CRV431 and CMX157: anti-HBV combination effects in vitro between a cyclophilin inhibitor and a nucleotide prodrug

POSTER PRESENTATIONS Results: Treatment with PX20606 caused a significant induction of miR-144 in uninfected animals ( p = 0.01) but not in HBV infected mice receiving the FXR-agonist. A similar behavior could be observed for miR-451, which is expressed in a cluster with miR-144. Simultaneous hybridization of miR-144 and human-specific mRNA confirmed the enhancement of miR-144 in human hepatocytes in uninfected mice treated with the FXR agonist. Conclusions: Chronic HBV infection hinders FXR-dependent cellular pathways, thus altering the transcription not only of genes involved in bile acid and cholesterol metabolism, but also of FXR-targeted microRNAs. Fluorescence in situ hybridization is a novel tool permitting sensitive and cell specific detection of miRNAs and host gene expression. SAT-180 CRV431 and CMX157: anti-HBV combination effects in vitro between a cyclophilin inhibitor and a nucleotide prodrug R.T. Foster1, D.J. Trepanier1, D.R. Ure1, J. Greytok1, J.L. Kulp2, P. Gallay3. 1 Contravir Pharmaceuticals Inc., Edison; 2Baruch S Blumberg Institute, Doylestown; 3The Scripps Research Institute, San Diego, United States E-mail: [email protected] Background and Aims: It is expected that a cure for HBV will require drug combinations that interact at more than one stage of viral replication and propagation. Our lead drug, CMX157, a tenofovir (TFV) prodrug, is a novel lipid acyclic nucleoside (NUC) phosphonate designed to deliver high intrahepatic concentrations of TFV, while minimizing off-target effects caused by high levels of circulating TFV. CRV431, our earlier-stage molecule, is a host targeting antiviral that inhibits cyclophilins, namely cyclophilin A (cypA), a peptidyl prolyl isomerase. As a cypA inhibitor, CRV431 reduces HBV DNA, suppresses HBsAg, inhibits viral uptake via NTCP and, more recently, has been shown to impede HBx-cypA binding. The aim of the current study was to investigate the combination of CMX157 and CRV431, measuring HBV DNA. Methods: The current study measured inhibition of intracellular HBV DNA at concentrations of CRV431 ranging from 0 to 320 nM alone, and in combination with CMX157 ranging from 0 to 640 nM. Both drugs were tested in vitro in AD38, DE19, and DES19 cells. Studies were each conducted twice, in triplicate, using DMSO as control. Drug concentration versus effect was evaluated using Prichard-Shipman MacSynergy. Additionally, CRV431 cytotoxicity was examined in a number of primary human cells and cell lines, utilizing DMSO and 0.5% saponin as negative and positive controls, respectively, comparing cyclosporine, alisporivir, and sanglifehrin A, to confirm cell viability in the assays. Results: Intracellular HBV DNA inhibition with CRV431 and CMX157, tested in combination with all three cell types, exhibited IC50 values in the low nanomolar range. Synergy scores for CRV431 with CMX157 indicated moderate to strong synergy. Further, cells were viable across all CRV431 concentrations tested, exceeding the viability of alisporivir, cyclosporine, and far exceeding sanglifehrin A. CRV431 exhibited CC50 values of approximately 24 µM. The selective index (SI) was widest for CRV431 compared with that of alisporivir, cyclosporine, and sanglifehrin A. Conclusions: CRV431 and CMX157, tested in combination, represents a viable therapeutic drug strategy for the cure of HBV. This strategy exploits the complementary modes of action of the two drugs, which allows for suppression of HBV DNA, HBsAg, HBeAg, inhibition of viral entry, and blocking of cypclophilin A binding to HBx. The complementary actions of CRV431 with CMX157 may reasonably extend to drugs with other modes of activity including, for example, core inhibitors.

SAT-181 The cyclophilin inhibitor CRV431 prevents both cyclophilin A-HBx complex formation and HBV replication R.T. Foster1, D.R. Ure1, D.J. Trepanier1, P. Gallay2. 1Contravir Pharmaceuticals Inc., Edison, NJ; 2The Scripps Research Institute, La Jolla, CA, United States E-mail: [email protected] Background and Aims: CRV431 is an anti-HBV drug candidate whose principal action is inhibition of cellular cyclophilins. Cyclophilins are peptidyl-prolyl isomerases that broadly regulate protein structure and function, and they are recruited into the life cycles of several viruses, including HBV, HCV, HIV-1, HPV, and coronaviruses. Although previous investigations have highlighted cyclophilin inhibition as a potential anti-HBV strategy, the mechanism(s) of action is largely unknown. One possibility suggested by some studies is that cyclophilins influence HBx-mediated regulation of restriction factors. However, a molecular interaction of cyclophilins and HBx has not yet been explored. The aim of this study is to determine whether CRV431 can demonstrate anti-HBV activity due by investigating the interaction between cyclophilin and HBx. Methods: The current study investigated whether cyclophilin A directly binds HBx, using purified, recombinant GST-human cyclophilin A and His-tagged HBx in pulldown and ELISA assays. Cyclophilin A-HBx binding specificity was tested by site-directed mutagenesis of cyclophilin A (H126Q) to inhibit the isomerase activity of the host protein, and further test the pharmacological disruption of putative cyclophilin A-HBx interaction with CRV431. In addition we explored the role of cyclophilin A in HBV replication in AD38 cells using cyclophilin A knockdown with siRNA and using the cyclophilin inhibitor CRV431. Results: GST-cyclophilin A pulldown assays revealed abundant binding of HBx by Western blot. The interaction was found to be dependent upon the cyclophilin isomerase active site, since the H126Q mutant cyclophilin A that is deficient in isomerase activity failed to bind HBx. CRV431, which interacts with cyclophilin A at its enzymatic active site, dose-dependently blocked cyclophilin A-HBx interaction in both pulldown (IC50 <100 nM) and ELISA (IC50 = 153 nM) assays. HBV replication was inhibited in HepAD38 cells both by cyclophilin A siRNA (IC50 = 42 nM) and CRV431 (IC50 = 31 nM), demonstrating that cyclophilin A is important in HBV replication in this cell type. Conclusions: Ongoing studies are examining the role of cyclophilin A-HBx interactions in HBV in vitro systems and the extent to which its disruption by CRV431contributes to the cyclophilin inhibitor antiHBV activity. By potentially blocking actions of the multifunctional HBx protein, use of CRV431 may represent an important novel approach for treating chronic HBV infection. SAT-182 Hepatitis B virus X RNA is present in cell culture-derived virus preparations and plasma from chronically infected patients R. Beran1, L. Li1, D. Burdette1, D. Ramakrishnan1, S. Lazerwith1, J. Du1, P. Morganelli1, W.E. Delaney, IV 1, S. Fletcher1. 1Gilead Sciences, Foster City, United States E-mail: [email protected] Background and Aims: The structural maintenance of chromosome 5/6 complex (Smc5/6) suppresses transcription from the covalently closed circular DNA (cccDNA) genome of hepatitis B virus (HBV). HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, it is not known how HBx is initially expressed if it is required for cccDNA transcription. We previously demonstrated that HBx RNA can be detected very early after HBV infection of primary human hepatocytes (PHH) and that Smc5/6 is degraded in the majority of cells by the time transcription of viral genes from cccDNA can be detected. In this study we performed RNA-Seq, Northern blot and 5″ RNA Ligase-Mediated

Journal of Hepatology 2017 vol. 66 | S543–S750

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