RG7128 alone or in combination with pegylated interferon-α2a and ribavirin prevents hepatitis C virus (HCV) Replication and selection of resistant variants in HCV-infected patients.

INTRODUCTION: RG7128 (prodrug of PSI-6130) shows potent antiviral efficacy in patients infected with hepatitis C virus (HCV) genotypes 1, 2, or 3, with mean viral load decreases of 2.7 and 5 log(10) IU/mL, respectively, associated with 1500-mg doses twice daily after monotherapy for 2 weeks and with 1000-mg and 1500-mg doses twice daily after treatment in combination with the standard of care (SOC) for 4 weeks. RESULTS: From 32 patients treated with RG7128 monotherapy for 2 weeks, marginal viral load rebound was observed in 3 HCV genotype 1-infected patients, whereas partial response was observed in 2 genotype 1-infected patients. From 85 patients receiving RG7128 in combination with SOC, 1 HCV genotype 1-infected patient experienced a viral rebound, and 2 genotype 3-infected patients experienced a transient rebound. Five genotype 1-infected patients had an HCV load of >1000 IU/mL at the end of 4-week treatment. No viral resistance was observed, per NS5B sequencing and phenotypic studies. PSI-6130 resistance substitution S282T needs to be present at levels of ≥90% within a patient's quasispecies to confer low-level resistance. No evidence of S282T was found by population or clonal sequence analyses. CONCLUSIONS: The requirement for a predominant S282T mutant quasispecies, its low replication capacity, and the low-level resistance it confers probably contribute to the lack of RG7128 resistance observed in HCV-infected patients.

Aza-peptidyl Michael acceptors. A new class of potent and selective inhibitors of asparaginyl endopeptidases (legumains) from evolutionarily diverse pathogens.

Aza-peptide Michael acceptors with the general structure of Cbz-Ala-Ala-AAsn- trans-CH=CHCOR are a new class of inhibitors specific for the asparaginyl endopeptidases (AE) (legumains). Structure-activity relationships (SARs) were characterized for a set of 31 aza-peptide Michael acceptors with AEs derived from three medically important parasites: the protist Trichomonas vaginalis, the hard tick Ixodes ricinus, and the flatworm Schistosoma mansoni. Despite arising from phylogenetically disparate organisms, all three AEs shared a remarkably similar SAR with lowest IC50 values extending into the picomolar range. The results suggest an evolutionary constraint on the topography of the prime side of the active site. SAR also revealed that esters in the P1' position are more potent than disubstituted amides and that monosubstituted amides and alkyl derivatives show little or no inhibition. The preferred P1' residues have aromatic substituents. Aza-asparaginyl Michael acceptors react with thiols, which provides insight into the mechanism of their inhibition of asparaginyl endopeptidases.

Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients.

OBJECTIVES: To characterize the effect of hepatitis C virus (HCV) polymerase intrinsic genetic heterogeneity on the inhibitory activity of nucleoside and non-nucleoside HCV polymerase inhibitors. METHODS: The sensitivity of genotype (GT) 1 HCV NS5B clinical isolates from treatment-naive patients to nucleoside and non-nucleoside polymerase inhibitors was assessed. The genetic diversity at the population level, as well as that of their quasispecies, was correlated with the observed reduced sensitivity to inhibitors. RESULTS: R1479 and NM107 (nucleoside analogues that have entered Phase 2 clinical trials as prodrugs R1626 and NM283, respectively) were similarly active across the tested clinical isolates. Resistance mutations to nucleoside analogues were not observed in any of the isolates. However, the activity of the non-nucleoside thumb II inhibitor NNI-1, palm I inhibitors NNI-2 and NNI-3, and palm II inhibitor HCV-796 was reduced across different isolates. This reduction in inhibitory activity for non-nucleoside inhibitors (NNIs) was, in most cases, correlated with the existence of known NNI resistance mutations in the NS5B polymerase population of the clinical isolates, as detected by population sequencing. Resistance mutations to NNIs were also observed at a low frequency within the clinical isolates' viral quasispecies that allowed for their rapid selection upon drug selective pressure. CONCLUSIONS: The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica.

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.