Structural and functional basis for pan-CoV fusion inhibitors against SARS-CoV-2 and its variants with preclinical evaluation.

The COVID-19 pandemic poses a global threat to public health and economy. The continuously emerging SARS-CoV-2 variants present a major challenge to the development of antiviral agents and vaccines. In this study, we identified that EK1 and cholesterol-coupled derivative of EK1, EK1C4, as pan-CoV fusion inhibitors, exhibit potent antiviral activity against SARS-CoV-2 infection in both lung- and intestine-derived cell lines (Calu-3 and Caco2, respectively). They are also effective against infection of pseudotyped SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.1.248 (Gamma) as well as those with mutations in S protein, including N417T, E484K, N501Y, and D614G, which are common in South African and Brazilian variants. Crystal structure revealed that EK1 targets the HR1 domain in the SARS-CoV-2 S protein to block virus-cell fusion and provide mechanistic insights into its broad and effective antiviral activity. Nasal administration of EK1 peptides to hACE2 transgenic mice significantly reduced viral titers in lung and intestinal tissues. EK1 showed good safety profiles in various animal models, supporting further clinical development of EK1-based pan-CoV fusion inhibitors against SARS-CoV-2 and its variants.

Identification of viral SIM-SUMO2-interaction inhibitors for treating primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.

Long-term functional maintenance of primary human hepatocytes in vitro.

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen.

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.

Rupintrivir is a promising candidate for treating severe cases of enterovirus-71 infection: evaluation of antiviral efficacy in a murine infection model.

Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.