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(Krieger
et al
., 2010), and agents that block endosomal acidification (Bartosch
et al
., 2003; Hsu
et al
., 2003; Koutsoudakis
et al
., 2006; Lavillette
et al
., 2005).
In
vivo
studies using humanized trimera mice model or human liver-u-PA-SCID
mice have also demonstrated prophylactic efficacy of monoclonal anti-E2 (Eren
et al
., 2008), respectively.
Some broad-spectrum antiviral drugs have also been tested (Pecheur
., 2006) and anti-CD81 antibodies (Meuleman
et al
., 2007),
but more recent studies have used the HCVpp system in order to undertake a
screening campaign that led to the discovery of a class of small molecule HCV-
specific inhibitors consisting of several structurally related compounds defined by
a common triazine core (Baldick
et al
., 2010). Inhibition of entry was confirmed
by using time-of-addition experiments to demonstrate that inhibitor activity is
confined to the first 3 h of infection, with inhibition occurring postattachment
and closely linked to the inhibition kinetics of the endosomal acidification
inhibitor bafilomycin.
Pseudoparticles can also be used to identify the mode of action of
molecules identified using a replication competent virus in a cell-based screening
system involving multiple rounds of infection in a 96-well format. After analysis
of a publicly available library of 446 clinically approved drugs, the impact of 33
identified compounds on viral entry was tested using HCVpp infection to
recapitulate HCV particle adsorption,
et al
internalization, and viral envelope-
mediated fusion (Gastaminza
., 2010). Many of the candidates were lysoso-
motropic compounds that inhibited HCV entry with differential efficacy.
Both pseudotype (Larson
et al
et al
., 2008) and infectious virus screening
(Bolken
., 2006) identified broadly active arenavirus entry inhibitors. Isola-
tion 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 to the interface between the C-terminal
stem and TMD domains. 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
(York
et al
., 2008).
Another alternative for developing entry inhibitor compounds is to
target endosomal protease necessary for entry of certain viruses. Inhibitors of
cathepsin L block viral entry of severe acute respiratory syndrome coronavirus
(SARS-CoV) and Ebola virus and impair conversion of HeV glycoprotein into
the mature, active form (Chandran
et al
et al
., 2005; Pager and Dutch, 2005; Simmons
et al
., 2005). With respect to the development of antiviral agents, inhibitors of
human cathepsin L are not subject to resistance arising from rapid mutations of
the viral genome (Shah
., 2010), making Cathepsin L an attractive target for
drug development. HTS for cathepsin L inhibitors identified a novel thiocarba-
zate compound exhibiting potent inhibition against cathepsin L (Beavers
et al
et al
.,
2008; Myers
et al
., 2008; Shah
et al
., 2008). This compound prevented 293T cell
