Interdomain Communication in Hepatitis C Virus Polymerase Abolished by Small Molecule Inhibitors Bound to a Novel Allosteric Site

Abstract

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3-and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small ␣-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen. The hepatitis C virus (HCV)1 is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans (1, 2). It is estimated that more than 170 million people worldwide are infected by this virus (3). There is no vaccine available for HCV, and the current therapy, based on interferon and ribavirin, is poorly tolerated and of limited efficacy. Therefore, there are intense research efforts toward the development of new drugs targeting essential HCV enzymes and in particular the polymerase. The HCV nonstructural protein 5B (NS5B) is the RNA-dependent RNA polymerase (RdRp) responsible for replication of the viral genome. NS5B can initiate RNA synthesis by two different mechanisms: primer-independent initiation from the 3Ј terminus of the viral genome, also known as de novo initiation (4, 5), and primer-dependent initiation using either DNA or RNA as primers (6). The de novo synthesis is likely used during virus replication in infected cells (7).The three-dimensional structures of soluble forms of the HCV polymerase genotype 1b, ⌬C21 and ⌬C55, lacking the last 21 and 55 C-terminal residues, respectively, have been reported (8 -10). These structures revealed a classical "right hand" shape formed by the palm, thumb, and fingers domains as initially defined in the Klenow fragment of Escherichia coli DNA polymerase I (11) and showed the presence of an extension in the fingers, the so-called fingertip subdomain, containing two loops, ⌳1 and ⌳2 (secondary structure nomenclature according to Ref. 9), which anchor the fingers to the thumb. As a result, the polymerase has a relatively closed and spherical appearance, and the active site cavity, to which the RNA template and the NTP substrates have access via two positively charged tunnels, is completely encircled. Structural studies have shown that other viral RdRps, such a...

Apolipoprotein E4: A causative factor and therapeutic target in neuropathology, including Alzheimer’s disease

“…Disruption of the intramolecular interaction within apoE4 and its conformation represents a reasonable therapeutic strategy. Recently, small molecules were shown to bind to the hepatitis C virus polymerase and inhibit RNA synthesis by altering intramolecular structure (158). Furthermore, small molecules correct the phenylalanine deletion mutation (⌬F508) in the cystic fibrosis transmembrane conductance regulator chloride channel, which is the common cause of cystic fibrosis (159).…”

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

“…Based on the mechanistic and resistance selection results, the authors proposed that the allosteric thumb site 1 inhibitors interact with the enzyme-RNA complex and impact a slow conformational transition, preceding nucleotide binding, which is required for the formation of productive initiation complexes. When co-crystal structures of NS5B and two structurally similar inhibitors confirmed the site of binding (17), the authors hypothesized that thumb site 1 inhibitors interfere with enzyme activity by preventing the formation of intramolecular contacts between fingers and thumb, precluding the coordinated movements required for RNA synthesis.…”

“…The site, referred to as thumb site 1, is occupied in the apoprotein by a protein loop (⌬1) within the fingers domain of the polymerase (14 -17). Co-crystal structures show that BMS-791325, like other thumb site 1 inhibitors, displaces the finger loop when it binds this pocket (17).…”

“…The slow isomerization step does not appear to be affected directly by the weaker finger-thumb interaction. The inability of L30S to substantially impact the affinity of the thumb site 1 binding pocket for BMS-791325 is not surprising in light of structural studies showing that displacement of the finger loop by thumb site 1 inhibitors causes no change in the structure of the thumb domain itself (17).…”

“…The fact that BMS-791325 is unable to block elongation suggests that the interaction between the fingers and thumb that occurs during the formation of active replication complexes is fundamentally different during elongation. It is possible that the fingers domain is less flexible during elongation and blocks the binding of BMS-791325 or that the shape of the thumb binding pocket is different and cannot bind inhibitor (17,38). Experiments with non-nucleoside inhibitors that bind to different polymerase structural domains are ongoing to further investigate the impact of the finger-thumb interaction on HCV NS5B replication.…”