Development of cell-based assays for in vitro characterization of hepatitis C virus NS3/4A protease inhibitors

Abstract

A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

FIG. 1.

Structures of pyrrolidine-5,5-trans-lactam compounds. Structure 1 is the parent for compounds 1a to 1q. The position of the R group in structure 1 is indicated on the left. R groups only are shown for compounds 1a to 1q. The complete structures of compounds 2 to 6 are given separately.

FIG. 2.

Metabolic labeling of vaccinia virus-infected BT7H cells in the presence and absence of compound 1a. BT7H cells were infected with rVV NS3-5. Compound 1a was added at 10 or 100 μM throughout the assay, and cells were metabolically labeled with [³⁵S]methionine at 19 to 21 h postinfection (see Materials and Methods). Cell lysates were analyzed by SDS-PAGE and autoradiography. Fully processed nonstructural proteins are indicated by arrows. P, precursor proteins. Due to the low molecular weight of NS4A, it is not visible on these gels. MW, molecular weight marker (in thousands).

FIG. 3.

Enantiomers, compounds 1a and 2, exhibit different activities in the gel-based assay. Compound (Cmpd) 1a was active at 10 μM, as demonstrated by the loss of fully processed nonstructural proteins NS3, NS4B, and NS5B, while its enantiomer, compound 2, was inactive at 100 μM. P, precursor proteins. MW, molecular weight marker (in thousands).

FIG. 4.

Effects of trans-lactams on transcription. Cytoplasmic RNA was extracted from BT7H cells that either were left uninfected (U) or were infected with rVV NS3-5 or wild-type vaccinia virus (WT). Cells infected with rVV NS3-5 were also treated with compound 1a or 2 at the concentrations shown. Northern blot analysis was carried out using a biotinylated oligonucleotide probe to the NS5B region. All lanes contain 0.2 μg of cytoplasmic RNA, except for the positive control (P), which contains 10 ng of the NS3-5 RNA transcript.

FIG. 5.

Protein expression in the presence of a trans-lactam. Compound 1b was added, at the concentrations indicated, to BT7H cells at different stages during the labeling process. The 21-h samples were continuously exposed to the compound. The 3-h sample was exposed to the compound from 18 to 21 h postinfection, during both the methionine starvation and the labeling phase. The 2-h sample was exposed to the compound from 19 to 21 h postinfection, during the metabolic labeling phase only. SDS-PAGE was carried out on the labeled samples. Western blot analysis with an in-house anti-NS3 polyclonal antibody was performed on parallel samples to assess the total amount of NS3 in the system. (A) Autoradiograph showing metabolically labeled proteins. MW, molecular weight marker (in thousands). P, precursor proteins. (B) Western blot analysis with the anti-NS3 antibody.

FIG. 6.

trans-lactam compounds can be ranked according to potency in the gel-based assay. Dose responses of compounds 1a and 3 were carried out at the concentrations indicated, from which end points can be determined. The end point is defined as the lowest concentration at which NS4B is no longer visible—in this case, 3 μM for compound 1a and 0.3 μM for compound 3. MW, molecular weight marker (in thousands).

FIG. 7.

Selection of the primary antibody for the rVV NS3-5 ELISA. (A) Commercially available murine monoclonal antibodies against HCV nonstructural proteins were assayed at 4 μg/ml against rVV NS3-5-infected BT7H cells (dark bars) and uninfected BT7H cells (light bars). Each bar represents the mean result for four (rVV NS3-5-infected cells) or three (uninfected cells) samples within the same experiment ± the standard error of the mean. Antibodies against the C-100 antigen (8958) and against NS5 (8808 and 1872) were selected for use in the ELISA (see Materials and Methods). (B) An example of an IC₅₀ curve for compound 1a using antibody 8958.

FIG. 8.

Use of a biotinylated trans-lactam probe for the detection of HCV NS3 in vaccinia virus-infected cells. Extracts of rVV NS3-5-infected cells were preincubated with compound (Cmpd) 1q, at the concentrations indicated, for 1 h prior to the addition of a biotinylated trans-lactam probe, compound 6. Samples were analyzed by Western blotting using an HRP-neutravidin probe and were visualized by using SuperSignal reagent. Wt, wild-type vaccinia virus-infected cell extract; NS3-5, rVV NS3-5-infected cell extract.