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

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