Hepatitis C virus-specific directly acting antiviral drugs.

The major targets for direct-acting antivirals (DAAs) are the NS3/4A protease, the NS5A protein, and the NS5B polymerase. The latter enzyme offers several target sites: the catalytic domain for nucleoside/nucleotide analogs and different allosteric sites for non-nucleoside inhibitors. Two protease inhibitors have already been approved and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development pipeline. Not only these agents can achieve very high cure rates when combined with PEG-IFN and RBV, but have also started to provide promising results when combined in IFN-free, all-oral combinations. In addition to the more canonical drug targets, new alternative viral targets for small molecule drug development are emerging, such as p7 or NS4B. Current research is focusing on defining the most efficacious DAA combination regimens, i.e., those which provide the highest rates of viral eradication, broadest spectrum of action, minimal or no clinical resistance, shortest treatment duration, and good tolerability.

In vitro selection and characterization of HCV replicons resistant to multiple non-nucleoside polymerase inhibitors.

BACKGROUND & AIMS: To delay or prevent the selection of HCV drug-resistant variants, combination therapy will be needed. Our aim was to determine the antiviral efficacy of various combinations of non-nucleoside polymerase inhibitors (NNI) (that have a different allosteric binding site) and the barrier towards resistance development of such combinations. METHODS: Short-term antiviral combination assays were performed in a checkerboard format. Resistance selection experiments employing HCV replicons were performed using two different protocols: (i) a short-term treatment with fixed concentrations and (ii) a long-term treatment with increasing concentrations. RESULTS: All pair-wise combinations of NNI resulted in an additive antiviral effect in short-term antiviral assays. Combination treatment of two NNIs markedly reduced or even prevented the emergence of double resistant colonies. However, double and even triple NNI-resistant variants emerged readily when relatively low starting concentrations were used in a long-term selection protocol. Genotyping confirmed the presence of the previously published resistance mutations. For some NNI, different signature mutations appeared depending on the other NNI in the particular combination. Remarkably, variants that were selected to be resistant to three different classes of NNIs [a thiophene carboxylic acid (TCA), a benzimidazole (JT-16), and a benzofuran (HCV-796)] proved resistant to yet a fourth class of NNIs (benzothiadiazines). CONCLUSIONS: Double and even triple NNI-resistant HCV replicons can be readily selected with a stepwise resistance selection protocol. Depending on the particular combination, different signature mutations may arise for some NNI. Resistance to three classes of NNI resulted in resistance to yet a fourth class.

Comparative study of the genetic barriers and pathways towards resistance of selective inhibitors of hepatitis C virus replication.

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.

Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase.

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and 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 ß-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 ß-hairpin in the thumb subdomain.

Statins potentiate the in vitro anti-hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development.

UNLABELLED: Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti-HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon-alpha (IFN-alpha) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short-term (3 days) antiviral assays. Neither statins, at a concentration of five-fold their median effective concentration (EC(50)) value, nor polymerase, protease inhibitors, or IFN-alpha, at concentrations 10- or 20-fold their EC(50) value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV-796. CONCLUSION: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors.

Substituted imidazopyridines as potent inhibitors of HCV replication.

BACKGROUND/AIMS: Following lead optimization, a set of substituted imidazopyridines was identified as potent and selective inhibitors of in vitro HCV replication. The particular characteristics of one of the most potent compounds in this series (5-[[3-(4-chlorophenyl)-5-isoxazolyl]methyl]-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine or GS-327073), were studied. METHODS: Antiviral activity of GS-327073 was evaluated in HCV subgenomic replicons (genotypes 1b, 1a and 2a), in the JFH1 (genotype 2a) infectious system and against replicons resistant to various selective HCV inhibitors. Combination studies of GS-327073 with other selective HCV inhibitors were performed. RESULTS: Fifty percent effective concentrations for inhibition of HCV subgenomic 1b replicon replication ranged between 2 and 50 nM and were 100-fold higher for HCV genotype 2a virus. The 50% cytostatic concentrations were > or = 17 microM, thus resulting in selectivity indices of > or = 340. GS-327073 retained wild-type activity against HCV replicons that were resistant to either HCV protease inhibitors or several polymerase inhibitors. GS-327073, when combined with either interferon alpha, ribavirin, a nucleoside polymerase or a protease inhibitor resulted in overall additive antiviral activity. Combinations containing GS-327073 proved highly effective in clearing hepatoma cells from HCV. CONCLUSIONS: GS-327073 is a potent in vitro inhibitor of HCV replication either alone or in combination with other selective HCV inhibitors.

Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors.

Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

Selective inhibitors of picornavirus replication.

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.

Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors.

We report here a comparative study of the anti-hepatitis C virus (HCV) activities of selected (i) nucleoside polymerase, (ii) nonnucleoside polymerase, (iii) alpha,gamma-diketo acid polymerase, (iv) NS3 protease, and (v) helicase inhibitors, as well as (vi) cyclophilin binding molecules and (vii) alpha 2b interferon in four different HCV genotype 1b replicon systems.

Synthesis of new benzimidazole-coumarin conjugates as anti-hepatitis C virus agents.

Nineteen new conjugated compounds were successfully synthesized by a one-flask method from benzimidazole and coumarin derivatives. A methylenethio linker was used to connect these two kinds of derivatives. In addition, substituted benzimidazol-2-thiones were also coupled with beta-D-glucose peracetate; the resultant glucosides were further converted to the corresponding 2-(methylthio)coumarin derivatives. Their activity against the hepatitis C virus was tested; two of the most potent compounds 2-[(6'-bromocoumarin-3'-yl)methylenethio]-5-fluorobenzimidazole (4i) and its derivative 1-[(2'',3'',4'',6''-tetra-O-acetyl)glucopyranos-1''-yl]-2-[(6'-bromocoumarin-3'-yl)methylenethio]benzimidazole (7c) showed EC(50) values of 3.4 microM and 4.1 microM, respectively. At a concentration of 5.0 microM, compound 7c inhibited HCV RNA replication by 90% and had no effect on cell proliferation. Given these data, a structure-activity relationship was established.

Rational design of antiviral drug combinations based on equipotency using HCV subgenomic replicon as an in vitro model.

Combination therapy of directly acting antivirals (DAA's) for the treatment of chronic HCV infections has proven to be a highly effective strategy to cure chronic infections with this virus. Here we studied, using HCV as an example, how to best design in vitro studies that explore the combined antiviral efficiency of combinations of three or more DAA's. To that end we used a HCV NS3 protease inhibitor, a NS5A targeting compound and two non-nucleoside NS5B polymerase inhibitors (each one targeting a different drug binding site). We demonstrate, employing HCV subgenomic replicon containing Huh 9-13 hepatoma cells, that quadruple therapy with these 4 different DAA's each at 1x their EC75, results in a highly efficient inhibition of viral replication. This is further reflected in the rapid clearance of the HCV replicon from the host cell. By contrast, neither equipotent combinations that consist of either molecules alone at 4x EC75 nor triple combinations at 1.33x the EC75 resulted in clearance. In contrast to the quadruple combo, drug-resistant variants emerged under mono-treatment and in most triple combo's. These data thus demonstrate that quadruple combinations at total suboptimal concentrations [i.e. concentrations at which neither mono- nor triple therapy is sufficiently potent] result rapidly in a pronounced antiviral efficacy. Altogether, this work provides an example as to how to design studies to explore the antiviral efficacy of combinations of more than two compounds.

Immune activation following cytomegalovirus infection: more important than direct viral effects in cardiovascular disease?

BACKGROUND: Over the last 30 years multiple micro-organisms have been associated with different types of vascular disease, like atherosclerosis, restenosis or transplant arteriosclerosis. Nonetheless, it is still ambiguous which molecular mechanisms are exactly involved in the exacerbating effect of microbes in these disorders. OBJECTIVES AND STUDY DESIGN: The present review summarizes sero-epidemiological, in vitro and animal data supporting the role of cytomegalovirus, a member of the herpes virus family, in vascular disease. Additionally, various ways by which the virus can potentially affect the disease will be discussed. RESULTS: Rodent models as well as in vitro studies suggested that CMV might enhance lesion formation in various ways, like augmentation of the oxLDL uptake, altering monocyte adhesion or increasing the production of pro-inflammatory cytokines. Nevertheless, recent data from our lab and others suggest that alternative mechanisms also may contribute to CMV induced. Inspired by this, we will hypothesise alternative mechanisms by which CMV might affect atherosclerosis. CONCLUSIONS: Although researchers have tried for many years to unravel the tactics by which micro-organisms, like CMV, aggravate atherosclerosis, so far most suggested mechanisms failed to fully explain both experimental and clinical observations. Therefore, new means to elucidate the observed effects are required.

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene.

To allow rapid identification of bacteria in pure cultures and blood culture bottles, an assay was developed which is based on real-time amplification and sequencing of bacterial 16 S rRNA genes. In principle, this assay allows identification of bacteria from pure cultures within 6.5 h, and from blood cultures within approximately 7 h.

Mouse cytomegalovirus antigenic immune stimulation is sufficient to aggravate atherosclerosis in hypercholesterolemic mice.

We have previously demonstrated that mouse cytomegalovirus (MCMV) infection aggravates atherosclerosis by stimulating the ongoing inflammatory process in the vascular wall. Here we investigated whether MCMV antigenic immune stimulation by UV-MCMV injection is sufficient to aggravate atherosclerosis. In addition we analyzed whether low viral doses are sufficient to stimulate atherosclerosis. Therefore, apoE(-/-) mice received a low dose injection with infectious virus (MCMV) or replication-deficient virus (UV-inactivated MCMV, UV-MCMV). Atherosclerosis progression, influx of inflammatory cells in atherosclerotic lesions and internal organs and the number of MCMV DNA copies in various organs were determined at 2 weeks after injection. After injection with infectious virus, MCMV DNA was present in internal organs, while no MCMV DNA could be detected after UV-MCMV injection. Interestingly, both MCMV and UV-MCMV significantly increased mean atherosclerotic lesion area and T cell number in the atherosclerotic lesions, while only MCMV infection increased T cell numbers in the internal organs. These data indicate that in apoE(-/-) mice both low dose infectious MCMV as well as MCMV antigenic injections are sufficient for atherosclerosis aggravation.

In vitro combinations containing Tegobuvir are highly efficient in curing cells from HCV replicon and in delaying/preventing the development of drug resistance.

Tegobuvir (GS-9190) is a non-nucleoside inhibitor of HCV RNA replication with proven antiviral activity in HCV-infected patients. The in vitro antiviral activity of Tegobuvir, when combined with one or two other direct acting antivirals (DAA) was assessed. When Tegobuvir was combined with either interferon α-2b, ribavirin, the protease inhibitor (PI) VX-950, the nucleoside polymerase inhibitor (NI) 2'-C-methylcytidine or various non-nucleoside polymerase inhibitors, an overall additive antiviral activity was observed. Adding Tegobuvir (at concentrations of 6, 30 or 150nM) to replicon-containing cells in the presence of suboptimal concentrations of the PI or of the various polymerase inhibitors either markedly delayed or completely prevented resistance development against these latter compounds. Tegobuvir (15nM), when combined with the PI, was able to cure replicon-containing cells from their replicon after a single passage, whereas either compound alone (at 2-fold higher concentration) was not. The triple combination of Tegobuvir (10nM), the PI and the NI resulted in clearance of replicon RNA after only two passages. In contrast, the inhibitors when used alone at 3-fold higher concentrations were not able to cure the cells from the replicon, after as long as 6 passages. Combinations containing low concentrations of Tegobuvir are thus highly effective in curing cells from HCV replicon and in delaying or preventing the development of resistance against other DAA.

Murine cytomegalovirus infection directs macrophage differentiation into a pro-inflammatory immune phenotype: implications for atherogenesis.

We have previously demonstrated that mouse cytomegalovirus (MCMV) aggravates atherosclerosis in apolipoprotein E knockout (apoE(-/-)) mice, most likely by enhancing both systemic and local (e.g. in the vascular wall) cytokine production. However, until now it was unclear which cell type is responsible for this enhanced pro-inflammatory cytokine production. In this study we focused on the macrophage (mPhi), which besides being an important source of such cytokines, is known to be an important player in both atherosclerosis and viral clearance. We investigated whether MCMV could induce a pro-inflammatory immune mPhi phenotype, which ultimately may contribute to the development of atherosclerosis. To this end, peritoneal exudate cells (PEC) were elicited in apoE(-/-) mice by either MCMV or thioglycolate injection, and mPhi were phenotyped at 1 week post-intraperitoneal injection. MCMV-induced peritoneal mPhi contained MCMV DNA but had limited MCMV mRNA expression, indicating latent infection. These mPhi showed increased production of interferon-gamma (IFNgamma), exclusive production of interleukin-18 (IL-18) and increased expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86, when compared with thioglycolate-induced mPhi. From these results, we conclude that intraperitoneal injection of MCMV induces an immune-responsive exudate in which at 7 days post-infection, MCMV-infected mPhi express a pro-inflammatory immune phenotype. As such, the MCMV-induced mPhi may be an important player in aggravating atherosclerosis through systemic and/or local immune activation.

Cytomegalovirus infection aggravates atherogenesis in apoE knockout mice by both local and systemic immune activation.

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.

Improved detection and quantification of mouse cytomegalovirus by real-time PCR.

During latent cytomegalovirus (CMV) infection, viral presence cannot be detected by plaque assay. Therefore, we assessed the applicability of real-time PCR for temporal determination of virus dissemination in two different mouse strains. Eight-week-old BALB/c and C57BL/6J mice were infected with mouse CMV (MCMV) and sacrificed at 1, 2, 4, 6, 14 and 28 days post infection. Real-time PCR was used to determine MCMV copy number in the heart, bone marrow cells, aorta and blood. In lung, liver, salivary gland and spleen the presence of MCMV was determined both by plaque assay and real-time PCR. In analogy with the plaque assay, the real-time PCR technique revealed higher numbers of MCMV genomic copies in all organs obtained from BALB/c mice when compared with C57BL/6J mice, demonstrating the applicability of the technique. A significant correlation was observed between both assays when a positive test result was seen with both assays. Nonetheless, lower viral infectivity titers were found compared to real-time PCR data. Thus, the real-time PCR technique is more sensitive in detecting the presence of MCMV and is therefore well suited for (dose-response) intervention studies aimed at studying virus eradication.

MCMV infection increases early T-lymphocyte influx in atherosclerotic lesions in apoE knockout mice.

BACKGROUND: Multiple epidemiological studies have suggested that cytomegalovirus (CMV) infection is associated with atherosclerotic disease. However, conclusive proof that the virus is directly related to the progression of the disease is still lacking. OBJECTIVES: The goal of this study was to investigate whether MCMV is able to exacerbate the atherosclerotic process in atherosclerosis-susceptible mice. STUDY DESIGN: apoE knockout mice kept on a chow diet were sacrificed at both 2 and 20 weeks post infection (p.i.). C57Bl/6J mice fed an atherogenic diet were sacrificed at 2 weeks p.i. Lesion area, lesion composition (endothelial cells and smooth muscle cells) and inflammatory influx (T-lymphocytes and macrophages) in lesions were determined. The former one was determined by means of a microscope coupled to a computer-assisted morphometry system. The latter ones were scored after immunohistochemical staining. RESULTS: In the chronic phase of the infection mean lesion size was significantly increased after MCMV infection in the apoE knockout mice. This increase could to a large extent be attributed to a significant increase in type V lesion area after MCMV infection. Also, a significant increase in T-lymphocyte influx was observed in the acute phase of the infection in lesions from apoE knockout mice after MCMV infection while this effect was absent in C57Bl/6J mice. After MCMV infection no increase was observed in macrophage, smooth muscle cell and endothelial cell number in lesions from both mice strains. CONCLUSIONS: MCMV infection may exacerbate the atherosclerotic process in apoE knockout mice by means of an acute lymphocytic inflammatory response. In contrast to the MCMV induced effect in apoE knockout mice, MCMV infection did not increase the influx of T-lymphocytes in atherosclerotic lesions of C57Bl/6J mice.

Selecting and characterizing drug-resistant hepatitis C virus replicon.

The current standard of care (SOC) for hepatitis C virus (HCV) genotype 1-infected patients consists of telaprevir or boceprevir in addition to pegylated interferon and ribavirin treatment. Other selective inhibitors of HCV replication are being developed. Drug pressure may, depending on the class of inhibitors, (rapidly) select for drug-resistant variants. Here we describe four different approaches to select in vitro for drug-resistant HCV subgenomic replicons.