In this phase 2 study of patients infected with HCV genotype 1 who had not been treated previously, one of the three telaprevir groups had a significantly higher rate of sustained virologic response than that with standard therapy. Response rates were lowest with the regimen that did not include ribavirin. (ClinicalTrials.gov number, NCT00372385.)

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Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.

These studies suggest that the initial antiviral response to telaprevir is due to a sharp reduction in wild-type virus, which uncovers pre-existing telaprevir-resistant variants. In patients given telaprevir alone, viral rebound can result from the selection of variants with greater fitness. However, the combination of telaprevir and PEG-IFN-alpha-2a inhibited both wild-type and resistant variants. In the present study, every patient who began PEG-IFN-alpha-2a and ribavirin after the 14-day dosing period had undetectable HCV RNA levels at 24 weeks, indicating that telaprevir-resistant variants are sensitive to PEG-IFN-alpha-2a and ribavirin.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...

The herpes simplex virus viion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (a)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and a mRNAs, whereas purified vhs-I mutant virions lack this activity. The functional half-life of many early ((3)-and late (y)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscrininately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection.Cells infected with herpes simplex viruses 1 and 2 proceed through the sequential transcription of several coordinately regulated groups of viral genes, including the a (immediate early), A3 (delayed early), and yj and y2 (late) genes (1-3). As suggested originally by Honess and Roizman (1), this transcriptional regulation must also be accompanied by a negative translational-regulatory scheme. Thus, the translation of host mRNA is turned off soon after virus entry into the cells, and the synthesis of a-and (3-viral polypeptides decreases at later intervals after infection.The early shutoff of host-protein synthesis (4) (15). Surprisingly, the vhs mutants are also altered with respect to a function that is present in the wild-type (wt) virus inoculum, which decreases the synthesis of a proteins from preexisting a mRNA (15). These findings suggest that a single virion-associated function might limit the translation of both host and a mRNAs.In this paper, we present data concerning the specificity of the host shutofffunction. We show that purified virions of the wt virus, but not those of the vhs-i mutant virus, carry a function that reduces the half-life of host and a mRNAs.Furthermore, the functional half-life of many (-and y-viral mRNAs is also prolonged in vhs-i-infected cells. These findings suggest that wt virions contain a function that indiscriminately reduces the half-life of the majority of infected-cell mRNAs. EXPERIMENTAL PROCEDURESCells and Viruses. Mouse Ltk-cells were obtained from B. Roizman (University of Chicago) and Vero monkey cells were obtained from S. Bachenheimer (University of North Carolina). The derivation of the vhs-i mutant was described previously (15). Virus stocks representing cell lysates were prepared by three cycles of freeze-thawing of infected cells (16), and virions were purified in dextran T 10 gradients (17,18).Drug Treatment and Analyses of Proteins. For cycloheximide reversal in the presence of actinomycin D (1), the cells were incubated fo...

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Infection with hepatitis C virus (HCV) is a major medical problem with over 170 million people infected worldwide. Substantial morbidity and mortality are associated with hepatic manifestations (cirrhosis and hepatocellular carcinoma), which develop with increasing frequency in people infected with HCV for more than 20 years. Less well known is the burden of HCV disease associated with extrahepatic manifestations (diabetes, B-cell proliferative disorders, depression, cognitive disorders, arthritis and Sjögren's syndrome). For patients infected with genotype 1 HCV, treatment with polyethylene glycol decorated interferon (peginterferon) α and ribavirin (PR) is associated with a low (40-50%) success rate, substantial treatment-limiting side effects and a long (48-week) duration of treatment. In the past 15 years, major scientific advances have enabled the development of new classes of HCV therapy, the direct-acting antiviral agents, also known as specifically targeted antiviral therapy for hepatitis C (STAT-C). In combination with PR, the HCV NS3-4A protease inhibitor telaprevir has recently been approved for treatment of genotype 1 chronic HCV in the United States, Canada, European Union and Japan. Compared with PR, telaprevir combination therapy offers significantly improved viral cure rates and the possibility of shortened treatment duration for diverse patient populations. Developers of innovative drugs have to blaze a new path with few validated sign posts to guide the way. Indeed, telaprevir's development was once put on hold because of its performance in a standard IC(50) assay. Data from new hypotheses and novel experiments were required to justify further investment and reduce risk that the drug might fail in the clinic. In addition, the poor drug-like properties of telaprevir were a formidable hurdle, which the manufacturing and formulation teams had to overcome to make the drug. Finally, novel clinical trial designs were developed to improve efficacy and shorten treatment in parallel instead of sequentially. Lessons learned from the development of telaprevir suggest that makers of innovative medicines cannot rely solely on traditional drug discovery metrics, but must develop innovative, scientifically guided pathways for success.