The HCV Non-Nucleoside Inhibitor Tegobuvir Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase Function

Abstract

Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.

Efficient Replication of Genotype 3a and 4a Hepatitis C Virus Replicons in Human Hepatoma Cells

“…To examine the inhibitory effect of directacting antivirals (DAAs), we exposed the replicons to various concentrations of danoprevir, an NS3 protease inhibitor, and 2=CMeA, an NS5B nucleoside polymerase inhibitor. Danoprevir and 2=CMeA are highly potent against genotype 1b replicons, with reported 50% effective concentrations (EC 50 s) of 1.8 nM and 0.2 to 0.5 M, respectively (20,52,54). Our results showed that genotype 2a and 4a replicons, with average EC 50 s of 65 and 1.2 nM, respectively, were sensitive to danoprevir, whereas the EC 50 for genotype 3a replicons was 900 nM, suggesting a lower inhibitory effect of danoprevir against genotype 3a (Fig.…”

A Review on Anti-HCV Agents Targeting Active Site and Allosteric Sites of Non-Structural Protein 5b [Ns5b]

“…The Palm II nonnucleoside inhibitors that are currently in different phases of development are given in Table 7. Tegobuvir has a unique mechanism of action and is distinct from other NNIs of NS5B [149]. Tegobuvir undergoes metabolic activation resulting in glutathione adducts which specifically interacts with NS5B thereby inhibiting viral replication [149].…”

“…Tegobuvir has a unique mechanism of action and is distinct from other NNIs of NS5B [149]. Tegobuvir undergoes metabolic activation resulting in glutathione adducts which specifically interacts with NS5B thereby inhibiting viral replication [149]. PPI-383 is a nonnucleoside inhibitor with pan-genotypic activity; it exhibits EC50 value of 8.3nM, 2.2nM against genotypes 1a and 1b respectively in cell-based replicon assays.…”

In vitro combinations containing Tegobuvir are highly efficient in curing cells from HCV replicon and in delaying/preventing the development of drug resistance

“…Cross-resistance analyses with the site III inhibitor A-782759 and site IV inhibitor HCV-796 revealed that Tegobuvir acts via a mechanism that is different from that of other non-nucleoside HCV polymerase inhibitors (Shih et al, 2011). Tegobuvir undergoes a CYP 1A-mediated intracellular activation step; the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with and inhibits NS5B (Hebner et al, 2012). When administered in combination with Peg-IFN/RBV in treatment naive GT1 patients, Tegobuvir (40 mg BID) increased RVR (rapid virological response) by $30% and cEVR (complete early virological reponse) by $20% over Peg-IFN/RBV treatment although, no improvement in SVR was observed in the final analysis (Lawitz et al, 2011).…”