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Jan 9, 2012

New Target and FDA-Approved Cholesterol-Lowering Drug Identified for HCV Therapy

  • Scientists have identified what they claim is a new target for preventing HCV infection, and also demonstrated that an existing FDA-approved cholesterol-lowering drug may fit the bill as a therapeutic agent against the disease. Building on previous research implicating viral cholesterol as a key factor in facilitating the entry of HCV into host cells, a team led by researchers at the University of Illinois-Chicago identified the cellular Neimann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor as a key HCV entry factor that is amenable to therapeutic intervention. Their work in addition showed that the FDA-approved NPC1L1 antagonist ezetimibe not only inhibits infection by all major HCV genotypes in vitro, but also delays the establishment of HCV genotype 1b infection in mice with human liver grafts.

    Susan L. Uprichard, M.D., and colleagues report their findings in Nature Medicine in a paper titled “Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.”

    HCV is believed to enter cells via receptor mediated endocytosis, a process that likely involves the interaction of multiple viral particle molecules with a series of cell surface receptors. For example, the researchers note, infectious HCV virus in cell culture (HCVcc) is enriched in cholesterol, which prior research has demonstrated is required for HCV cell entry and infectivity.

    This phenomen led the team to postulate whether cholesterol-uptake receptors other than SR-BI and LDLR might play a role in HCV cell entry. They focused on the cellular NPC1L1 cholesterol uptake receptor, a 13-transmembrane-domain cell surface cholesterol-sensing receptor that is expressed on the apical surface of intes­tinal enterocytes and human hepatocytes, including Huh7 cells.

    Initial analyses indicated that, as with other HCV entry factors, NPC1L1 was downregulated in HCVcc-infected Huh7 cultures, and there was a marked reduction in NPC1L1 protein levels as early as four days after infection. Moreover, when NPC1L1 expression in Huh7 cells was silenced using a targeted siRNA, the cells were much less susceptible to HCVcc infection. Reduced susceptibility to HCVcc infection was also observed in Huh7 cells transfected wtih siRNA targeting either of the known HCV entry factors CD81 or SR-BI. This reduction in susceptibility to infection by HCVcc associated with siRNA transfection of Huh7 cells was found to be HCV- and HPC1L1-specific. Interestingly, though, NPC1L1 silencing had no effect on HCV subgenomic RNA replication, full-length infectious HCVcc RNA replication, or secretion of de novo HCVcc.

    The results thus far indicated that HCV infection was inhibited at a step before RNA replication or secre­tion, so the team next evaluated the impact of antibody-mediated blocking of cell surface NPC1L1. Compared with cells treated using control IgG antibodies, those treated using antibodies specific to either NPC1L1 or the known HCV cell entry factor CD81 demonstrated notably reduced infection by HCVcc, as measured by intracellular HCV RNA levels. Using antibodies specific to each of the three large extracellular loops (LELs) of NPC1L1, the researchers were also able to determine that HCV infection was reduced only when NPC1L1 LEL1, but not LEL2 or LEL3, was blocked.

    They then evaluated treatment using the FDA-approved cholesterol-lowering drug ezetimibe (which acts as a direct inhibitor of NPC1L1) either before, during, or after viral inoculation. The effects were measured using HCVcc foci-reduction assays, to quantify foci after ezetimibe treatment. The results showed that the drug reduced HCVcc foci formation in a dose-dependent manner when it was administered either before infection and then removed, or during virus inoculation, but not when treatment was carried out after inoculation.

    Dose-responsive, time-of-addition–dependent inhibition of HCV infection was also evi­dent when HCV RNA levels were measured, and ezetimibe sensitivity was seen across a panel of HCVcc clones representative of HCV genotypes 1-7. “Hence, the data support the conclusion that direct pharmacological inhibition of NPC1L1 reduces HCV infection by directly inhibiting viral cell entry,” the authors write.

    Further studies indicated that while although HCV can efficiently still bind to ezetimibe-treated cells, the drug blocks a post-binding step, and indeed prevents HCVcc cell entry either at or before virion-host cell fusion. 

    To confirm whether the dependence of HCV cell entry on NPC1L1 is actually related to the cholesterol in the HCV virions, the researchers devised a study to evaluate the dependence on NPC1L1 of viruses containing different amounts of cholesterol. This demonstrated that the cholesterol-rich strain of HCVcc was strongly dependent on NPC1L1 for cell entry, and was hypersensitive to ezetimibe-mediated inhibition. In contrast, when NPC1L1 was silenced or inhib­ited by ezetimibe, the cholesterol-scarce strain showed NPC1L1-independent cell entry and insensitivity to ezetimibe inhibition. “Together, these data reveal a correlation between the amount of virion-associated cholesterol and dependence on NPC1L1 for HCV cell entry,” they write.

    In a final set of experiments the authors evaluated the involvement of NPC1L1 in HCV cell entry in a mouse model of acute HCV infection. Animals were treated with ezetimibe either two weeks, one week, or two days before challenge with HCV genotype 1b-positive serum. In the mice pretreated using the drug for two weeks before infection, HCV infection was significantly delayed, whereas ezetimibe was less effective at delaying infection in animals pretreated for just one week. In contrast, ezetimibe treatment was completely ineffective at delaying infection in mice receiving the drug just two days before challenge with HCV-infected serum.

    More specifically, while all control mice were serum-positive for HCV by one week after challenge, 71% and 43% of mice treated with ezetimibe for two weeks and one week before infection were HCV negative, respectively. And of the five mice in the two-week ezetimibe pretreatment group that were HCV negative at week one, two were completely protected, remaining HCV negative at weeks two and three after infection, the authors note.

    “Our finding that ezetimibe can delay the establishment of HCV genotype 1 infection in mice confirms the involvement of NPC1L1 in HCV infection in vivo and highlights the therapeutic potential of further pursuing the refinement or develop­ment of anti-NPC1L1 therapies for the treatment of HCV,” they conclude. What does remain to be determined, the team admits, is whether NPC1L1 directly interacts with HCV or indirectly participates in HCV entry by removing virion-associated cholesterol, possible to reveal protected viral glycoprotein binding sites or confer a required conformational change. Nevertheless, they state, “we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.”


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