Development and evaluation of a FACS-based medium-throughput assay for HCV entry inhibitors.

The interaction between the hepatitis C virus (HCV) envelope glycoprotein E2 and the human tetraspanin protein CD81 is one of the key events involved in HCV cell entry. Therefore, compounds that interfere with this interaction may be useful tools for basic research and potential drugs for the treatment of HCV infection. The authors describe a medium-throughput assay for ligands of the E2 binding site on the CD81 receptor. In the assay, human hepatoma cells are incubated with the test compounds and stained with a fluorescently labeled anti-CD81 antibody (JS81). Flow cytometry is used to detect the level of bound antibody, reflecting the inhibitory potencies of the compounds. Eighty percent of compounds active in the assay show efficacy in an infection assay using luciferase reporter genome in cell culture. Thus, the assay can be used as a fast screening system for inhibitors of interaction of viral E2 to host cell CD81-LELs.

Identification of terfenadine as an inhibitor of human CD81-receptor HCV-E2 interaction: synthesis and structure optimization.

Terfenadine (4-[4-(hydroxydiphenylmethyl)-1-piperidyl]-1-(4-tert-butylphenyl)-butan-1-ol) was identified in a biological screening to be a moderate inhibitor (27% inhibition) of the CD81-LEL-HCV-E2 interaction. To increase the observed biological activity, 63 terfenadine derivates were synthesized via microwave assisted nucleophilic substitution. The prepared compounds were tested for their inhibitory potency by means ofa fluorescence labeled antibody assay using HUH7.5 cells. Distinct structure-activity relationships could be derived. Optimization was successful, leading to 3g, identified as the most potent compound (69 % inhibition). Experiments with viral particles revealed that there might be additional HCV infection reducing mechanisms.

Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.

BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.