Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor

Journal Pre-proof Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor Eunji Jo, Dong-Kyun Ryu, Alexander König, Soonju Park, Yoojin Cho, Sang-Hyun Park, Tae-Hee Kim, Seung Kew Yoon, Wang-Shick Ryu, Jonathan Cechetto, Marc P. Windisch PII:

S0166-3542(19)30603-5

DOI:

https://doi.org/10.1016/j.antiviral.2020.104709

Reference:

AVR 104709

To appear in:

Antiviral Research

Received Date: 22 October 2019 Revised Date:

3 January 2020

Accepted Date: 9 January 2020

Please cite this article as: Jo, E., Ryu, D.-K., König, A., Park, S., Cho, Y., Park, S.-H., Kim, T.-H., Yoon, S.K., Ryu, W.-S., Cechetto, J., Windisch, M.P., Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor, Antiviral Research (2020), doi: https://doi.org/10.1016/ j.antiviral.2020.104709. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Identification and Characterization of a Novel Hepatitis B Virus Pregenomic RNA Encapsidation Inhibitor Eunji Joa,#, Dong-Kyun Ryua,1,#, Alexander Königa, Soonju Parkb, Yoojin Choa, Sang-Hyun Parka, TaeHee Kimb, Seung Kew Yoonc, Wang-Shick Ryud,2, Jonathan Cechettob, and Marc P. Windischa,e,* a

Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea b Screening Discovery Platform, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnamsi, Gyeonggi-do, South Korea c Catholic University Liver Research Center, The Catholic University of Korea, Seoul, South Korea d Department of Biochemistry, Yonsei University, Seoul, South Korea e Division of Bio-Medical Science and Technology, University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, South Korea 1 Present address: Celltrion, Inc., Incheon, South Korea 2 Present address: Institut Pasteur Korea #

These authors contributed equally to this research.

*

Corresponding author:

Marc P. Windisch, PhD Applied Molecular Virology Laboratory Discovery Biology Department Institut Pasteur Korea Gyeonggi-do, 463-400, South Korea Phone: +82-31-8018-8180 Fax: +82-31-8018-8014 Email: [email protected]

Abbreviations: hepatitis B virus (HBV), high-throughput screening (HTS), polymerase (Pol), epsilon (ε), pregenomic RNA (pgRNA), (Z)-2-(allylamino)-4-amino-N’-cyanothiazole-5-carboximidamide (AACC), dose-response curve (DRC), cell culture-derived HBV (HBVcc), patient-derived HBV (HBVpt), genotype (gt), primary human hepatocytes (PHHs), sodium taurocholate transporting polypeptide (NTCP)

Abstract Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5' epsilon (ε) stem-loop structure, identifying (Z)-2(allylamino)-4-amino-N’-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB. Keywords: hepatitis B virus, high-throughput drug screening, pregenomic RNA, epsilon signal, encapsidation, polymerase

1. Introduction Hepatitis B virus (HBV) has chronically infected more than 350 million individuals worldwide (World Health Organization, 2017). Although various vaccines and medications are available, chronic hepatitis B (CHB) infection is still a major cause of liver diseases such as cirrhosis and hepatocellular carcinoma. At present, there are two classes of FDA-approved therapeutic strategies to treat CHB. The immunomodulator interferon-alpha (IFN-α) has limited antiviral efficacy and severe side effects. As direct-acting antivirals, nucleos(t)ide analogs (NAs) are administered to inhibit viral reverse transcriptase (RT), thereby preventing HBV replication and resulting in viral load suppression. Neither strategy is curative and prolonged therapy is a risk for the development of drug resistance and adverse effects. To increase the cure rate of CHB and shorten treatment duration with fewer side effects, novel anti-HBV drugs targeting distinct viral or cellular factors involved in the HBV life cycle are urgently needed. The HBV pregenomic RNA (pgRNA) plays a pivotal and versatile role within the viral life cycle; pgRNA serves as a template for the translation of both the HBV polymerase (Pol) and core protein. Subsequently, HBV Pol uses its own pgRNA template to replicate the viral DNA genome through a unique mechanism of reverse transcription. The pgRNA 5' end-located epsilon (ε) RNA stem-loop structure, which is recognized by HBV Pol, plays a crucial role in the initiation of replication. The binding of HBV Pol to ε triggers HBV core protein subunit assembly and encapsidation of the complex, and mediates protein priming to synthesize minus-strand DNA (Seeger and Mason, 2000). Several reports have suggested that HBV Pol plays a pivotal role as a molecular switch from pgRNA translation to replication and encapsidation, demonstrating that HBV Pol suppresses pgRNA translation and thereby facilitates HBV genome packaging into capsids (Ryu et al., 2008)(Ryu et al., 2010). Within the nucleocapsid, HBV Pol carries out multiple enzymatic functions: reverse transcription of minus-strand DNA from the plus-strand pgRNA template, degradation of pgRNA via its RNaseH domain, and synthesis of a DNA plus-strand, resulting in a partially doublestranded HBV genome (Tavis et al., 2013) (Bartenschlager et al., 1990; Junker-Niepmann et al., 1990; Nassal and Rieger, 1996). As small molecules are highly attractive drug targets, numerous studies of novel small molecules that

target viral replication using different modes of action (MoA) have been reported. For instance, core protein allosteric modulators (CpAMs), such as AT-61 (King et al., 1999), AT-130 (Delaney et al., 2002; Feld et al., 2007), Bay41-4109 (Berke et al., 2017; Finn et al., 2005), and NVR3-778 (Lam et al., 2018), regulate nucleocapsid assembly and/or disassembly, thereby suppressing viral replication. Another new class of inhibitors are N-hydroxyisoquinoline-diones (Edwards et al., 2017), which target the HBV Pol RNaseH domain, preventing the degradation of pgRNA and terminating plusstrand DNA synthesis. In addition, it was reported that hemin blocks the duck hepatitis B virus (DHBV) ε-Pol interaction and priming activity by directly targeting DHBV Pol (Lin and Hu, 2008). Recently, rosmarinic acid was shown to specifically inhibit the binding of ε-RNA and HBV Pol in vitro, resulting in the suppression of viral replication (Tsukamoto et al., 2018). In this study, we devised and applied a high-throughput screening (HTS) assay to identify chemicals that inhibit the HBV ε-Pol interaction. We also describe the in vitro characterization of a novel antiHBV inhibitor class (Z)-2-(allylamino)-4-amino-N’-cyanothiazole-5-carboximidamide (AACC) using physiologically relevant in vitro assay systems.

2. Materials and Methods 2.1. Plasmids The epsilon-luciferase (ε-Luc) reporter plasmid and HBV Pol-overexpressing plasmid were provided by Dr. Ryu (Yonsei University, South Korea) (Ryu et al., 2008). Full-length HBV plasmids with wildtype (WT) genotype (gt) B and gtD (1.3-mer ayw) were provided by Drs. Glebe (Giessen University, Germany) and Ryu, respectively. The WT gtC and TDF/LMV-resistant plasmids were provided by Dr. Kim (Konkuk University, South Korea) (Park et al., 2019). CpAM-resistant plasmids were constructed by inserting previously reported mutations into gtD 1.3-mer ayw (Zhou et al., 2017).

2.2. Cell culture and transfection HEK293T and HepG2 cells were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium (Welgene, Gyeongsangbuk-do, South Korea) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and penicillin-streptomycin (Gibco, Waltham, MA, USA). HepG2.2.15 and HepAD38 cells were kindly provided by Dr. Seeger (Fox Chase Center, USA). HepG2-NTCPsec+ cells were generated and cultured, as previously described (König et al., 2019). For transfection of HEK293T and HepG2 cells, polyethylenimine (Sigma-Aldrich, St Louis, MO, USA) and X-tremeGENE HP (Roche Applied Science, Penzberg, Germany) were used, respectively.

2.3. Inhibitors The inhibitors used were as follows: 17-DMAG [heat shock protein 90 (hsp90) inhibitor; InvivoGen, San Diego, CA, USA], myrcludex B (MyrB; PSL GmbH, Heidelberg, Germany), Bay41-4109 (Medchem Express, NJ, USA), lamivudine (LMV, Selleckchem, Houston, TX, USA), and tenofovir (TDF; Selleckchem).

2.4. Screening of compound libraries using the HBV ε-Pol assay and hit confirmation In-house compound libraries, composed of 110,000 small molecules from TimTec, ChemBridge, ChemDiv, Cerep, Prestwick, etc. were used for screening. HEK293T cells were seeded into a T175 flask and co-transfected with 4.5 µg ε-Luc and 18 µg HBV Pol using PEI at a 1:2 ratio. At 16 h posttransfection (hpt), 5 × 103 cells were transferred into 384-well plates using an automatic microplate dispenser (Matrix Wellmate; Thermo Scientific, Waltham, MA, USA). Prior to cell dispensing, compounds were added to the plates using an automated liquid handler (Hummingbird; Digilab, USA) to achieve a final concentration of 10 µM. At 40 hpt, the Bright-Glo reagent (Promega, Madison, WI, USA) was added using an automated laboratory workstation (cell:explorer; PerkinElmer, Waltham, MA, USA); after a 10-min incubation, luciferase activity was analyzed using an EnVision Multilabel Plate Reader (PerkinElmer). Hits were confirmed using a 10-point dose-response curve (DRC) analysis of 2-fold serially diluted compounds, starting at a concentration of 20 µM.

2.4. Cell viability assay To measure cell viability in the presence of the compounds, cells were prepared in two independent plates for every experiment (one plate to determine activity and the other to determine cell viability). CellTiter-Glo (Promega) was added to the cells, and the resulting signal, which represents cellular ATP levels, was measured using the Victor III Multilabel Plate Reader (PerkinElmer).

2.5. Nucleocapsid-captured qRT-PCR encapsidation assay Nucleocapsid-captured qRT-PCR was performed as previously described (Ryu et al., 2015). Briefly, after lysis of HepG2.2.15 cells, the supernatants were clarified by centrifugation and transferred to 96well capture plates coated with anti-HBcAg antibodies. The pgRNA of the captured nucleocapsids was further purified using the CellAmp Direct RNA Prep Kit (Takara Bio Inc., Shiga, Japan) in the

presence of DNase I and proteinase K (Takara Bio Inc.), and analyzed by qRT-PCR.

2.6. HBV particle gel assay and Western blot analysis HBV core particles were analyzed as previously described (Yan et al., 2017). Briefly, the cell lysates were electrophoresed through a 1% native agarose gel and transferred onto a nylon membrane, and the membrane was soaked in 2.5% formaldehyde and 50% methanol. Membranes were incubated first with anti-HBcAg antibodies (Dako, Santa Clara, USA) and then with horseradish peroxidaseconjugated antibodies. HBV capsids were visualized using the ECL Western blot detection system (Amersham, Buckinghamshire, UK). For Western blot analysis, cell lysates were clarified and mixed with loading buffer (3M Applied Science, St. Paul, Minnesota, USA) and subjected to SDS-PAGE. After incubation with anti-HBcAg or anti-tubulin antibodies (Abcam, Cambridge, UK), proteins were visualized as described above. To measure capsid-associated DNA levels, HBV core particles on the membrane were denatured and hybridized to a DIG-labeled HBV probe and detected using a DIG Luminescent Detection kit (Roche Applied Science, Penzberg, Germany).

2.7. HBV DNA analysis by qPCR and Southern blot Cell lysates and culture supernatants were incubated with proteinase K buffer (100 mM Tris-HCl, pH 8.5, 2 mM EDTA, 1% SDS, and 400 µg/mL proteinase K) at 56°C for 3 h, followed by inactivation at 70°C for 10 min. HBV DNA was analyzed by qPCR using Premix Ex Taq (Takara Bio Inc.) containing 5% Tween-20 to neutralize the SDS. Probe and primer sequences are as follows: forward primer for gtC and gtD: 5'-ACTCACCAACCTCTTGTCCT-3', forward primer for gtB: 5'ACTCACCAACCTGTTGTCCT-3', reverse primer: 5'-GACAAACGGGCAACATACCT-3', and probe: 5'-FAM-TATCGCTGGATGTGTCTGCGGCGT-TAMRA-3' (Liu et al., 2007). For the Southern blot assay, HBV DNA was extracted from HepG2.2.15 cells and subjected to agarose gel electrophoresis. Following membrane transfer, the DNA was hybridized to a P32-labeled HBV probe

and visualized using a phosphoimager as previously described (Ko et al., 2014).

2.8. HBV infection and progeny passaging assay HBV infection and a progeny passaging assay were performed as previously described (König et al., 2019) with minor modifications. Briefly, HepG2-NTCPsec+ cells were inoculated with 5,000 GEq/cell of HepAD38-derived HBV (cell culture-derived HBV or HBVcc; gtD) and treated with various compounds (p1 infection). At 7 days postinfection (dpi), cell culture supernatants were transferred to naïve HepG2-NTCPsec+ cells in the presence of 4% PEG8000 and incubated for another 7 days (p2 infection). At 7 dpi, cells were fixed, and HBV infection was monitored by immunofluorescence analysis (IFA) using anti-HBcAg antibodies. HepG2-NTCPsec+ cells were infected with 5 µL crude serum from a CHB patient (gtC) and treated with compounds once per week up to 4-weeks postinfection (wpi). At 4 wpi, HBV DNAs extracted from the cell lysates and culture supernatants were analyzed by qPCR.

2.9. Primary human hepatocyte (PHH) infection Infection of PHHs (Yecuris, Tualatin, OR, USA) was performed as previously described. Briefly, 2 days after seeding the cryopreserved PHHs in collagen-coated plates, the cells were inoculated with 5,000 GEq/cell of HBVcc in the presence of 4% PEG. Inocula were washed at 1 dpi, and compounds were added to the cells at 3-day intervals until 15 dpi. The culture supernatants were harvested at 11 and 15 dpi, and secreted viral DNA was analyzed by qPCR. Cell viability was measured as described above.

2.10. Drug-drug combination assay To assess the effects of drug combinations, HepG2.2.15 cells were treated with AACC individually or

in combination with DAAs, including LMV, TDF, and Bay41-4109, every 3 days for 9 days. Selected concentrations of compounds for treatment were as follows: 8-fold EC50, 4-fold EC50, 2-fold EC50, EC50, 0.5-fold EC50, 0.25-fold EC50, and 0.125-fold EC50. Supernatants were collected, and HBV DNA was extracted and measured using qPCR as described above. Data were analyzed using CompuSyn software (CompuSyn, Inc., Lake Geneva, WI, USA). The combination index (CI) was determined as previously described (Chou, 2006).

2.11. Statistical analysis The quality of the HTS performed was calculated using the percent coefficient of variation (%CV) and Z´ factor (which evaluates the robustness of the HTS: 0.5 ≤ Z < 1 indicates a high-quality assay). All of the experiments were performed at least twice, and data are presented as the mean ± standard deviation (SD) by normalizing the values of the inhibitors to the value of a control for each experiment. Median effective and cytotoxic concentrations (EC50 and CC50, respectively) were calculated using Prism 6.0 software (GraphPad, Inc., La Jolla, CA, USA). Statistical significance was determined using the Student’s t test.

3. Results 3.1. Development of an HTS assay to identify inhibitors of the HBV ε-Pol interaction To identify novel inhibitors disrupting the ε-Pol interaction, we devised an HTS assay, as shown in Figure 1A. HEK293T cells were co-transfected with HBV Pol and ε-Luc reporter plasmids. After the expression of HBV Pol, the protein binds to the ε mRNA structure (ε-Pol interaction), thereby terminating the translation of the luciferase mRNA template and suppressing luminescence (Luc activity, inactive state). However, we hypothesized that certain compounds could interrupt the HBV εPol interaction, restoring the translation of luciferase (active state). As a proof of concept, to demonstrate that HBV Pol will suppress luminescence, we transfected increasing amounts of the HBV Pol expression plasmid and observed a dose-dependent reduction in luciferase activity (Fig. 1B). We also transfected a Renilla reporter construct, which lacks an upstream ε-structure, to monitor general protein translation; no inhibition of luminescence was observed except at the highest HBV Pol levels, indicating a highly specific ε-Pol interaction with good assay window (fold-difference) of 7.6 at a 1:4 ε-Luc:HBV Pol plasmid ratio. Applying this strategy, we developed a fully automated HTS assay for the identification of small molecules that could inhibit the ε-Pol interaction; we validated the HTS 2times independently, by determining separate luciferase counts of either ε-Luc- or ε-Luc + HBV Poltransfected cells (Fig. 1C). By quantifying luminescence in three control 384-well microtiter plates that contained cells transfected with either ε-Luc or ε-Luc + HBV Pol and then treated 0.5% DMSO (v/v), and six control plates containing cells transfected with ε-Luc + HBV Pol and treated with 0.5% DMSO (v/v), single ε-Luc-transfected cells were clearly separated from ε-Luc + HBV Pol-transfected cells. The robustness and reproducibility of the assay were confirmed by an R2 value of 0.98, a 9– 14%CV, an average assay window of 9- to 12-fold, and a Z´ factor of 0.5–0.6 (Table 1).

3.2. Screening campaign and hit confirmation of HBV ε-Pol interaction inhibitors After the HTS assay validation, we screened compound libraries composed of approximately 110,000 small molecules at a single and final concentration of 10 µM. Applying low stringency hit selection criteria, 450 primary hits were identified, according to a statistical cut-off with compounds increasing ε-Luc activity by more than 130% compared to 0.5% DMSO (v/v), which was set as 100% (Fig. 2A). Next, the 450 hits were retested in a 10-point DRC analysis, and 32 compounds were confirmed to increase ε-Luc activity in a dose-dependent manner. The ε-Luc activities of the four highest concentrations are presented in Table 2. Decreased ε-Luc activity at high concentrations of the compounds might be due to the cytotoxicity of the compounds. We then aimed to verify the antiviral efficacies of the 32 hits using stable HBV-replicating hepatoma cells (HepG2.2.15), which mimic authentic HBV replication. DRC analysis was used to calculate the median effective and cytotoxic concentrations (EC50 and CC50) as well as the selectivity index (SI; CC50/EC50) (Table 2, right). The SI values of the 31 compounds were less than 5, which were considered inactive; only compound 1, a (Z)-2-(allylamino)-4-amino-N’-cyanothiazole-5-carboximidamide (AACC) (Fig. 2B), efficiently reduced secreted HBV DNA level (SI 5.24). Next, we sought to confirm that AACC targets the ε-Pol interaction in HepG2 cells, the most commonly used hepatoma cell line. As a result, we confirmed that AACC increased ε-Luc activity in a dose-dependent manner, but only in the presence of HBV Pol, indicating that AACC specifically restored ε-Luc activity that was abolished by HBV Pol binding to ε (Fig. 2C, left). As a negative control, LMV, an RT inhibitor, was not found to interfere with the ε-Pol interaction (Fig. 2C, middle). Interestingly, 17-DMAG, a geldanamycin derivative that was previously shown to interfere with the HBV ε-Pol interaction by disrupting hsp90, increased ε-Luc activity in both the presence and absence of HBV Pol, indicating possible off-target effects (Fig. 2C, right) (Hu et al., 2004).

3.3. AACC inhibits HBV replication by preventing pgRNA encapsidation Having shown that AACC interrupts the ε-Pol interaction and prevents the secretion of HBV genomes,

we next investigated the MoA of AACC in the viral life cycle. As the ε-Pol interaction is part of a signaling complex that triggers HBV core protein subunit assembly and the formation of pgRNAcontaining nucleocapsids (encapsidation), we investigated whether AACC could inhibit the encapsidation of HBV pgRNA using a nucleocapsid-captured quantitative RT-PCR encapsidation assay, as described by (Ryu et al., 2015). The encapsidated pgRNA of HBV capsids was analyzed by qRT-PCR. Results showed that AACC reduced the levels of nucleocapsid-associated pgRNA in a dose-dependent manner, approaching a 90% reduction at the highest non-cytotoxic concentration of 5 µM; AACC was determined to have an EC50 of 0.83 µM and a CC50 of 9.3 µM (SI 11.2) (Fig. 3A). To determine whether AACC interferes with the assembly of HBV capsids, we performed an HBV particle gel assay. AACC inhibited capsid formation and capsid-associated DNA in a dose-dependent manner without reducing either total core protein or cellular tubulin expression (Fig. 3B). In contrast, Bay41-4109, an HBV CpAM, inhibited capsid formation and reduced total core protein levels. As expected, LMV exclusively inhibited the synthesis of capsid DNA, but did not interfere with either HBV capsid formation or core protein level. Taken together, these data demonstrate that AACC inhibits pgRNA encapsidation, HBV capsid formation, and viral replication. In addition, we confirmed that AACC in HepG2.2.15 cells had EC50s of 0.74 µM and 0.48 µM for the reduction of intra- and extracellular HBV DNA, respectively, a CC50 of 7.2 µM, and an SI of 9–15 (Fig. 3C). A reduction in viral replication at 1 µM of AACC was further corroborated by Southern blot analysis, which demonstrated a 77% reduction in rcDNA levels, whereas 10 µM LMV inhibited rcDNA levels by 96% (Fig. 3D). Next, we demonstrated that AACC had cross-activity in various HBV genotypes. HepG2 cells transiently transfected with replicating HBV plasmids containing gtB, gtC, or gtD were treated with 0.5 or 2 µM AACC, or with reference inhibitors (LMV and TDF), and intracellular HBV DNA levels were quantified by qPCR. Importantly, AACC inhibited viral replication of all tested HBV genotypes; however, the maximum inhibition varied among these genotypes. GtD was the most susceptible to AACC [gtD (~90% inhibition) > gtB (70%) > gtC (40%)] (Fig. 3E).

3.4. AACC inhibits propagation of HBVcc and patient-derived HBV (HBVpt) in infected HepG2-NTCPsec+ cells and PHHs To investigate whether AACC is active in infectious HBV cell culture systems, which mimic authentic viral infection and enable the monitoring of the entire HBV life cycle, we utilized HepG2-NTCPsec+ cells infected with HepAD38-derived HBV (König et al., 2019). The experimental scheme is shown in Figure 4A. During p1 infection, the early HBV life cycle (entry to viral translation) can be monitored, whereas during p2 infection, cccDNA-driven viral replication and secretion of progeny can be assessed after supernatant passage to naïve cells. In the absence of inhibitors, IFA of the viral core protein (HBc) revealed that approximately 51% and 43% of p1 and p2 cells, respectively, were HBV positive (Fig. 4B, top). As controls, MyrB, an HBV entry inhibitor, almost completely prevented p1 and p2 infections, whereas replication inhibitor LMV exclusively inhibited p2 infection by 98% (Fig. 4B, middle), indicating that p2 infection is dependent on newly produced viral progeny in the supernatant from p1-infected cells. AACC, at a non-cytotoxic concentration of 4 µM, was exclusively active in p2 cells, inhibiting HBV infection by 80% (Fig. 4B, bottom). DRC analysis in p2 cells revealed that AACC had an EC50 of 0.81 µM and an SI of 8.0, which are consistent with the values obtained in HepG2.2.15 cells (Fig. 4C, bottom). EC50, CC50, and SI values obtained for MyrB and LMV were similar to previously reported values (König et al., 2019) (Fig. 4C, top and middle). Importantly, no inhibitory effects were observed when AACC was added to hepatitis C virus (HCV)or human immunodeficiency viruses (HIV)-infected cells, demonstrating the specificity of AACC for HBV (Supplementary Fig. 1). To further confirm the antiviral effects of AACC in a more natural setting, HepG2-NTCPsec+ cells were infected with HBVpt (gtC); treated with AACC, MyrB, or TDF; and harvested at 4 wpi, as shown in Figure 5A. Intra- and extracellular HBV DNA was extracted from cell lysates and culture supernatants, and analyzed by qPCR. As expected, MyrB (entry inhibitor) treatment efficiently prevented de novo infection of HBVpt, reducing intra- and extracellular HBV DNA levels by 28- and 13-fold, respectively (Fig. 5B). Treatment with TDF, an RT inhibitor, reduced intra- and extracellular HBV DNA by up to 60- and 16-fold, respectively. At 4 µM, AACC reduced intra- and extracellular

HBV DNA by up to 31- and 17-fold, respectively in a dose-dependent manner. To confirm the inhibitory effects of AACC on HBV replication in a more physiologically relevant in vitro system, we infected PHHs with HBVcc, physiologically the most relevant infection system for HBV (Fig. 5C). HBV secreted from infected PHHs at 11 and 15 dpi were harvested, and DNA levels were analyzed by qPCR. The results showed that LMV, TDF, and AACC inhibited the secreted HBV genome to the same extent without significant cytotoxicity. Over time, the GEq/mL of HBV increased by two-fold, indicating the generation of viral progeny (Fig. 5D).

3.5. AACC inhibits replication of nucleoside- and capsid inhibitor-resistant HBV To further investigate the mechanistically distinct properties of AACC, as compared to NAs and CpAMs, we evaluated the activity of AACC on LMV- and CpAM-resistant (RS) HBV variants. HepG2 cells were transfected with these resistant replicons or WT HBV, and viral replication was assessed by qPCR. As controls, LMV and Bay41-4109 significantly inhibited WT HBV replication; however, the antiviral activities of LMV and Bay41-4109 were abrogated when used to treat LMV-RS and CpAM-RS, respectively (Fig. 6). In contrast, AACC inhibited the replication of LMV-RS and CpAM-RS HBV variants by 50%, which was comparable to the inhibition of WT. These data clearly indicate a novel MoA of AACC that is distinct from HBV NAs and CpAMs.

3.6. AACC is synergistic with NAs and a CpAM Antiviral agents that act on different targets are often used in combination to improve clinical outcomes. Therefore, to demonstrate that AACC could complement existing HBV therapies, we performed an in vitro drug-drug combination study. Antiviral activities of two selected inhibitor classes, NAs (LMV and TDF) and CpAM (Bay41-4109), in combination with AACC, were evaluated. HepG2.2.15 cells were treated with various concentrations of LMV, TDF, and Bay41-4109 either alone or in combination with serially diluted AACC for 9 days, followed by the determination of HBV

DNA levels in the supernatant by qPCR and the calculation of the combination index (CI) (Chou, 2006). AACC showed considerable synergy with all tested drugs, as indicated by the CI values (< 1), suggesting that AACC is a suitable alternative drug that could be used in combination therapy with approved DAAs (Table 3).

4. Discussion The development of anti-HBV drugs has mainly focused on the HBV Pol RT, with the successful clinical use of five NAs that inhibit DNA synthesis as well as the immune modulator PEG-IFN-α. However, none of these treatments is curative in most cases, and life-long treatment increases the risk for the development of drug resistance and severe side effects. To improve current therapies for CHB patients, new therapeutic options with different MoAs are required to either replace or be added to HBV therapies. Current therapies using NAs are already targeting the RT activity of HBV Pol; however, as a multifunctional protein, other domains of HBV Pol are also attractive drug targets. Besides its RT function, HBV Pol is active as an RNaseH, and is involved in protein priming and the synthesis of HBV plusstrand DNA during viral replication. In addition to its versatile enzymatic activities, HBV Pol plays an essential role in the initiation of pgRNA packaging into capsids (encapsidation) via its interaction with the pgRNA ε stem-loop and host cellular factors like hsp90 (Hu et al., 2004; Hu and Seeger, 2002). In addition, the ε-Pol interaction leads to the termination of the ribosomal pgRNA translation of core and polymerase proteins that are thought to be prerequisites for encapsidation. Herein, we aimed to identify small molecules that are able to interrupt the HBV ε-Pol interaction. In the present study, we established a cell-based, luciferase reporter HTS assay for the identification of novel inhibitor(s) targeting the HBV ε-Pol interaction. By transiently overexpressing HBV Pol and a ε-Luc reporter in the HEK293T kidney cell line, we screened ~110,000 small molecules, identified 450 hits and confirmed 32 by DRC analysis. However, HBV is a hepatotropic virus and can only establish a persistent infection in hepatocytes because of its dependence on host cell-specific proviral

factors for viral entry and replication. Interestingly, when using hepatocytes replicating the full HBV genome, only one out of 32 hits showed sufficient antiviral activity (SI > 5), underlining the relevance of physiological cell culture models for early drug discovery. Presumably, host cell-specific factors and/or the expression patterns of viral factors strongly influence the identification of chemical inhibitors. After all, we identified AACC as a specific inhibitor of the HBV ε-Pol interaction in HEK293T cells as well as in an HBV-transfected, liver-derived hepatoma cell line (HepG2). To further investigate the MoA of AACC, mechanistic studies revealed that AACC targets the ε-Pol interaction and thereby prevents downstream steps in the HBV life cycle: We observed strongly reduced encapsidated pgRNA, capsid assembly, and capsid-associated DNA replication in HepG2.2.15 cells. A similar MoA was described for CpAM anti-HBV reagents, like the phenylpropenamide derivatives AT-61 and AT130, Bay41-4109, and NVR3-778; however, this class of inhibitors directly targets the HBV core protein and thereby blocks encapsidation and HBV replication (Delaney et al., 2002). By directly comparing Bay41-4109 and AACC, we observed clear differences in the MoAs to prevent encapsidation. While Bay41-4109 suppressed capsid formation and the total level of detectable HBc, AACC prevented capsid formation without affecting HBc levels (Figs. 3B and 4B). Furthermore, AACC showed synergistic effects with Bay41-4109, LMV, and TDF, and inhibited viral replication of both WT and mutant HBV, which are resistant to NAs and CpAMs (Park et al., 2019) (Zhou et al., 2017). Both data sets provide strong evidence that AACC possesses distinct features from Bay41-4109 and LMV. These observations suggest that drugs with an MoA as described for AACC may be used to treat patients with drugresistant HBV. For other inhibitors that target HBV Pol, that is, hemin and porphyrin, it was reported that disrupting the ε-Pol RNP complex inhibits protein priming in both DHBV and HBV in vitro (Lin and Hu, 2008). Carbonyl J acid derivatives were shown to block protein priming via the targeting of Pol activity in an in vitro assay using DHBV (Wang et al., 2012). More recently, rosmarinic acid was shown to inhibit the HBV ε-Pol interaction (Tsukamoto et al., 2018). Together, these reports suggest that the HBV εPol interaction is a promising new drug target, thereby leading to the suppression of protein priming

and encapsidation, distinct from the mechanism of existing anti-HBV drugs and most experimental drug candidates. Furthermore, we confirmed that AACC acts on multiple HBV genotypes and inhibits HBV propagation after long-term therapeutic treatment of HepG2-NTCPsec+ cells persistently infected with HBVpt as well as in PHHs infected with HBVcc, which are used as the gold standards for HBV in vitro studies. In conclusion, to our knowledge, this is the first approach to successfully identify an inhibitor of the HBV ε-Pol interaction using a target- and cell-based HTS approach. We discovered AACC as an HBV inhibitor targeting the ε-Pol interaction with a different MoA than NAs and CpAMs. However, a structure-activity relationship study to further improve the drug-like properties of AACC is required. Furthermore, it is unknown whether AACC inhibits the ε-Pol interaction by disrupting ε-Pol binding or by preventing the priming activity of HBV Pol. In addition, the direct cellular or viral target of AACC must be elucidated. The synergistic effects of AACC with LMV, TDF, and Bay41-4109 provide new opportunities as novel combinational therapeutic options for CHB patients to overcome the emergence of drug resistance and the potential destabilization of cccDNA to potentially cure CHB.

Conflicts of interest All authors declare that they have no conflicts of interest to this work.

Acknowledgements: This

study

was

supported

by

the

National

Research

Foundation

of

Korea

(MSIT

2017M3A9G6068246 and NRF-2014R1A2A1A11052535) and the Gyeonggi Provincial Government.

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Ryu, D.K., Ahn, Y., Ryu, W.S., Windisch, M.P., 2015. Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR. Biotechniques 59, 287–293. https://doi.org/10.2144/000114354 Ryu, D.K., Kim, S., Ryu, W.S., 2008. Hepatitis B virus polymerase suppresses translation of pregenomic RNA via a mechanism involving its interaction with 5′ stem-loop structure. Virology 373, 112–123. https://doi.org/10.1016/j.virol.2007.11.010 Seeger, C., Mason, W.S., 2000. Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. Tavis, J.E., Cheng, X., Hu, Y., Totten, M., Cao, F., Michailidis, E., Aurora, R., Meyers, M.J., Jacobsen, E.J., Parniak, M.A., Sarafianos, S.G., 2013. The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes. PLoS Pathog. https://doi.org/10.1371/journal.ppat.1003125 Tsukamoto, Y., Ikeda, S., Uwai, K., Taguchi, R., Chayama, K., Sakaguchi, T., Narita, R., Yao, W.L., Takeuchi, F., Otakaki, Y., Watashi, K., Wakita, T., Kato, H., Fujita, T., 2018. Rosmarinic acid is a novel inhibitor for hepatitis b virus replication targeting viral epsilon RNA-polymerase interaction. PLoS One 13, 1–16. https://doi.org/10.1371/journal.pone.0197664 Wang, Y.-X., Wen, Y.-M., Nassal, M., 2012. Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids. J. Virol. 86, 10079–10092. https://doi.org/10.1128/jvi.00816-12 World Health Organization, 2017. Global Hepatitis Report, 2017, World Health Organisation. https://doi.org/10.1016/j.poly.2012.11.010 Yan, R., Cai, D., Liu, Y., Guo, H., 2017. Detection of hepatitis B virus particles released from cultured cells by particle gel assay, in: Methods in Molecular Biology. https://doi.org/10.1007/978-1-4939-6700-1_15 Zhou, Z., Hu, T., Zhou, X., Wildum, S., Garcia-Alcalde, F., Xu, Z., Wu, D., Mao, Y., Tian, X., Zhou,

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Figure Legends Fig. 1. Validation of the HBV ε-Pol assay. (A) Schematic of the assay. HEK293T cells were cotransfected with plasmids encoding ε-Luc and HBV Pol. No luciferase activity is measured when the interaction is intact (inactive). In the presence of compounds that interfere with the ε-Pol interaction, luciferase can be measured (active). (B) Proof of concept in the absence of compounds with different ratios of ε-Luc and HBV Pol, as indicated. (C) Adaptation and validation of an automated ε-Pol HTS assay. ε-Luc activity was measured and analyzed by relative luminescence units (RLU). ε-Luc activity in the absence or presence of HBV Pol is plotted as black and gray dots, respectively, from two independent HTS simulations (validations 1 and 2).

Fig. 2. HBV ε-Pol HTS and hit confirmation. (A) HTS campaign. Compounds were added at a concentration of 10 µM, and data were normalized to percent ε-Luc activity. Primary hits (450; blue) among 110,000 compounds (gray) were selected if 130% ε-Luc activity was exceeded. (B) Structure and chemical name of AACC. (C) The ε-Pol assay in HepG2 cells. HepG2 cells were transfected with ε-Luc with or without HBV Pol and treated with serially diluted compounds (highest concentration, factor of dilution): LMV (1.25 µM, 4-fold), 17-DMAG (0.039 µM, 2-fold), and AACC (2.5 µM, 2fold). ε-Luc activity and cell viability were measured and normalized to DMSO-treated cells set as

100% activity and cell viability.

Fig. 3. MoA studies with AACC. (A–D) HepG2.2.15 cells were seeded into 96-well plates (A and C) or 6-well plates (B and D) and treated with compounds at the indicated concentrations every 3 days for 9 days. (A) Nucleocapsid-captured qRT-PCR encapsidation assay. Encapsidated pgRNAs (black) and cell viability (gray) were analyzed in the presence of AACC as indicated. (B) The level of capsid formation was determined by a particle gel assay. Subsequently, capsid-associated DNA was analyzed by hybridization to a DIG-labeled HBV probe. In parallel, the level of core proteins was assessed by Western blot analysis. Alpha-tubulin was used as an internal loading control. LMV (10 µM) and Bay41-4109 (4 µM) were used as positive controls. (C) DRCs of intra- and extracellular HBV DNA. Data were normalized to DMSO (0% HBV inhibition and 100% cell viability). (D) Inhibition of HBV replication by AACC was visualized by Southern blot analysis. Intensities of bands shown in B and D were quantified using ImageJ software and presented as a percentage at the bottom of the images (DMSO 100%). RC, DL, and M represent relaxed circular, duplex linear, and size marker, respectively. (E) Cross-genotypic antiviral assay. HepG2 cells were transiently transfected with gtB, gtC, and gtD replicons, plated in a 96-well plate, and treated with inhibitors as indicated at 1 and 3 days posttransfection (dpt). TDF and LMV were used for reference. At 5 dpt, intracellular HBV DNA was analyzed by qPCR. For normalization, 10 µM LMV set as 100% inhibition. Data are shown as the mean ± SD.

Fig. 4. Evaluation of AACC using HBV-infected HepG2-NTCPsec+ cells. (A) The scheme of HBV infection and the progeny passaging assay. HepG2-NTCPsec+ cells were infected with 5,000 GEq/cell of HBVcc. MyrB and AACC were added 2 h before inoculation. LMV was added at 3 and 6 dpi. HBV replication and viral progeny release were monitored during p2 infection. (B) Representative images of HBV infection by IFA. HBV-infected cells (HBcAg positive) and cell nuclei are shown in green and blue, respectively. HBV infection (%) is presented as the mean ± SD in black boxes. (C) DRC

analysis of compounds. Data were normalized as follows: DMSO treatment (0% inhibition), 0.6 µM MyrB (100% inhibition in p1), and 10 µM LMV (100% inhibition in p2). HBV inhibition (%) in p1 and p2 cells indicated by black circles and diamonds, respectively. Cell viability (gray triangles) was analyzed by counting the total cell number. EC50, CC50 (µM), and SI values are shown in the table on the right. Representative data of three independent experiments are shown.

Fig. 5. Evaluation of AACC using patient-derived HBV and PHHs. (A) Schematic of the long-term infection assay in HepG2-NTCPsec+ cells. Cells were infected with 5 µL crude serum from a CHB patient and treated with inhibitors (MyrB: 0.6 µM; TDF: 0.1 µM; AACC: 0.5, 1, 2, and 4 µM) as indicated for 4 wpi. (B) qPCR analysis of intra- (black) and extracellular (gray) HBV DNA. Discontinuous lines represent the lowest level of HBV DNA detectable by TDF treatment. (C) Scheme of the PHH infection assay. PHHs infected with HBVcc were treated with 10 µM LMV, 0.1 µM TDF, and 1 µM AACC. Cell culture supernatants were harvested at 11 (black) and 15 (light gray) dpi. (D) qPCR analysis of extracellular HBV DNA. Cell viability was determined at 15 dpi (gray). Representative data of two independent experiments are shown as the mean ± SD in triplicate. Statistical significance was determined using the Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 6. Effects of AACC on NAs- and CpAM-RS HBV. (A) NA-RS HBV. HepG2 cells were transiently transfected with WT HBV and LMV-RS HBV replicons, and treated with the indicated concentrations of inhibitors at 1 and 3 dpt. At 5 dpt, intracellular HBV DNA was harvested and analyzed by qPCR. Data were normalized to 4 µM Bay41-4199, which was set as 100% inhibition of HBV DNA. (B) CpAM-RS HBV. Experiment and analysis using CpAM-resistant HBV was conducted as described above. Data were normalized to 10 µM LMV set as 100% inhibition of HBV DNA. Representative data of two independent experiments are shown as the mean ± SD.

Appendix A. Supplementary Data. Fig. S1. Evaluation of AACC using infectious HCV and HIV. (A) HCV infection. Huh7-cells were treated with sofosbuvir (an HCV inhibitor) or AACC 2 h before inoculation with HCVcc (Gt2-E2p7NS5A-GFP) (Lee et al., 2017). At 3 dpi, HCV infection and cell viability were analyzed by determining GFP expression and counting cell nuclei using confocal microscopy, respectively. (B) HIV infection. HeLa-LTR-GFP cells (Gervaix et al., 1997) were treated with nevirapine (an HIV inhibitor) or AACC during the inoculation with HIV-1 IIIB (DayMoon Industries, Inc.). At 5 dpi, HIV infection was analyzed using confocal microscopy.

Table 1. Table 1. HTS assay validation. Validation 1

CV(%)

Validation 2

- Pol

+ Pol

- Pol

+ Pol

12

9

13

14

Window

9

12

Z'

0.6

0.5

Table 2. Table 2. Activity of hit compounds. Primary assay: ε-Luc activity (% of control) at

Secondary assay: Extra-cellular HBV DNA (HepG2.2.15)

No.

2.5 µM

5 µM

10 µM

20 µM

EC50 [µM]

CC50 [µM]

SI*

1

141.1

131.3

182.4

214.2

2.8 ± 1.9

14.9 ± 1.5

5.24

2

110.8

108.5

147.5

182.2

85

100

1.18

3

130.4

126.1

132.3

179.2

-

>100

-

4

101.1

103.8

100.2

119.5

25.07

30.75

1.23

5

102.7

126.9

165.4

114

15.41 ± 9.7

18.2 ± 0.7

1.18

6

94.3

78

178.3

279.3

6.27

6.42

1.02

7

126.7

184.89

89.3

87.9

6.37

4.41

0.69

8

117.8

148.1

350.2

312.8

10.95

3.83

0.35

9

134

143.7

143.4

179.3

-

70

-

10

125.1

144.4

164.5

166.7

39.34

92.8

1.42

11

108.1

123.2

152.2

171.2

3.6 ± 0.14

10.3 ± 0.3

2.86

12

111.9

111.54

139.1

210.9

-

12.79

-

13

93.9

101.8

100.8

169.9

20

19.57

0.98

14

90.1

97.4

113.6

161.8

6.8

15.48

2.28

15

98.8

92.8

98.6

148.5

-

>100

-

16

96.5

96.8

125.7

169.2

13.01

19.9

1.53

17

155.5

142.3

138.8

117.2

-

>100

-

18

100.9

122

135.8

142.8

-

>100

-

19

163.2

158.3

142.8

127.2

17.52

10.32

0.59

20

97.4

88.2

85.4

178.7

84.2

43.1

0.51

21

143.4

157.1

171.8

179.2

12.1

13.98

1.16

22

94

97.5

85.4

158.8

-

74.63

-

23

132.5

141.9

156.1

248.1

33.8 ± 4.4

51.8 ± 3.9

1.72

24

121.2

132.2

135.8

164.5

46.2 ± 14.1

65.94 ± 4.72

1.43

25

112.7

122.7

135.6

154.9

-

>100

-

26

150.5

167.9

151

116.6

-

>100

-

27

122.8

153

170.7

154.5

28.86 ± 24.3

46.15 ± 16.03

1.92

28

114.9

147.5

146.8

127.2

-

>100

1

29

126.3

143.9

143.7

165.1

62.9 ± 72.6

48.9 ± 12.9

0.73

30

163.1

155.8

140.6

125.7

-

100

-

31

17

164.9

220.7

214.5

-

>100

-

32

91.8

114.5

132.2

114.2

45.15

59.61

1.32

*Selectivity index: CC50/EC50

Table 3. Table 3. Drug-Drug combinations.

Drug 1

Drug 2

Combination ratio

CI valuesa,b at inhibition of 50%

75%

90%

95%

Weighted average CI valuesa

Assigned symbolc

Description

Lamivudine

AACC

1 : 25

0.95

0.49

0.29

0.22

0.37

+++

Synergism

Tenofovir

AACC

1 : 25

2.31

0.92

0.43

0.29

0.66

+++

Synergism

Bay414109

AACC

1 : 2.5

0.97

0.8

0.73

0.71

0.76

++

Moderate synergism

a

Weighted (wt) average combination index (CI) values were determined as CIwt = [CI50 + 2xCI75 +3xCI90 +4xCI95] / 10. Data analysis was performed using CompuSyn software (ComboSyn, Inc.). Data represent mean of the average of three independent experiments (n=3). c Degree of synergism or antagonism is based on the ranges of CI values as described previously (Ting-Chao Chou, 2006). b

Figure 1.

A

HBV Pol ε-Luc Compound Pol Pol Cap

Luc

p(A)

Cap

INACTIVE

Luc

p(A)

ACTIVE

B ε-Luc reporter Renilla reporter

120

Luc activity (%)

100 80 60 40 20 0 ε-Luc : HBV Pol Window Z´ factor

1:0 1.0

1:1 2.2 0.2

1:2 3.6 0.4

1:3 4.9 0.5

1:4 7.6 0.6

1:8 17.5 0.6

C ε-Luc ε-Luc + HBV Pol

RLU (validation 2)

1,300,000 1,100,000 900,000 700,000 500,000 300,000 100,000 0

0

500,000

1,500,000

2,500,000

RLU (validation 1)

3,500,000

Figure 2.

A

B 800

ε- Luc activity (%)

AACC

Primary hits Compounds

700 600 500 400 300 200

Z-2-(allylamino)-4-amino-N’cyanothiazole-5-carboximidamide

100 0 0

20,000

40,000

60,000

80,000

100,000

Number of compounds

C

-1.5 -0.5 0.5 Log10 Concentration [µM]

Lamivudine

-2.0 -1.0 0 Log10 Concentration [µM]

300 250 200 150 100 50 0

300 250 200 150 100 50 0

17-DMAG

-2.5 -2.0 -1.5 Log10 Concentration [µM]

300 250 200 150 100 50 0

Cell viability (%)

300 250 200 150 100 50 0

ε-Luc activity (%)

300 250 200 150 100 50 0

Cell viability (%)

AACC

ε-Luc activity (%)

300 250 200 150 100 50 0

Cell viability (%)

ε-Luc activity (%)

ε-Luc ε-Luc + HBV Pol Cell viability

Figure 3.

100

100

80

80

60

60

40

40

20

20

0

LMV

120

120

Cell viability (%)

Encapsidated pgRNA Relative to non-treated (%)

Encapsidated pgRNA Cell viability

Bay41-4109

B DMSO

A

0.5

100

0

86

44.1 28.7 7.7 (%)

100

0

6.8 76.4 30.6 0.1 (%)

AACC 1.0 2.0 [μM]

Capsid

Capsid-DNA

Core protein (21 kD)

0

a-tubulin

C

D M LMV

100

100

50

50

0

0 -1

0

1

100

50

50

0

0 1

2

Log10 Concentration [µM]

Cell viability (%)

Inhibition of HBV DNA (%)

100

0

E

V ML M 01 F D T M  1. 0

120

2 2 K PI M  2

AACC

-1

4.35 100 102 33.7 (%)

2 2 K PI M  5. 0

Log10 Concentration [µM]

-2

1 [μM]

3.2 kb

Inhibition of HBV replication (%)

-2

0.1

DL

LMV

-3

AACC RC

Cell viability (%)

Inhibition of HBV DNA (%)

Intra-cellular HBV DNA Extra-cellular HBV DNA Cell viability

DMSO

AACC Concentration [μM]

10 μM LMV 0.1 μM TDF 0.5 μM AACC 2 μM AACC

100 80 60 40 20 0

Genotype B

Genotype C

Genotype D

Figure 4.

AACC LMV

MyrB

C Inhibition (p1 infection) Inhibition (p2 infection) Cell viability

HBV Day 0 1

3

6

9

HepG2NTCPsec+

Supernatant passage

p1 infection

p2 infection

p1 infection

p2 infection

MyrB 100

100

50

50

0

0

DMSO

-3.0

EC50 (p2) 0.005 ± 0.001 CC50

>0.6

SI

>75

0.0

LMV 100

100

50

50

0

0 -2.0

-1.0

Cell viability (%)

0.91 ± 0.63

HBV inhibition (%)

0.6 μM MyrB

2.74 ± 0.08

42.5 ± 2.14

-3.0

EC50 (p1) >2.5 EC50 (p2) 0.015 ± 0.009 CC50

>2.5

SI

>167

0.0

Log10 Concentration [µM]

53.09 ± 2.43

50.93 ± 2.93

1.77 ± 0.2

9.77 ± 1.42 HBcAg/Nuclei

HBV inhibition (%)

10 μM LMV

-1.0

EC50 (p1) 0.008 ± 0.002

Log10 Concentration [µM]

54.8 ± 1.53

4 μM AACC

-2.0

AACC 100

100

50

50

0

0 -0.5 0.0 0.5 1.0 1.5 Log10 Concentration [µM]

Cell viability (%)

B

HBV inhibition (%)

-2h

Cell viability (%)

A

EC50 (p1) >25 EC50 (p2) 0.81 ± 0.4 CC50

6.5 ± 0.8

SI

8.0

Figure 5. A

B MyrB

MyrB, TDF, AACC

day -1-2h 0

1

7

14

HepG2HBVpt NTCPsec+

21

28

Harvest cell & supernatant

C

HBV DNA (GEq/mL)

109

1.2 x 9.0 x 108 6.0 x 108 3.0 x 108 4.0 x 107 3.0 x 107 2.0 x 107 1.0 x 107 0

Intra-cellular HBV DNA Extra-cellular HBV DNA

1.46 x 107 1.54 x 106

0.5 1.0 2.0 4.0 AACC Concentration [μM]

D LMV, TDF, AACC 0

1

4

7

11

1.5 x 108

15

150

*

HBVcc

Harvest supernatant

* *

1.0 x 108

100 ** ** **

5.0 x 107

0

50

0

Cell viability (%)

PHH

HBV DNA (GEq/mL)

day -2

HBV DNA (11 dpi) HBV DNA (15 dpi) Cell viability (15 dpi)

Figure 6. B

150

DMSO 10 μM LMV 4 μM Bay41-4109 1 μM AACC

Inhibition of HBV replication (%)

Inhibition of HBV replication (%)

A

100

50

0

-50 WT

LMV-RS

150

DMSO 10 μM LMV 4 μM Bay41-4109 1 μM AACC

100

50

0

-50 WT

CpAM-RS

Highlights Target-based HTS identifies HBV encapsidation inhibitors interfering with the interaction of HBV polymerase and pgRNA. AACC blocks capsid formation, suppresses viral replication, and has cross-genotypic activity. AACC inhibits HBV replication of cell culture- and patient-derived HBV in infected HepG2-NTCP cells and PHHs. AACC inhibits drug-resistant viruses and is synergistic with nucleoside analogs and a capsid inhibitor.