Identification and Biological Characterization of Heterocyclic Inhibitors of the Hepatitis C Virus RNA-dependent RNA Polymerase

Abstract

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC 50 of ϳ0.5 M, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.Hepatitis C virus (HCV), 1 a positive strand RNA virus of the Flaviviridae family, is the major etiological agent of post-transfusion and sporadic non-A, non-B hepatitis (1). An estimated 2-3% of the world population is chronically infected with HCV, which causes significant liver disease, cirrhosis, and can eventually lead to the development of hepatocellular carcinoma. In infected cells, translation of the viral RNA yields a 3011-residue polyprotein chain (2-4), which is subsequently cleaved to generate envelope and core proteins, for assembly of new virus particles and nonstructural enzymes essential for viral replication (5-7). Studies using recombinant NS5B polymerase have provided direct evidence for RNA-dependent RNA polymerase activity (8, 9), and this catalytic activity has been confirmed to be required for infectivity in chimpanzees (10).NS5B polymerase contains a hydrophobic C-terminal domain thought to be responsible for anchoring the protein to mammalian cell membranes. Removal of the C-terminal 21 residues has been reported to facilitate protein isolation from Escherichia coli without compromising RdRp activity (11). The HCV RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12, 13-15) but lacks specificity for HCV RNA in vitro, because it readily utilizes heterologous nonviral templates (8). Based on crystallographic studies of the enzyme containing C-terminal truncations (16, 17), the hydrophobic tail present in the full-length ...

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

“…The benzo-1,2,4-thiadiazines (compounds 1 and 4) were shown to be potent inhibitors of the HCV RdRp with biochemical IC 50 values between 80 and 100 nM and cell-based IC 50 values around 500 nM (1). Calorimetry studies confirmed a direct interaction of these compounds with the viral polymerase and a window of selectivity for nucleic acid interaction.…”

“…: 610-240-8207; Fax: 610-240-4064; E-mail: rsarisky@cntus.jnj.com. 1 The abbreviations used are: HCV, hepatitis C virus; NS5B, nonstructural protein 5B; RdRp, RNA-dependent RNA polymerase; nt, nucleotide(s); SPR, surface plasmon resonance; RU, relative units; PFA, phosphonoformic acid; TNTase, terminal transferase; 3Ј-rdGTP, 3Ј-ribodeoxy-GTP. tion, a direct initiation assay, was established.…”

“…At the same time that heparin was added, 15 M 3Ј-ribodeoxy-GTP (3Ј-rdGTP), compound 1 (C 20 H 19 N 3 O 4 S) or compound 4 was also added. The reaction was continued for an additional 60 min and then terminated, processed, and analyzed as described previously (1).…”

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

“…The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction.…”

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

“…One of the reasons for the inefficient immunoprecipitation of the NS5B may also be the ability of the NS5B protein to oligomerize. Glu 18 and His 502 residues are critical for the oligomerization process of HCV RdRp (39).…”