Small Molecules Targeting Hepatitis C Virus-Encoded NS5A Cause Subcellular Redistribution of Their Target: Insights into Compound Modes of Action

Abstract

Hepatitis C virus (HCV) is a global health concern; recent estimates suggest that 2.2 to 3% of the world's population, equivalent to 130 to 170 million individuals, are chronically infected with the virus (13, 31). These patients are at risk of developing debilitating liver diseases such as cirrhosis and hepatocellular carcinoma (1). Furthermore, current models suggest that the burden of HCV-associated disease is set to rise for the next 20 years (6). There is no HCV vaccine; the current standard of care (SOC) involves lengthy treatments with ribavirin and injected pegylated interferon, which exhibit variable efficacies and are associated with severe, and sometimes lifethreatening, side effects. Encouragingly, many direct-acting antiviral (DAA) molecules are in clinical development, and the most advanced (telaprevir and boceprevir) will probably be used to treat HCV-infected patients in 2011 (19,29,42,43,61). However, caution should be employed against overoptimism; attrition rates are high during drug development, and the first drugs will be given in combination with, not instead of, the current SOC. Therefore, the continued development of additional treatments is needed, especially since it is widely acknowledged that to limit the emergence of drug-resistant viral variants, effective therapeutic strategies for HCV will consist of multiple DAAs (50).A multitude of screening campaigns has revealed many diverse and interesting chemical compounds capable of specifically inhibiting HCV RNA replication. Many of these compounds target the HCV-encoded nonstructural (NS) proteins (NS3, NS4A, NS4B, NS5A, and NS5B), which are required for HCV genome synthesis (3, 37). To instigate HCV genome replication, the NS proteins interact with viral genomes and certain host-encoded factors to form multiprotein assemblies termed "replication complexes" (RCs), which are sites of viral RNA synthesis derived from the endoplasmic reticulum (ER) (8,14,45,53). In HCV-infected cells, RCs are juxtaposed to intracellular lipid storage organelles termed lipid droplets (LDs), which are coated with the HCV capsid protein (core) and probably serve as platforms to accept replicated genomes from RCs to initiate virion assembly (26,44,53). Of considerable interest are inhibitors that target the HCV-encoded NS5A protein. These inhibitors were originally discovered from the screening of cells containing HCV subgenomic replicons against libraries of small molecules and were identified as NS5A inhibitors by utilizing a strategy termed "chemical genetics" (12, 32). NS5A-targeting inhibitors are notable for their unprecedented potency in cell-based HCV replication assays: 50% inhibitory concentrations (IC 50 s) in the low-picomolar

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…A recent study showed that both Domains I and II of NS5A exhibit RNA binding activities (20). However, the ability to bind specifically to uridine-and guanosine-rich RNA resides in domain I and the adjacent linker region (33). An alternative structure of Domain I has been determined that is also dimeric in nature, although the interface lacks the large groove or any obvious RNA interaction surface (31).…”

“…The dimer is oriented in such a way that a large groove exists between the two monomers and has been proposed as an RNA binding site (29). NS5A has RNA binding activity that requires a dimer based on glutaraldehyde-cross-linking experiments (30,33). Unfortunately, a connection between the NS5A RNA binding activity and a specific aspect of RNA replication or virion assembly remains to be established (30).…”

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors. [12][13][14] Furthermore, NS5A inhibitors have been shown to induce redistribution of NS5A from endoplasmic reticulum-derived foci, [12][13][14] possibly to lipid droplets, 12 and limit hyperphosphorylation of NS5A, [14][15][16] although these effects may be indirect. Finally, it has been suggested that NS5A inhibitors may disrupt interactions of NS5A with HCV RNA, other viral proteins, and/or host factors that are coopted by NS5A during the HCV life cycle.…”

“…In this context, the class-defining resistance site Y93 is located at opposing, membrane-proximal surfaces of the dimer interface for both "back-to-back" and "clam-like" alternative domain I (genotype 1b) crystal structures. 10,11 Third, biotin-tagged DCV derivatives enable precipitation of NS5A from pretreated HCV replicon-harboring cells, 6,12 although interestingly fail to precipitate NS5A from replicon lysates or pretreated NS5A-overexpressing cells. 12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors.…”

“…10,11 Third, biotin-tagged DCV derivatives enable precipitation of NS5A from pretreated HCV replicon-harboring cells, 6,12 although interestingly fail to precipitate NS5A from replicon lysates or pretreated NS5A-overexpressing cells. 12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors. [12][13][14] Furthermore, NS5A inhibitors have been shown to induce redistribution of NS5A from endoplasmic reticulum-derived foci, [12][13][14] possibly to lipid droplets, 12 and limit hyperphosphorylation of NS5A, [14][15][16] although these effects may be indirect.…”