There remains a significant unmet need for a targeted, efficacious chemotherapy against HCV genotype 1. HCV genotype 1 is prevalent worldwide, especially in the United States, Europe, and Japan, but the current therapy is poorly tolerated and is not sufficiently effective in HCV genotype 1-infected patients (36
). Two protease inhibitors (Telaprevir and Boceprevir) were already launched for HCV treatment in 2011. The treatment outcome was improved by these new drugs but not to a sufficient extent and, furthermore, drug-resistant variant viruses emerged easily in the clinical trials.
We have demonstrated here that JTK-853 produces potent and selective inhibition of the HCV RdRp of genotypes 1a and 1b. Notably, the 316th amino acid residue of HCV polymerase is polymorphic, as seen with Con1 with cysteine (C316) and BK with asparagine (N316). JTK-853 showed inhibitory activities against HCV polymerases of Con1 and BK strains in the same range, suggesting that JTK-853 has inhibitory activity against both HCV polymerases regardless of the amino acid polymorphism at amino acid 316.
The attenuation of the antiviral activity of JTK-853 by the addition of HS to the HCV replicon cells was very small. The protein binding levels of JTK-853 to 100% HS or 40% HS plus 10% FBS by equilibrium analysis were 99.87% and 99.74%, respectively. Therefore, free JTK-853 from protein binding was 0.13% and 0.26%, suggesting the protein shift of JTK-853 in 100% HS is almost comparable to that in 40% HS plus 10% FBS.
Similarly, the physiological concentrations of major HS proteins (4.4% HSA plus 0.08% AAG) produced just a slight attenuation of the antiviral activity of JTK-853, with an EC90
of 0.449 μM. The results of the protein shift with JTK-853 in 100% HS and the EC90
value of JTK-853 in the physiological concentration of human serum protein strongly demonstrate that the antiviral activity of JTK-853 might be maintained even upon protein binding. This is in clear agreement with the demonstration of the compound's antiviral activity in HCV-infected patients (30
We previously demonstrated that the HCV polymerase C-terminal residues LWF (Leu547, Trp550, and Phe551) have an autoinhibitory effect on RdRp activity (1
). This inhibition is caused by occupation of a putative RNA-binding cleft in HCV polymerase by the LWF residues per se
. The LWF residue-binding region has been designated the LWF pocket and is located in the palm and thumb domain. Our structural analysis demonstrated that JTK-853 interacts with the LWF pocket in the palm site as well as with the β-hairpin region, by a distinctly different mode from that of other palm site-binding HCV polymerase inhibitors. Particularly, the association of JTK-853 with the LWF pocket of HCV polymerase is characteristic, because JTK-853 occupies the LWF pocket and displaces the LWF residues of HCV polymerase at the LWF pocket, suggesting the strong inhibitory activity of JTK-853 on HCV polymerase.
Our in vitro resistance selection study of JTK-853 identified the amino acid substitutions C316Y, M414T, Y452H, and L466V at the palm site of the HCV polymerase. Importantly, the antiviral activity of JTK-853 remained unchanged by the resistance mutations from other DAAs. In addition, JTK-853 showed an additive combination effect with other DAAs. These results suggest that JTK-853 could be used in combination with DAAs, such as other class polymerase inhibitors, protease inhibitors, and NS5A inhibitors.
In conclusion, we have presented the results of a preclinical in vitro
study of JTK-853 and demonstrated a potent and specific antiviral activity against HCV genotype 1. In fact, JTK-853 administered in a twice-daily dosing regimen has demonstrated potent antiviral activity in genotype 1 HCV-infected patients (30
), supporting its beneficial use as an oral antiviral agent for hepatitis C.