18. KapadiaSB

BarthH

BaumertT

McKeatingJA

ChisariFV

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

19. BartoschB

VitelliA

GranierC

GoujonC

DubuissonJ

2003 Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278 41624 41630

20. CormierEG

TsamisF

KajumoF

DursoRJ

GardnerJP

2004 CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 101 7270 7274

21. LavilletteD

TarrAW

VoissetC

DonotP

BartoschB

2005 Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus. Hepatology 41 265 274

22. ZhangJ

RandallG

HigginbottomA

MonkP

RiceCM

2004 CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol 78 1448 1455

23. ZeiselMB

KoutsoudakisG

SchnoberEK

HaberstrohA

BlumHE

2007 Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 46 1722 1731

24. DreuxM

Dao ThiVL

FresquetJ

GuerinM

JuliaZ

2009 Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains. PLoS Pathog 5 e1000310

25. CataneseMT

AnsuiniH

GrazianiR

HubyT

MoreauM

2010 Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants. J Virol 84 34 43

26. CataneseMT

GrazianiR

von HahnT

MoreauM

HubyT

2007 High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein. J Virol 81 8063 8071

27. EvansMJ

von HahnT

TscherneDM

SyderAJ

PanisM

2007 Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446 801 805

28. LiuS

YangW

ShenL

TurnerJR

CoyneCB

2009 Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol 83 2011 2014

29. PlossA

EvansMJ

GaysinskayaVA

PanisM

YouH

2009 Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457 882 886

30. ZhengA

YuanF

LiY

ZhuF

HouP

2007 Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus. J Virol 81 12465 12471

31. AndreP

Komurian-PradelF

DeforgesS

PerretM

BerlandJL

2002 Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76 6919 6928

32. MeertensL

BertauxC

DragicT

2006 Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol 80 11571 11578

33. CodranA

RoyerC

JaeckD

Bastien-ValleM

BaumertTF

2006 Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis. J Gen Virol 87 2583 2593

34. BlanchardE

BelouzardS

GoueslainL

WakitaT

DubuissonJ

2006 Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80 6964 6972

35. TscherneDM

JonesCT

EvansMJ

LindenbachBD

McKeatingJA

2006 Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80 1734 1741

36. ModisY

OgataS

ClementsD

HarrisonSC

2004 Structure of the dengue virus envelope protein after membrane fusion. Nature 427 313 319

37. BressanelliS

StiasnyK

AllisonSL

SturaEA

DuquerroyS

2004 Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. Embo J 23 728 738

38. StiasnyK

BressanelliS

LepaultJ

ReyFA

HeinzFX

2004 Characterization of a membrane-associated trimeric low-pH-induced Form of the class II viral fusion protein E from tick-borne encephalitis virus and its crystallization. J Virol 78 3178 3183

39. KoutsoudakisG

KaulA

SteinmannE

KallisS

LohmannV

2006 Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol 80 5308 5320

40. KielianM

2006 Class II virus membrane fusion proteins. Virology 344 38 47

41. MukhopadhyayS

KuhnRJ

RossmannMG

2005 A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3 13 22

42. GarryRF

DashS

2003 Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins. Virology 307 255 265

43. YagnikAT

LahmA

MeolaA

RoccaseccaRM

ErcoleBB

2000 A model for the hepatitis C virus envelope glycoprotein E2. Proteins 40 355 366

44. FlintM

ThomasJM

MaidensCM

ShottonC

LevyS

1999 Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 73 6782 6790

45. LavilletteD

PecheurEI

DonotP

FresquetJ

MolleJ

2007 Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol 81 8752 8765

46. KreyT

d'AlayerJ

KikutiCM

SaulnierA

Damier-PiolleL

2010 The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog 6 e1000762

47. BartoschB

DubuissonJ

CossetFL

2003 Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197 633 642

48. HsuM

ZhangJ

FlintM

LogvinoffC

Cheng-MayerC

2003 Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100 7271 7276

49. MeanwellNA

2006 Hepatitis C virus entry: an intriguing challenge for drug discovery. Curr Opin Investig Drugs 7 727 732

50. YiM

MaY

YatesJ

LemonSM

2007 Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81 629 638

51. PietschmannT

KaulA

KoutsoudakisG

ShavinskayaA

KallisS

2006 Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci U S A 103 7408 7413

52. WakitaT

PietschmannT

KatoT

DateT

MiyamotoM

2005 Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11 791 796

53. SmeeDF

MorrisonAC

BarnardDL

SidwellRW

2002 Comparison of colorimetric, fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds. J Virol Methods 106 71 79

54. SunJH

LemmJA

O'BoyleDR

2nd

RacelaJ

ColonnoR

2003 Specific inhibition of bovine viral diarrhea virus replicase. J Virol 77 6753 6760

55. LinP-F

Nowicka-SansB

TerryB

ZhangS

WangC

2008 Entecavir Exhibits Inhibitory Activity against Human Immunodeficiency Virus under Conditions of Reduced Viral Challenge. Antimicrob Agents Chemother 52 1759 1767

56. GaoM

NettlesRE

BelemaM

SnyderLB

NguyenVN

2010 Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465 96 100

57. TimpeJM

StamatakiZ

JenningsA

HuK

FarquharMJ

2008 Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47 17 24

58. McPheeF

GoodAC

CampbellJA

SheafferA

WangAX

2009 The discovery and early development of the HCV NS3 protease inhibitor BMS-605339. HEP DART. Kohala Coast, Hawaii

59. ChouTC

2006 Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58 621 681

60. KoutsoudakisG

HerrmannE

KallisS

BartenschlagerR

PietschmannT

2007 The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol 81 588 598

61. WitteveldtJ

EvansMJ

BitzegeioJ

KoutsoudakisG

OwsiankaAM

2009 CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J Gen Virol 90 48 58

62. HelleF

WychowskiC

Vu-DacN

GustafsonKR

VoissetC

2006 Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans. J Biol Chem 281 25177 25183

63. BroeringTJ

GarrityKA

BoatrightNK

SloanSE

SandorF

2009 Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus. J Virol 83 12473 12482

64. SchofieldDJ

BartoschB

ShimizuYK

AllanderT

AlterHJ

2005 Human monoclonal antibodies that react with the E2 glycoprotein of hepatitis C virus and possess neutralizing activity. Hepatology 42 1055 1062

65. YuMY

BartoschB

ZhangP

GuoZP

RenziPM

2004 Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Proc Natl Acad Sci U S A 101 7705 7710

66. HabersetzerF

FournillierA

DubuissonJ

RosaD

AbrignaniS

1998 Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties. Virology 249 32 41

67. LawM

MaruyamaT

LewisJ

GiangE

TarrAW

2008 Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14 25 27

68. OwsiankaA

TarrAW

JuttlaVS

LavilletteD

BartoschB

2005 Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J Virol 79 11095 11104

69. MeulemanP

HesselgesserJ

PaulsonM

VanwolleghemT

DesombereI

2008 Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo. Hepatology 48 1761 1768

70. MassonD

KosekiM

IshibashiM

LarsonCJ

MillerSG

2009 Increased HDL cholesterol and apoA-I in humans and mice treated with a novel SR-BI inhibitor. Arterioscler Thromb Vasc Biol 29 2054 2060

71. YeJ

2007 Reliance of host cholesterol metabolic pathways for the life cycle of hepatitis C virus. PLoS Pathog 3 e108

72. BurloneME

BudkowskaA

2009 Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90 1055 1070

73. BradleyD

McCaustlandK

KrawczynskiK

SpelbringJ

HumphreyC

1991 Hepatitis C virus: buoyant density of the factor VIII-derived isolate in sucrose. J Med Virol 34 206 208

74. NielsenSU

BassendineMF

BurtAD

MartinC

PumeechockchaiW

2006 Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients. J Virol 80 2418 2428

75. ReyFA

HeinzFX

MandlC

KunzC

HarrisonSC

1995 The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375 291 298

76. KanaiR

KarK

AnthonyK

GouldLH

LedizetM

2006 Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes. J Virol 80 11000 11008

77. NayakV

DessauM

KuceraK

AnthonyK

LedizetM

2009 Crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion. J Virol 83 4338 4344

78. YuX

QiaoM

AtanasovI

HuZ

KatoT

2007 Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles. Virology 367 126 134

79. CiczoraY

CallensN

PeninF

PecheurEI

DubuissonJ

2007 Transmembrane domains of hepatitis C virus envelope glycoproteins: residues involved in E1E2 heterodimerization and involvement of these domains in virus entry. J Virol 81 2372 2381

80. DrummerHE

PoumbouriosP

2004 Hepatitis C virus glycoprotein E2 contains a membrane-proximal heptad repeat sequence that is essential for E1E2 glycoprotein heterodimerization and viral entry. J Biol Chem 279 30066 30072

81. StiasnyK

FritzR

PangerlK

HeinzFX

2009 Molecular mechanisms of flavivirus membrane fusion. Amino Acids

82. ZhouZ

KhaliqM

SukJE

PatkarC

LiL

2008 Antiviral compounds discovered by virtual screening of small-molecule libraries against dengue virus E protein. ACS Chem Biol 3 765 775

83. WangQY