Lakhasly

Hepatitis C virus (HCV) is the most frequent cause of chronic
viral hepatitis, with about 3% of the world’s population infected.1
In the majority of acutely infected individuals, HCV evades the immune response and establishes a chronic infection associated with
liver cirrhosis and in some cases hepatocellular carcinoma.2 There
is currently no broadly effective therapy, making the development
of HCV-specific antiviral agents an urgent need.3,4
HCV is a small, enveloped, single stranded positive RNA virus in
the Flaviviridae family. The genome is approximately 10,000 nucleotides and encodes a single polyprotein of about 3000 amino acids.
This polyprotein comprises the structural (C, E1 and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins that
are required for replication and packaging of the viral genomic
RNA. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The nonstructural protein NS5A is an active component of HCV replicase5,6, possibly involved in regulation
of viral replication and resistance to the antiviral effect of interferon.7,8 NS5A is a large phosphoprotein (56–58 kDa) organized into
three domains, including an amphipathic a-helix at its amino terminus that promotes membrane association and a zinc binding
domain. Mutations disrupting either the membrane anchor9 or zinc
binding10 of NS5A have been shown to be lethal for RNA replication.
Thus, NS5A is a potential target for antiviral therapy.
Whilst HCV replicative enzymes, such as NS3 protease and
NS5B polymerase, have been extensively explored, efforts to target
NS5A have largely been hampered by an incomplete understanding
of the role of this multifunctional protein in replication—and a
resultant lack of a screening protocol for enzyme inhibition.
A few years ago, the development of subgenomic replicons that
replicate in a human hepatoma cell line provided a system to test
or screen compounds that inhibit genome replication11,12, and thus
make it possible to identify structural classes that disrupt the function of any of the viral proteins involved in replication—including
those with no known enzymic activity (such as NS5A).
In this report, we describe a class of piperazinyl-N-(aryl)benzamides (identified from screening the in-house compound collection) as potent inhibitors of HCV replication in such a surrogate
cell-based assay (Fig. 1). Resistant replicon mutants raised to this
class show mutations that map to domain IA of NS5A—implicating
modulation of NS5A function as a potential MOA for this class.
The compounds described herein were assessed for their ability
to inhibit replication of subgenomic HCV RNA, measured in HUH-7
cells using a modification of the procedure of Bartenschlager.