HCV entry represents an attractive target for drug discovery from a mechanistic view, with opportunities to prevent multiple virus-receptor interactions and to interfere with virus-cell membrane fusion 
. Each of these steps, although not completely defined, is likely mediated by the HCV E1 and/or E2 envelope glycoproteins. In vitro
, proof-of-concept for inhibiting the HCV entry process has been demonstrated using cyanovirin-N that targets the N
-linked glycans of the viral envelope proteins and prevents E2-CD81 interaction 
, neutralizing antibodies directed against the HCV E1 and E2 proteins 
, antibodies against cellular receptors CD81 
and SR-BI 
, and agents that block endosomal acidification 
. In vivo
studies using human liver-u-PA-SCID mice have also demonstrated prophylactic efficacy of anti-CD81 antibodies 
. In the present study, we used the HCVpp system in order to isolate the entry pathway from other HCV replication functions, and undertook a screening campaign that led to discovery of a class of small molecule HCV-specific inhibitors, exemplified by EI-1. Inhibition of entry was confirmed by using time-of-addition experiments to demonstrate that EI-1 activity is confined to the first 3 hours of infection, with inhibition occurring post-attachment and closely linked to the inhibition kinetics of the endosomal acidification inhibitor bafilomycin.
EI-1 does not inhibit entry of VSVpp, which also undergoes receptor-mediated endocytosis and pH-dependent endosomal fusion, thus making cellular factors required for internalization unlikely targets of this compound. Furthermore, although EI-1 inhibited all 31 genotype 1a and 1b isolates in our HCVpp panel, activity towards isolates with envelope genotypes 2–5 was greatly diminished. This result also argues against a cellular protein as the target for EI-1 as such an inhibitor should display similar activity across genotypes. Lastly, genotype 1 HCVcc resistance to EI-1 is conferred by a V719F/G change in the C-terminal TMD region of HCV E2, supporting the concept that EI-1 blocks HCV entry by inhibiting the function of the HCV envelope glycoproteins. It is tempting to speculate that EI-1 binds to E2 in part through an interaction with the valine or isoleucine residue 719. Alternatively, E2:V719 may represent an allosteric site, whereby changes induce a conformational alteration of E2 and/or E1 that prevent EI-1 binding. However, the E2 protein of genotypes 2–5 in our HCVpp panel also contains valine or leucine, yet these isolates are not susceptible to EI-1suggesting the determinant(s) for the intrinsic resistance of non-genotype 1 HCV may lay elsewhere. Indeed, further HCVcc resistance selection experiments suggest that changes to residues within E1 can also modulate susceptibility to other members of the EI-1 chemotype (data not shown). Ultimately, conclusive evidence for the target of EI-1 awaits biophysical experiments designed to demonstrate a direct compound-protein interaction.
It was critical to determine whether the EI-1 entry inhibitor prevented infection by HCVcc as well as HCVpp. While experimental findings obtained with the HCVpp model have generally extended to those with the HCVcc system, this is not always the case. For example, while a small molecule targeting SR-BI 
potently inhibits HCVcc infection, it does so at a markedly reduced potency in the HCVpp system (unpublished observations). More importantly, however, it was unclear if small molecule inhibitors discovered through the HCVpp system could prove to inhibit HCVcc infection, especially since much of the HCVcc infection occurs through a cell-to-cell transmission route that is shielded from neutralizing antibodies 
and bypasses the requirement for the CD81 receptor 
. Since it is assumed that cell-to-cell infection is an important feature of viral pathogenesis, inhibitors that operated through both prevention of cell-free virus infection and cell-to-cell spread of virus would logically be needed for therapy. Our results demonstrate that EI-1 is potent at blocking genotype 1 HCVpp and HCVcc entry, as well as direct cell-to-cell spread of HCVcc. However, because circulating HCV in patients is highly associated with lipoprotein particles 
, it will be important to determine the efficacy of EI-1 and similar HCV entry inhibitors in cell culture systems using serum-derived HCV or in human liver chimeric mouse model systems 
The structure of either of the HCV envelope proteins has yet to be solved. However, high-resolution structural models for the related flavivirus class II envelope glycoproteins of dengue virus, tick-borne encephalitis virus, and West Nile virus have been reported in both the pre- and post-fusion states 
. It is unclear how the HCV E1 and E2 proteins perform the functions of the homologous proteins in other flaviviruses. However, structural features characteristic of class II viral fusion proteins, such as a membrane proximal heptad repeat and a putative hydrophobic fusion peptide have been identified within both E1 and E2 
. In addition, other laboratories have used mutational analysis to ascribe E1-E2 heterodimerization, entry, and membrane fusion functions to residues in the E2 stem and TMD 
. These results have lead to the hypothesis that flavivirus glycoproteins form intermolecular hairpin motifs projecting hydrophobic fusion peptides that facilitate the final fusion of viral envelopes with cellular membranes 
. Further defining the target of EI-1 and elucidating the mechanism of inhibition may contribute to understanding the functional roles of the HCV envelope proteins.
A dengue virus entry inhibitor, the detergent n-octyl-β-D-glucoside (β-OG) was found to bind to a hydrophobic pocket formed in a postulated hinge region between domains I and II in the viral envelope E protein 
. Several other inhibitors of dengue virus entry were found based on an exercise of modeling candidate compounds into this pocket 
. Modeling of domains I, II and III of the dengue E protein with HCV E1 and E2 proteins suggested that the β-OG site between domains II and III localized to the HCV E2 protein and not to E1. While β-OG did not inhibit HCV entry in our hands (data not shown), it is unclear what portion of HCV E1 or E2 may be analogous to the hinge region. In contrast to the β-OG binding site, which is within the soluble fragment of dengue virus E protein, HCV resistance to the EI-1 compound described here maps to the second amino acid of the putative TMD region of genotype 1 E2. Perhaps multiple binding sites within the HCV entry proteins exist, accessible during the numerous conformational states that may operate during receptor binding and fusion. Consistent with this concept is the finding that during our own discovery efforts, several HCV entry inhibitors with diverse structural characteristics and resistance mapping were identified (data not shown). Downstream implications of these findings are the possibility that multiple, diverse inhibitors of HCV entry could contribute to combination therapy for HCV.
Similarities between the HCV entry inhibitors described here and diverse compounds inhibiting the entry of arenaviruses into cells are intriguing. Both pseudotype 
and infectious virus screening 
identified broadly active arenavirus entry inhibitors. Isolation and mapping of resistant viruses, as well as chimeras between sensitive and resistant strains, mapped the target of activity to the GP2 subunit of the G envelope protein complex, specifically the interface between the C-terminal stem and TMD domains 
. These results are strikingly similar to those described in the current work in that resistance to EI-1 occurs in the same region of the genotype 1 HCV E2 envelope glycoprotein Mechanistic studies showed that these arenavirus inhibitors prevented low-pH-induced fusion by blocking reorganization between the GP2 stem with N-terminal domains of the G protein complex 
. By analogy, perhaps the HCV entry inhibitors described here prevent pH-induced reorganization of the HCV E1E2 complex that mediates fusion 
The activity surrounding the search for antivirals targeting HCV is considerable. Each antiviral therapy is accompanied by a unique set of challenges for its development. Although HCV entry inhibitors could be a valuable component of therapy, their development will provide differences from those of replication inhibitor compounds, currently in clinical development. Preclinical development of entry inhibitors require infection assay formats, with pseudotyped or full-length HCV, such as those described here. While capable experimental systems have been developed, these are not as robust as many virus systems such as HIV, influenza or herpesviruses. HCV replication inhibitor assays, on the other hand, require assays with the more facile stable, transformed, replicon cell line. The inherent genetic variation in viral envelope proteins also presents a unique target for entry inhibitors. We have addressed these issues with various assay systems and an HCVpp genotype panel assembled from patient isolates, which demonstrated the genotype 1 specificity of EI-1. Furthermore, as noted above, it has been reported that the viral envelope lipoprotein content differs between cell culture HCV and virus isolated from patients. Additionally, cellular receptors and entry processes may vary from the transformed cells used here and primary hepatocytes. For these reasons, it will be important to evaluate the efficacy of small molecule HCV entry inhibitors in primary cells, using patient-derived virus, and potentially in in vivo model systems. Finally, current HCV inhibitor clinical studies have been limited in duration to prevent the development of resistance. The efficacy of these replication inhibitors, however, can quickly be assessed through circulating levels of HCV RNA produced by chronically-infected cells. Since entry inhibitors will prevent new infections of uninfected cells, they will have no immediate impact on the levels of circulating virus in the blood. Thus, more protracted clinical studies may be required.
Several small molecule inhibitors have been advanced to the clinic, and some have progressed after an initial high attrition rate 
. Unfortunately, both the high replication and error rates of the viral polymerase leads to exceeding diversity of viral sequences, thus resulting in preexisting and rapidly emerging resistance 
. Despite potent efficacy, it is now well understood that combinations of inhibitors, including both small molecules targeting the virus and interferon regimens acting through host targets, will be required for optimal treatment 
. By analogy to HIV, safe, potent inhibitors of multiple viral targets will be needed to prevent resistance from emerging or for optimal management of patients with resistance. We have shown the EI-1 entry inhibitor functions additively or synergistically with other HCV replication inhibitors and IFN-α in the HCVcc cell culture assay system. Entry inhibitors, such as those described here, by virtue of their distinct, relatively new targets, may provide a valuable component in the eventual optimal therapy for HCV infection.