The Novel Immunosuppressive Protein Kinase C Inhibitor Sotrastaurin Has No Pro-Viral Effects on the Replication Cycle of Hepatitis B or C Virus

The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients

Characterization of the inhibition of hepatitis C virus entry by in vitro-generated and patient-derived oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) has been reported as an inhibitor of hepatitis C virus (HCV) cell entry, making it the only known component of human lipid metabolism with an antiviral effect on HCV. However, several questions remain open, including its effect on full-length cell-culture-grown HCV (HCVcc) of different genotypes or on other steps of the viral replication cycle, its mechanism of action, and whether endogenous oxLDL shares the anti-HCV properties of in vitro-generated oxLDL. We combined molecular virology tools with oxLDL serum measurements in different patient cohorts to address these questions. We found that oxLDL inhibits HCVcc at least as potently as HCV pseudoparticles. There was moderate variation between genotypes, with genotype 4 appearing the most oxLDL sensitive. Intracellular RNA replication and assembly and release of new particles were unaffected. HCV particles entering target cells lost oxLDL sensitivity with time kinetics parallel to anti-SR-BI (scavenger receptor class B type I), but significantly earlier than anti-CD81, suggesting that oxLDL acts by perturbing interaction between HCV and SR-BI. Finally, in chronically HCV-infected individuals, endogenous serum oxLDL levels did not correlate with viral load, but in HCV-negative sera, high endogenous oxLDL had a negative effect on HCV infectivity in vitro. Conclusion: oxLDL is a potent pangenotype HCV entry inhibitor that maintains its activity in the context of human serum and targets an early step of HCV entry

Combination of sotrastaurin with calcineurin inhibitors.

<p>(A, B) Effect of different concentrations of sotrastaurin plus (A) CsA or (B) tacrolimus on HCV Jc1-Luc replication at 48 h after electroporation.</p

Effects of sotrastaurin on the HBV lifecycle.

<p>(A) Differentiated HepaRG cells were infected with HBV virions. Sotrastaurin 5 or 10 µg/ml was present either during inoculation (initial 16 h), during inoculation and infection (throughout), or after inoculation (from 16 h onwards). An inhibitory fragment from the HBV L-protein (HBV pre-S/2-48my) applied during inoculation served as a positive control. All reactions were adjusted to contain the same final concentration of DMSO. HBeAg levels secreted into the cell culture supernatant were determined at day 9 after infection. (B) Cytotoxic effect of sotrastaurin on differentiated HepaRG cells as measured by and LDH release assay. (C) HBV antigens released from AD38 harboring a replicating HBV genome after 72 hours of sotrastaurin treatment. IU – international units; S/CO - signal to cutoff ratio. (D) AD38 cells were treated with increasing amounts of sotrastaurin. After 72 h cells were stained with a live/dead cell stain kit and analyzed by FACS.</p

Effect on early HCV replication cycle stages.

<p>(A) Infection of Huh-7.5 cells with Jc1-Luc produced in the absence of sotrastaurin. The drug was added for at least 4 h during infection. (B) Strain H77 HCVpp infection of Huh-7.5 cells in the presence or absence of sotrastaurin. Pseudoparticles bearing the glycoprotein of the vesicular stomatitis virus (VSVG) served as controls. (C) Pseudoparticle infection of Huh-7.5 cells in the presence of increasing concentrations of BIM-I. pcDNA represents pseudoparticles devoid of viral glycoproteins.</p

Sotrastaurin does not affect cell-to-cell spread of HCV.

<p>(A) HCV-positive donor cells were mixed with HCV-negative target cells under an agarose overlay preventing cell-free virus spread in the presence or absence of sotrastaurin. Target cells contained a tagRFP-reporter that assumes a cytosolic or nuclear localization depending whether the cells are uninfected or infected with HCV. After 96 hours of co-culture nuclei were co-stained with DAPI. (B) Percentage of infected target cells after 96 hours of co-culture.</p