Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479

Abstract

PSI-6130 (beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine) is a selective inhibitor of hepatitis C virus (HCV) replication that targets the NS5B polymerase. R7128, the prodrug of PSI-6130, has shown antiviral efficacy in patients chronically infected with HCV genotype 1a (GT-1a) and GT-1b. We observed that the compound exhibited potent in vitro activity against laboratory-optimized HCV replicons as well as against a panel of replicons containing NS5B HCV polymerases derived from GT-1a and GT-1b clinical isolates. We used the HCV replicon cell system to examine the emergence of variants with reduced sensitivity to PSI-6130. Short-term treatment of cells harboring the HCV subgenomic replicon with PSI-6130 cleared the replicon without generating resistant variants. Long-term culture of the cells under the compound selection generated the S282T substitution in a complex pattern with other amino acid substitutions in the NS5B polymerase. The presence of the coselected substitutions did not increase the moderate three- to sixfold loss of sensitivity to PSI-6130 mediated by the S282T substitution; however, their presence enhanced the replication capacity compared to the replication levels seen with the S282T substitution alone. We also observed a lack of cross-resistance between PSI-6130 and R1479 and demonstrated that long-term culture selection with PSI-6130 in replicon cells harboring preexisting mutations resistant to R1479 (S96T/N142T) results in the emergence of the S282T substitution and the reversion of S96T to wild-type serine. In conclusion, PSI-6130 presents a high barrier to resistance selection in vitro, selects for variants exhibiting only low-level resistance, and lacks cross-resistance with R1479, supporting the continued development of the prodrug R7128 as a therapeutic agent for the treatment of HCV infection.

FIG. 1.

Chemical structures of HCV inhibitors used in the study. PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine; R7128, PSI-6130 prodrug; RO2433, PSI-6130 uridine metabolite; NM107, 2′-C-methylcytidine; R1479, 4′-azidocytidine; NNI-1, thiophene-2 caboxylic acid.

FIG. 2.

PSI-6130 exhibits potent activity against GT-1b and GT-1a NS5B clinical isolates. The inhibitory activity of PSI-6130 was evaluated using a panel of transient replicons containing the NS5B coding region derived from clinical isolates of treatment-naïve HCV GT-1a- or GT-1b-infected patients. After the amplified NS5B region was cloned from a patient serum into the corresponding genotype transient shuttle replicon, 96 individual molecular clones were pooled for each clinical isolate to mimic the natural polymorphism in the patient. The activity of PSI-6130 was assessed using the HCV transient replicon assay.

FIG. 3.

PSI-6130 and NM107 clear the HCV replicon. Cells harboring the stable HCV replicon were treated with 10× the EC₅₀ of PSI-6130, NM107, or NNI-1 in the absence of G418 for 29 days (clearance phase). The inhibitors were then removed, and G418 was added for 14 days (rebound phase). HCV replicon levels were monitored using qPCR during the clearance and rebound phases.

FIG. 4.

Replication capacity of transient replicons containing the selected NS5B amino acid substitutions. The amino acid substitutions observed in the selection with PSI-6130 or R1728 were introduced into the GT-1b Con1 transient HCV replicon. In vitro-transcribed RNA was transfected into Huh-7 cells, and the replication capacity was calculated (error bars represent the standard errors of the means of the results of three to five independent experiments). Clone C5 was generated by cloning the NS5B coding sequence from the selected cells into the transient replicon and was tested as described above.

FIG. 5.

Characterization of the observed amino acid substitutions in NS5B in the transient replicon assay. The amino acid substitutions observed in the selection with PSI-6130 were introduced into the GT-1b Con1 transient HCV replicons by site-directed mutagenesis. In vitro-transcribed RNA was transfected into Huh-7 cells, and the sensitivity of the replicons to PSI-6130 (A), R7128 (B), NM107 (C), and NNI-1 (D) was measured at 72 h following treatment (error bars represent the standard errors of the means of the results of three to five independent experiments). A value representing a severalfold shift in EC₅₀ compared to the WT replicon value (determined by dividing the EC₅₀ mutant value by the EC₅₀ WT value) is presented above each bar graph point.

FIG. 6.

Stable cells containing the replicons obtained from the cells selected with PSI-6130 show moderate reduced sensitivity to PSI-6130 compared to NM107 results. Whole cellular RNA was isolated from the cells selected with PSI-6130 and transfected into naïve Huh-7 cells. Stable cell lines were generated from selection set 2 (5R32) and from untreated cells (RC57). The generated stable cell lines were tested for sensitivity to PSI-6130, NM107, and NNI-1 by the use of qPCR (error bars represent the standard errors of the means of the results of three independent experiments). The shift values were calculated based on the untreated cell line value, which was set to 1.

FIG. 7.

The R1479-resistant mutant S96T reverts to WT under conditions of treatment with PSI-6130. Cells harboring replicons with the S96T/N142T mutations were passaged in the presence of PSI-6130. Clonal sequencing analysis of NS5B was performed on samples collected at passages 12, 16, and 20.

FIG. 8.

PSI-6130 and NM107 adopt similar 3′-end conformations. X-ray crystal structures for PSI-6130 and NM107 are shown.