A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals
| Autor: | David Shum, Seung Kew Yoon, Marc Peter Windisch, Soonju Park, Eva Zusinaite, Eunji Jo, Denis E. Kainov, Jaewon Yang, Pil Soo Sung, Alexander König |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
GEq
genome equivalents DRC dose–response curve NTCP sodium taurocholate cotransporting polypeptide %CV percent coefficient of variation RC799-869 PF-rcDNA protein-free relaxed circular DNA medicine.disease_cause HTS high-throughput screening LHB HBV large surface protein Virion secretion HBV Immunology and Allergy Cytotoxic T cell CpAM core protein allosteric modifiers dpi days post-infection Gastroenterology High-throughput screening iPSCs induced pluripotent stem cells MoA mechanism of action MyrB myrcludex B cccDNA Diseases of the digestive system. Gastroenterology LMV lamivudine Fludarabine %Imax percent maximum inhibition High-content screening TI therapeutic index CHB chronic hepatitis B cccDNA covalently closed circular DNA medicine.drug Research Article FDA-approved drugs Supernatant transfer HCS high content screening Entry Replication CC50 50% cytotoxic concentration SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 Virus HID N-hydroxyisoquinolinedione Viral life cycle HLCs hepatocyte-like cells Internal Medicine medicine HepG2-NTCP IFNα interferon alpha TDF tenofovir disoproxil fumarate Patient-derived HBV PEG polyethylene glycol Hepatitis B virus Hepatology p1 passage 1 business.industry SOP standard operation procedure pgRNA pregenomic RNA HBVpt patient-derived HBV Virology FDA Food and Drug Administration digestive system diseases Small-molecule inhibitors IFA immunofluorescence analysis Cell culture p2 passage 2 business HCC hepatocellular carcinoma IFNλ interferon lambda |
| Zdroj: | JHEP Reports JHEP Reports, Vol 3, Iss 4, Pp 100296-(2021) |
| Popis: | Background & Aims Chronic hepatitis B is an incurable disease. Addressing the unmet medical need for therapies has been hampered by a lack of suitable cell culture models to investigate the HBV life cycle in a single experimental setup. We sought to develop a platform suitable to investigate all aspects of the entire HBV life cycle. Methods HepG2-NTCPsec+ cells were inoculated with HBV. Supernatants of infected cells were transferred to naïve cells. Inhibition of infection was determined in primary and secondary infected cells by high-content imaging of viral and cellular factors. Novel antivirals were triaged in cells infected with cell culture- or patient-derived HBV and in stably virus replicating cells. HBV internalisation and target-based receptor binding assays were conducted. Results We developed an HBV platform, screened 2,102 drugs and bioactives, and identified 3 early and 38 late novel HBV life cycle inhibitors using infectious HBV genotype D. Two early inhibitors, pranlukast (EC50 4.3 μM; 50% cytotoxic concentration [CC50] >50 μM) and cytochalasin D (EC50 0.07 μM; CC50 >50 μM), and 2 late inhibitors, fludarabine (EC50 0.1 μM; CC50 13.4 μM) and dexmedetomidine (EC50 6.2 μM; CC50 >50 μM), were further investigated. Pranlukast inhibited HBV preS1 binding, whereas cytochalasin D prevented the internalisation of HBV. Fludarabine inhibited the secretion of HBV progeny DNA, whereas dexmedetomidine interfered with the infectivity of HBV progeny. Patient-derived HBV genotype C was efficiently inhibited by fludarabine (EC50 0.08 μM) and dexmedetomidine (EC50 8.7 μM). Conclusions The newly developed high-content assay is suitable to screen large-scale drug libraries, enables monitoring of the entire HBV life cycle, and discriminates between inhibition of early and late viral life cycle events. Lay summary HBV infection is an incurable, chronic disease with few available treatments. Addressing this unmet medical need has been hampered by a lack of suitable cell culture models to study the entire viral life cycle in a single experimental setup. We developed an image-based approach suitable to screen large numbers of drugs, using a cell line that can be infected by HBV and produces large amounts of virus particles. By transferring viral supernatants from these infected cells to uninfected target cells, we could monitor the entire viral life cycle. We used this system to screen drug libraries and identified novel anti-HBV inhibitors that potently inhibit HBV in various phases of its life cycle. This assay will be an important new tool to study the HBV life cycle and accelerate the development of novel therapeutic strategies. Graphical abstract Highlights • We developed a high-content screening assay suitable to monitor the entire HBV life cycle and eligible to discriminate between early and late viral life cycle inhibition. • We screened FDA-approved drugs and bioactives. • We confirmed antiviral activity in primary and secondary assays, using stably virus replicating cells and cell culture- and patient-derived HBV. • Novel HBV inhibitors prevent receptor binding, virus internalisation, replication, or egress of viral progeny. |
| Databáze: | OpenAIRE |
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