A novel small molecule inhibitor of hepatitis C virus entry.
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.
Baldick
Carl J
CJ
Bristol-Myers Squibb, Research and Development, Wallingford, Connecticut, United States of America. joe.baldick@bms.com
Wichroski
Michael J
MJ
Pendri
Annapurna
A
Walsh
Ann W
AW
Fang
Jie
J
Mazzucco
Charles E
CE
Pokornowski
Kevin A
KA
Rose
Ronald E
RE
Eggers
Betsy J
BJ
Hsu
Mayla
M
Zhai
Weixu
W
Zhai
Guangzhi
G
Gerritz
Samuel W
SW
Poss
Michael A
MA
Meanwell
Nicholas A
NA
Cockett
Mark I
MI
Tenney
Daniel J
DJ
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D000998:Antiviral Agents / Q000302:isolation & purification / Q000627:therapeutic use*; D002478:Cells, Cultured; D024882:Drug Resistance, Viral; D004357:Drug Synergism; D016174:Hepacivirus / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D006526:Hepatitis C / Q000188:drug therapy* / Q000235:genetics / Q000821:virology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D054852:Small Molecule Libraries / Q000032:analysis / Q000378:metabolism*; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D053586:Virus Internalization / Q000187:drug effects*
D015703:Antigens, CD; D000998:Antiviral Agents; C554798:CD81 protein, human; C087035:E1 protein, Hepatitis C virus; D054852:Small Molecule Libraries; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D007372:Interferons