The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of ␤-D-2-deoxy-2-fluoro-2-C-methyluridine-5-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ؎ 0.050 M (mean ؎ standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 M). Crossresistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.Hepatitis C virus (HCV) currently affects more than 170 million people worldwide. Approximately 70% of infected individuals develop chronic hepatitis, among whom about 20% will develop liver cirrhosis and fibrosis and up to 5% will progress to hepatocellular carcinoma (2). The current standard of care (SOC), which combines pegylated alpha interferon (PegIFN-␣) and ribavirin (RBV), has limited efficacy in providing a sustained virological response (SVR), especially in individuals with HCV genotype 1 (ϳ50%), the most prevalent genotype in Western countries (8,11,35). The impact of genetic diversity of HCV in patients receiving SOC therapy has been reviewed (26): SVR rates are higher in patients infected with genotype 2 or 3 (ϳ80%), patients infected with genotype 4 appear to have a slightly better SVR rate (ϳ60%) than patients infected with genotype 1, and patients infected with genotypes 5 and 6 may achieve an SVR at a level between those of genotypes 1 and 2/3. In addition to the variability in efficacy, the lengthy treatment (24 to 48 w...

In budding yeast, the MLH1-PMS1 heterodimer is the major MutL homolog complex that acts to repair mismatches arising during DNA replication. Using a highly sensitive mutator assay, we observed that Saccharomyces cerevisiae strains bearing the S288c-strain-derived MLH1 gene and the SK1-strain-derived PMS1 gene displayed elevated mutation rates that conferred a long-term fitness cost. Dissection of this negative epistatic interaction using S288c-SK1 chimeras revealed that a single amino acid polymorphism in each gene accounts for this mismatch repair defect. Were these strains to cross in natural populations, segregation of alleles would generate a mutator phenotype that, although potentially transiently adaptive, would ultimately be selected against because of the accumulation of deleterious mutations. Such fitness ''incompatibilities'' could potentially contribute to reproductive isolation among geographically dispersed yeast. This same segregational mutator phenotype suggests a mechanism to explain some cases of a human cancer susceptibility syndrome known as hereditary nonpolyposis colorectal cancer, as well as some sporadic cancers.colorectal cancer ͉ incompatibility T he highly conserved mismatch repair (MMR) system contributes to genome stability by repairing errors that occur during DNA replication (1). In Escherichia coli, MMR is initiated by the binding of MutS protein to DNA mismatches. MutL interacts with the MutS-mismatch complex and activates downstream repair factors. Multiple MutS homologs (MSH) and MutL homologs (MLH) have evolved in eukaryotes that form heterodimers with specialized functions in DNA repair and recombination (2, 3). In Saccharomyces cerevisiae, MSH2-MSH3 and MSH2-MSH6 function in mismatch recognition, and MLH1-PMS1 is the primary MLH heterodimer in postreplicative MMR. Mutations in MSH and MLH genes that act in MMR elevate mutation rate, as measured in reversion and forward mutation assays, and reduce spore viability of diploid cells due to the accumulation of recessive lethal mutations (4-6). In addition, MMR proteins act to prevent recombination between divergent DNA sequences. This activity has been shown to prevent chromosomal rearrangements (7,8) and to enforce reproductive barriers between species (9, 10).Previously we created 60 alleles of the S. cerevisiae MLH1 gene from the S288c strain (cMLH1) in which clusters of charged residues were simultaneously changed to Ala (11). These alleles were tested for defects in MMR in the S288c (12) and SK1 (13) strains. More than one-third of the mutation set conferred a more severe MMR defect in SK1 strains than in S288c strains. Two mutations, cmlh1-29 and cmlh1-56, conferred wild-type-like phenotypes in S288c but null-like phenotypes in SK1. Introduction of the S288c PMS1 gene into the SK1 strain suppressed the mutator phenotype of these mutants, suggesting that the MMR phenotype was due to incompatibility, or negative epistasis, between MLH components (11).The influences of epistatic interactions on a wide variety of traits and proc...

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.In eukaryotes, mismatch repair plays a critical role in mutation avoidance and is carried out by the MutSLH family of proteins (for reviews, see references 13, 36, and 40). During vegetative growth, these proteins recognize and bind DNA mispairs that result primarily from replication errors or DNA damage. In Escherichia coli, MutS binding to DNA mispairs results in the recruitment of MutL, a matchmaker protein that functions in postreplicative mismatch repair by interacting with both the MutH endonuclease and UvrD helicase (22, 23). These interactions coordinate mispair recognition with DNA strand-specific signals so that mispairs are removed via excision and resynthesis steps that occur on the newly replicated strand.Eukaryotes contain multiple MutS (Msh) and MutL (Mlh) homologs, with six Msh and four Mlh homologs present in Saccharomyces cerevisiae (36). Genetic and biochemical studies have shown that the eukaryotic homologs display specialized functions with respect to the types of DNA substrates on which they act (10, 36, 40). In S. cerevisiae, the Mlh proteins form heterodimers (Mlh1p-Pms1p, Mlh1p-Mlh3p, and Mlh1p-Mlh2p) that display unique functions. Mlh1p is considered a central member of this group because heterodimers have not been identified among the other members (45, 70). The Mlh1p-Pms1p complex plays a major role in postreplicative mismatch repair, while the other two Mlh complexes appear to be redundant with Mlh1p-P...

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC 50 = 1.4 μM), suramin sodium salt (IC 50 = 3.6 μM), NF 023 hydrate (IC 50 = 6.2 μM) and tyrphostin AG 538 (IC 50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors ( , only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.Hepatitis C virus (HCV) infects more than 170 million people worldwide, causing chronic hepatitis, cirrhosis, hepatocellular carcinoma, and/or liver failure (12). Clinical HCV isolates are categorized into different genotypes that display up to 30% sequence variation. Response rates to all interferon-based HCV therapies are genotype dependent, with patients infected with HCV genotype 1 having about one-half the chance of a sustained virological response as patients infected with other genotypes (37). Current HCV therapies combine pegylated alpha interferon and ribavirin (1-␤-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). A number of viral factors have been proposed to contribute to the different response rates to interferon (reviewed in reference 59), but less is known about the effects of viral variation on sensitivity to ribavirin. Here, we employ biochemical methods to examine the simple hypothesis that as a nucleoside analog, ribavirin may affect the enzymes encoded by HCV, and that variation among the enzymes from different genotypes could affect ribavirin response rates. We also examine whether HCV genetic variation influences the sensitivities of HCV enzymes to other nucleoside analogs similar to those currently in development as HCV-specific drugs.The ϳ9,600-nucleotide HCV genome harbors a single open reading frame that encodes a polyprotein, which is processed into 10 distinct proteins by both host and v...

Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B (Mol. Cell 19:111). We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidylprolyl isomerase activity, is not required for stimulation of RNA synthesis.

We examined mismatch repair (MMR)-defective diploid strains of budding yeast grown for $160 generations to determine whether decreases in spore viability due to the uncovering of recessive lethal mutations correlated with an increase in gross chromosomal rearrangements (GCRs). No GCRs were detected despite dramatic decreases in spore viability, suggesting that frameshift and/or other unrepaired DNA replication lesions play a greater role than chromosomal instability in decreasing viability in MMRdefective strains.

Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.