studies

696A AASLD ABSTRACTS HEPATOLOGY, October, 2015
993
Analysis of AL-516, a Novel Potent Nucleotide Analog,
Combination Drug Interactions in the Hepatitis C Virus
(HCV) Subgenomic Replicon System
Hua Tan, Kenneth Shaw, vivek Rajwanshi, Gary Wang, Leo Beigelman,
David B. Smith, Lawrence M. Blatt, Julian A. Symons;
Alios, South San Francisco, CA
Background: Nucleotide analogs have emerged as an important
component of interferon (IFN)-free combination therapies
for the treatment of chronic hepatitis C (CHC) based on their
potent activity and high barrier to the generation of viral resistance.
AL-516, a novel monophosphate prodrug of a guanosine-based
nucleotide analog, has been identified as a potent
and selective inhibitor of NS5B-directed HCV RNA replication
in the cell based replicon system. In this study, inhibition of the
HCV replicon by AL-516 was examined in pairwise combinations
with multiple compounds either registered for the treatment
of CHC or currently in clinical development. Methods: Studies
were performed using a Huh-7 cell line expressing a Firefly
luciferase-encoding HCV 1b subgenomic replicon. Compounds
were added to cells in a checkerboard fashion and inhibition
of HCV replication measured by luminescence. Data were analyzed
using two drug interaction models; Isobologram analysis
using the Loewe additivity model and the Bliss-Independence
model using Pritchard’s MacSynergy II software. Results: In
the HCV 1b replicon, AL-516 exhibits potent antiviral activity
with an EC 50
of 6.5 nM. When tested in pairwise combinations
with representative members of the NS3/4A protease
inhibitors, NS5A inhibitors, nucleoside/tide and non-nucleoside
polymerase inhibitors, cyclophilin A inhibitors, type I and
type III IFN’s or RBV, AL-516 demonstrated significant synergy
(>100 mM2%), synergy (25-100 mM2%) or additivity (0-25
mM2%), no antagonistic effects were observed. For approved
compounds, combination of AL-516 with the NS3/4A protease
inhibitor, simeprevir, exhibited significant synergy with
a synergy volume of 114.1 mM2%. AL-516 also exhibited
significant synergistic interactions with the HCV NS5A inhibitor,
daclatasvir, (117.1 mM2%) and additive-to-synergistic
interactions with the investigational uridine-based nucleoside
polymerase inhibitor, AL-335, (33.5 mM2%). Conclusions:
Future IFN-free therapy for CHC will require a combination
of compounds with different mechanisms of action. AL-516
demonstrates an in vitro antiviral profile that suggests it may
become an important component of IFN-free combination therapy.
To this end, AL-516 is currently advancing towards human
clinical trials for CHC.
Disclosures:
Kenneth Shaw - Employment: Alios Biopharma
David B. Smith - Employment: Alios BioPharma
Lawrence M. Blatt - Management Position: Alios BioPharma
Julian A. Symons - Employment: Alios BioPharma
The following authors have nothing to disclose: Hua Tan, vivek Rajwanshi, Gary
Wang, Leo Beigelman
994
Regulation of Hepatitis C Virus Infection by Long
Non-Coding RNAs
Tetsuro Shimakami, Masao Honda, Takayoshi Shirasaki, Fanwei
Liu, Masaya Funaki, Kazuhisa Murai, Shuichi Kaneko; Kanazawa
University, Kanazawa, Japan
Background Recently, large numbers of non-coding RNAs
have been identified that reportedly have a wide range of
functions. Non-coding RNAs are categorized into two groups:
the first is composed of short non-coding RNAs, such as siRNA
and miRNA, and the second contains long non-coding RNAs,
which are generally more than 200 ntds in length and possess
a 5ʹ cap and 3ʹ poly-A tail. The role of short non-coding RNAs
in hepatitis C virus (HCV) infection, such as miR-122, has been
characterized considerably more than that of long non-coding
RNAs. Here, we studied the role of long non-coding RNAs
in HCV infection. Method To identify long non-coding RNAs
whose expression is specifically altered by HCV infection,
Huh-7.5 cells were infected with the HJ3-5 virus, which is a
Ia/IIa chimera. Total cellular RNAs were extracted, followed
by amplification of only poly-A tail RNAs, and subjected to
sequence analysis using a next-generation sequencer. We
selected 26 candidate long non-coding RNAs whose expression
was specifically and significantly increased by HCV infection.
Next, we designed 2–3 siRNAs for each candidate and
tested the effect of knockdown of each long non-coding RNA in
HCV cell culture. Result The knockdown of 4 long non-coding
RNAs significantly suppressed HCV replication. From these,
we focused on lncRNA-H, which has already been registered
in the lncRNA database and whose expression in human livers
has been confirmed by several reports; however, its role in
HCV infection remains unknown. The expression of lncRNA-H
was induced by HCV infection in a multiplicity of infection-dependent
manner in HCV cell culture, and NS5A seemed to be
the inducer of lncRNA-H expression. This induction was also
observed in an HCV-infected chimpanzee as well as HCV-infected
chimeric mice. Furthermore, when we compared the
expressions of lncRNA-H in human liver between pre- and
post-eradication of HCV, the expression of lncRNA-H was significantly
decreased by the eradication of HCV. The knockdown
of lncRNA-H by siRNAs suppressed HCV replication
as well as infectious virus production, in accordance with the
knockdown of lncRNA-H. This effect seemed to be through the
suppression of HCV-IRES-dependent translation. Furthermore,
we compared the amount of lncRNA-H in HCV-infected human
livers and serums derived from IL28B genotype (rs8099917)
major and minor allele patients. Interestingly, the amount of
lncRNA-H from minor allele patients was significantly higher
than in patients with the major both in livers and serums. Discussion
These results suggest that lncRNA-H could be a new
target for the treatment of HCV infection as well as a new
biomarker for interferon-based therapies.
Disclosures:
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Tetsuro Shimakami, Masao
Honda, Takayoshi Shirasaki, Fanwei Liu, Masaya Funaki, Kazuhisa Murai
995
Iron chelation restores mitophagy suppressed by hepatitis
C virus
Yuichi Hara 1 , Sohji Nishina 1 , Tasuku Hirayama 2 , Hideko Nagasawa
2 , Keisuke Hino 1 ; 1 Kawasaki Medical College, Kurashiki,
Japan; 2 Gifu Pharmaceutical University, Gifu, Japan
Background and aim: Oxidative stress is present in chronic
hepatitis C to a greater degree than in other inflammatory
liver disease and closely related to disease progression. We
recently reported that hepatitis C virus (HCV) core protein
suppresses mitophagy by interacting with Parkin (Am J Pathol
2014), which may amplify and sustain HCV-induced oxidative
stress. Therefore, the restoration of mitophagy is a critical therapeutic
intervention for preventing disease progression including
liver cancer development in chronic hepatitis C. On the other