Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

The Drosophila TNF ortholog eiger is required in the fat body for a robust immune response.

Eiger is the sole TNF family member found in Drosophila melanogaster. This signaling molecule is induced during infection and is required for an appropriate immune response to many microbes; however, little is known about where eiger is produced. Here, we show that eiger is made in the fly's fat body during a Salmonella typhimurium infection. Using tissue-specific knockdown, we found that eiger expression in the fat body is required for all of the phenotypes we observed in eiger null mutant flies. This includes reduced melanization, altered antimicrobial peptide expression and reduced feeding rates. The effect of eiger on feeding rates alone may account for the entire phenotype seen in eiger mutants infected with S. typhimurium.

Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus.

The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2'-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61-109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC(50)) regardless of genotype or subtype. Importantly, we observed a significant correlation (p<0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.

Drosophila eiger mutants are sensitive to extracellular pathogens.

We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger.