Picornaviruses cover a large family of pathogens that have a major impact on human but also on veterinary health. Although most infections in man subside mildly or asymptomatically, picornaviruses can also be responsible for severe, potentially life-threatening disease. To date, no therapy has been approved for the treatment of picornavirus infections. However, efforts to develop an antiviral that is effective in treating picornavirus-associated diseases are ongoing. In 2007, Schering-Plough, under license of ViroPharma, completed a phase II clinical trial with Pleconaril, a drug that was originally rejected by the FDA after a New Drug Application in 2001. Rupintrivir, a rhinovirus protease inhibitor developed at Pfizer, reached clinical trials but was recently halted from further development. Finally, Biota's HRV drug BTA-798 is scheduled for phase II trials in 2008. Several key steps in the picornaviral replication cycle, involving structural as well as non-structural proteins, have been identified as valuable targets for inhibition. The current review aims to highlight the most important developments during the past decades in the search for antivirals against picornaviruses.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Hepatitis C virus (HCV) represents a major health burden. An estimated 170 million to 180 million people worldwide are chronically infected with this virus and are at increased risk of developing liver cirrhosis and/or hepatocellular carcinoma (64). The current standard of care for chronic hepatitis C consists of pegylated alpha interferon (IFN) in combination with ribavirin (RBV) (12). This therapy is, however, associated with serious side effects and results in a sustained virological response in only 50 to 60% of patients (depending on the genotype). There is thus an urgent need for more effective and better-tolerated drugs.Selective inhibitors of HCV replication that target the NS3 protease and the NS5B RNA-dependent RNA polymerase (RdRp) in particular have been pursued as potential new therapies (38). BILN 2061 (culprivir), a peptidomimetic inhibitor of the HCV NS3 protease (the first selective inhibitor of HCV to be administered to patients), resulted in a rapid and pronounced decline in the level of viral replication in patients chronically infected with HCV genotype 1. Its clinical development was, however, halted because of cardiotoxicity (22,31). Currently, four NS3 protease inhibitors are in clinical development, i.e., VX-950 (telaprevir), SCH-503034 (boceprevir), and TMC435350 (2,51,56). Telaprevir and boceprevir are at the most advanced stages of development and are being evaluated in combination with the standard therapy in phase III and phase II clinical trials, respectively (3,39,45,50). Several nucleoside polymerase inhibitors (NIs) and nonnucleoside polymerase inhibitors (NNIs) are or have been in development. Nucleoside analogue inhibitors of HCV replication basically act as chain terminators of the polymerization process once they have been phosphorylated to their 5Ј-triphosphate metabolite (13).

Worldwide approximately 170 million people (or almost 3% of the global population) are chronically infected with HCV. Chronically infected patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. 1 In Western countries, infection with HCV is the most common cause of liver transplantation. The current standard therapy for chronic hepatitis C consists of the combination of pegylated interferon alpha (IFN-␣) and ribavirin. This therapy is only effective in 50% to 60% of infected patients and is associated with serious side effects. 2 There is thus an urgent need for more selective, potent, and better-tolerated therapies for chronic hepatitis C.Most antiviral drugs that are currently in clinical trials are inhibitors of the viral polymerase or serine protease. The design of antiviral drugs that inhibit the function of the HCV protease and polymerase therefore appears to be logical. The first HCV nonstructural (NS)3/4A serine protease inhibitor to enter clinical trials was

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GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitroand has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the ␤-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the ␤-hairpin in the thumb subdomain.Hepatitis C virus (HCV) is a major cause of morbidity, affecting approximately 170 million people worldwide with an estimated 3 to 4 million additional new infections occurring each year (36). HCV is a positive-strand RNA virus with six major genotypes that are further divided into multiple subtypes. Due to the error-prone nature of its replication enzyme, a myriad of different viral quasispecies exists within an infected individual (32). With this high degree of viral variability, the current treatment regimen, which consists of weekly injections of pegylated alpha interferon (PEG-IFN) and twice-daily oral doses of ribavirin (RBV), is of limited efficacy and, in addition, carries significant side effects (8, 23). Although the HCV NS3/4A protease inhibitors telaprevir and boceprevir for treatment of chronic HCV infection will soon be available, these compounds will still need to be combined with the current standard of care (PEG-IFN/RBV) to be efficacious and will not cure all infected individuals (10,14,30). Therefore, the development of additional direct antiviral agents with diverse resistance profiles is necessary, with the ultimate goal of developing all-oral antiviral combinations that can achieve superior ...

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Hepatitis C virus (HCV) inhibitors include direct-acting antivirals (DAAs) such as NS3 serine protease inhibitors, nucleoside and nonnucleoside polymerase inhibitors, and host-targeting antivirals (HTAs) such as cyclophilin inhibitors that have been developed in recent years. Drug-resistant HCV variants have been reported both in vitro and in the clinical setting for most classes of drugs. We report a comparative study in which the genetic barrier to drug resistance of a representative selection of these inhibitors is evaluated employing a number of resistance selection protocols. The NS3 protease inhibitors VX-950 and BILN 2061, the nucleoside polymerase inhibitor 2-C-methylcytidine, three nonnucleoside polymerase inhibitors (thiophene carboxylic acid, benzimidazole, and benzothiadiazine), and DEB025 were included. For each drug and passage in the selection process, the phenotype and genotype of the drug-resistant replicon were determined. For a number of molecules (BILN 2061 and nonnucleoside inhibitors), drug-resistant variants were readily selected when wild-type replicon-containing cells were directly cultured in the presence of high concentrations of the inhibitor. Resistance to DEB025 could be selected only following a lengthy stepwise selection procedure. For some DAAs, the signature mutations that emerged under inhibitor pressure differed depending on the selection protocol that was employed. Replication fitness of resistant mutants revealed that the C445F mutation in the RNA-dependent RNA polymerase can restore loss of fitness caused by a number of unfit resistance mutations. These data provide important insights into the various pathways leading to drug resistance and allow a direct comparison of the genetic barriers of various HCV drugs.Hepatitis C virus (HCV) is a positive single-stranded RNA virus and the only member of the Hepacivirus genus within the Flaviviridae family. An estimated 170 million people are chronically infected worldwide. Three million to four million people become newly infected each year (57). Chronically infected patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. In Western countries, infection with HCV is the most common reason for liver transplantation. The current standard of care for the management of chronic hepatitis C virus infection consists of the combination of pegylated alpha interferon (pegIFN-␣) and ribavirin. This therapy is effective in only 50 to 60% of infected patients and is associated with serious side effects (44). Therefore, more tolerable, highly potent inhibitors of HCV replication are urgently needed and are currently also being developed. Antivirals that specifically target viral proteins are referred to as "direct-acting antivirals" (DAAs) for HCV. A number of NS3/NS4A protease inhibitors are currently in clinical development. The first HCV NS3/4A serine protease inhibitor to enter clinical trials was ciluprevir (BILN 2061) (54), but clinical development was halted because of cardiotoxicity. Other protease inhib...