Cell experiment:

Replicon-containing cells are trypsinized and seeded in cell culture media without G418 in white 96-well plates for EC50 analysis. Stable replicon carrying cell lines are seeded at a density of 5,000 cells per well. Serial threefold dilutions (10 concentrations) of compounds are performed in DMSO followed by further dilution in cell culture media and subsequent addition to cell plates. Compound-treated cells are incubated 72 hours at 37℃ in a 5% CO2 incubator.

Tegobuvir (TGV) is a non-nucleoside of HCV RNA replication EC50s of ＜ 16 nM against genotype 1 and ＞100 nM for other genotypes. [1, 2]

Tegobuvir inhibited HCV replication by targeting the NS5B polymerase. The NS5B polymerase is an RNA polymerase which is responsible for HCV replication and is RNA-dependent RNA polymerase.[3, 4] The HCV NS5B contains canonical thumb, finger, and palm subdomains.[3]

Tegobuvir has potent activity against genotypes 1a and 1b HCV in HCV replicons in vitro, and in HCV genotype 1-infected patients and GT 1a EC50 is 17-fold higher than that observed in GT 1b.[5] However, It exhibited reduced activity against GT2a replicons and GT2a infectious virus. Furthermore, the C445F mutation selected by tegobuvir caused a 7.1 fold increase in EC50.[2] Thus, it indicated that the mechanism of tegobuvir is related to HCV NS5B. The data from different mutation of NS5B including C316Y, C445F, and Y452H indicated that the inhibitory effect is due to the interaction with the hairpin in the thumb subdomain.[2] But, The mechanism of TGV still has not been clearly defined. TGV does not have any inhibition effect on NS5B enzymatic activity with recombinant NS5B proteins.[5] A decrease in antiviral potency of TGV was observed when a CYP1A inhibitor was combined used in antiviral assays. It demonstrated that TGV binds to the NS5B polymerase after undergoing a multistep metabolic activation pathway.[5] The activity of TGV also is related to the specific glutathione adducts.[5] Besides the sub-type differences, TGV also is less potent against the GT 1b replicon in the HeLa cell line with EC50＞10?M.

When individual patients treated only with tegobuvir with 8 days, it demonstrated median reductions of 1.5 log10 IU/mL in HCV RNA. But, rates of RVR (HCV RNA< 25 IU/mL at week 4) was enhanced when treated with Peg-IFN and RBV at the same time.[5]

References:
[1].  Zeuzem S, Andreone P, Pol S, Lawitz E, Diago M, Roberts S, Focaccia R, Younossi Z, Foster GR, Horban A et al: Telaprevir for retreatment of HCV infection. N Engl J Med 2011, 364(25):2417-2428.
[2].  Wong KA, Xu S, Martin R, Miller MD, Mo H: Tegobuvir (GS-9190) potency against HCV chimeric replicons derived from consensus NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a. Virology 2012, 429(1):57-62.
[3].  Shih IH, Vliegen I, Peng B, Yang H, Hebner C, Paeshuyse J, Purstinger G, Fenaux M, Tian Y, Mabery E et al: Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase. Antimicrob Agents Chemother 2011, 55(9):4196-4203.
[4].  Behrens SE, Tomei L, De Francesco R: Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 1996, 15(1):12-22.
[5].  Hebner CM, Han B, Brendza KM, Nash M, Sulfab M, Tian Y, Hung M, Fung W, Vivian RW, Trenkle J et al: The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS One 2012, 7(6):e39163.