Document text
Principal Investigator: T. Jake Liang
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2023
Award: $2,198,134
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of several direct-acting antivirals. However, most of the DAAs in clinical use or clinical trials target the same stage of HCV replication cycle and are associated with rapid emergence of drug-resistant viral mutations. In addition, different HCV genotypes and clinical conditions may also require adjustment of treatment regimen. Therefore, there is still an ongoing need to develop new HCV inhibitors that target different stages of the HCV replication cycle, such as entry and assembly.
Hepatitis B virus (HBV) infects hepatocytes and causes immune-mediated liver damage, leaving chronically infected patients with a high risk of developing liver cirrhosis and hepatocellular carcinoma. Current treatments for chronic HBV infection are effective but have many limitations, creating an urgent need for the development of new therapies. In this study, we identified novel anti-HBV agents via a high throughput screen, validated these compounds, and are now determining their mechanisms of inhibition. First, the Amplified Luminescence Proximity Homogeneous Assay-linked Immunosorbent Assay (AlphaLISA) was established for detection of hepatitis B e antigen (HBeAg), a marker of HBV infection. In a high throughput format, HepG2-NTCP cells were infected and treated with a library of 14,402 small molecule compounds. AlphaLISA and an ATP-based cell viability assay were used to measure inhibition and cytotoxicity, respectively. From the high throughput screen, twenty hits showing max inhibition >80% and CC50>5uM were selected for further validation. Using normal cell culture format, the anti-HBV activities and cytotoxic profiles of the selected hits were further titrated in HepG2.215 cells, virus-infected HepG2-NTCP, and virus-infected primary hepatocytes of human origin (PXB cells). Collectively, a majority of the compounds showed consistent inhibition of HBeAg and HBV DNA in HepG2.215 cells and virus-infected HepG2-NTCP and PXB cells, with the primary cell model being more sensitive to the antiviral treatment. After validation, based on the potential mode of action and the antiviral efficacy, compounds of high interest are under investigation for the detailed molecular mechanisms of their anti-HBV effects.
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a serious threat to global public health, underscoring the urgency and high priority to develop effective vaccines and therapies. Therapeutic, and more specifically, antiviral development, is still very much in its infancy. Currently, no clinically approved therapies or vaccines are available for this disease, with the exception of remdesivir for severely ill patients with Covid-19. The overall goal of this project is to identify and develop effective antivirals against the SARS-CoV-2, either by repurposing existing pharmaceuticals or developing new drugs. We are establishing non-infectious cell-based model systems to study various stages of SARS-CoV-2 infection and replication cycle, to develop high-throughput platform based on these model systems to screen large small-molecule libraries for anti-SARS-CoV-2 compounds, and to conduct extensive preclinical studies of highly active and nontoxic compounds from the screen for further drug development.
Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. We developed multiple entry assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike proteins of various human CoVs and spike-mediated syncytia formation to examine the efficacy and define the mechanism of these inhibitors. Both compounds were effective with half maximal effective concentration (EC50) values in the single-digit micromolar range. The antiviral effects were confirmed in live SARS-CoV-2 infection systems. These compounds were equally effective against recently emerging spike variants with N439K, Y453F, E484K, N501Y, D614G, or P681H mutation. Structural modeling suggests that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants and other pathogenic CoVs.
Since the emergence of the Omicron variants at the end of 2021, they quickly became the dominant variants globally. The Omicron variants may be more easily transmitted compared to the earlier Wuhan and the other variants. In this study, we aimed to elucidate mechanisms of the altered infectivity associated with the Omicron variants. We systemically evaluated mutations located in the S2 sequence of spike and identified mutations that are responsible for altered viral fusion. We demonstrated that mutations near the S1/S2 cleavage site decreased S1/S2 cleavage, resulting in reduced fusogenicity. Mutations in the HR1 and other S2 sequences also affected cell-cell fusion. Based on nuclear magnetic resonance (NMR) studies and in silico modeling, these mutations affect fusogenicity possibly at multiple steps of the viral fusion. Our findings reveal that the Omicron variants have accumulated mutations that contribute to reduced syncytial formation and hence an attenuated pathogenicity.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Affect><Antiviral Agents><Antiviral Drugs><Antivirals><Assay><Attenuated><B.1.1.529><Binding><Bioassay><Biologic Assays><Biologic Models><Biological Assay><Biological Models><COVID infected patient><COVID patient><COVID positive patient><COVID-19><COVID-19 infected patient><COVID-19 infection><COVID-19 patient><COVID-19 positive patient><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID-19 virus infection><COVID19><COVID19 infection><COVID19 patient><COVID19 positive patient><COVID19 therapy><COVID19 treatment><COVID19 virus><CV-19><CV19><Cell Body><Cell Culture Techniques><Cell Survival><Cell Viability><Cell fusion><Cell model><Cells><Cellular model><Chronic><Chronic Hepatitis><Chronic Hepatitis B><Clinical><Clinical Trials><CoV-2><CoV2><Coronaviridae><Coronavirus><Detection><Development><Digit><Digit structure><Disease><Disorder><Drug resistant viral><GS-5734><Genetic Alteration><Genetic Change><Genetic defect><Genotype><Giant Cells><Goals><HBV><HBeAg><HCV><HCV infection><HCV therapy><HCV treatment><HCoV><Hep G2><HepG2><HepG2 cell line><Hepatic Cells><Hepatic Cirrhosis><Hepatic Parenchymal Cell><Hepatitis B Virus><Hepatitis B e Antigens><Hepatitis Be Antigens><Hepatitis C><Hepatitis C Therapeutics><Hepatitis C Therapy><Hepatitis C Virus Treatment><Hepatitis C treatment><Hepatitis C virus><Hepatitis C virus infection><Hepatitis, Viral, Non-A, Non-B, Parenterally-Transmitted><Hepatitus C><Hepatocarcinoma><Hepatocellular Carcinoma><Hepatocellular cancer><Hepatocyte><Hepatoma><High Throughput Assay><Homologous Serum Hepatitis Virus><Human><Hydrophobicity><Immune><Immunes><Immunoadsorbents><Immunosorbents><Infection><Injury to Liver><Investigation><Libraries><Link><Liver Cells><Liver Cells Carcinoma><Liver Cirrhosis><Lytotoxicity><Measures><Mediating><Model System><Modeling><Modern Man><Molecular><Molecular Interaction><Multinucleated Giant Cells><Mutation><Normal Cell><Nuclear Magnetic Resonance><Omicron variant><Pathogenicity><Patients><Peptides><Pharmaceutical Agent><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Polykaryocytes><Population><Primary carcinoma of the liver cells><Proteins><Public Health><Reporting><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 B.1.1.529><SARS-CoV-2 infected patient><SARS-CoV-2 infection><SARS-CoV-2 omicron><SARS-CoV-2 omicron variant><SARS-CoV-2 patient><SARS-CoV-2 positive patient><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome related corona virus 2><Site><Structural Models><Syncytium><System><Technology><Therapeutic><Titrations><Transmission><Treatment Protocols><Treatment Regimen><Treatment Schedule><Tzanck Cell><VSV><Vaccines><Validation><Variant><Variation><Veklury><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Activity><Viral Diseases><Viral Function><Viral Physiology><Viral hepatitis><Virus><Virus Diseases><Virus Inhibitors><Virus Replication><Wuhan coronavirus><access to vaccination><access to vaccines><anti-hepatitis B><anti-viral agents><anti-viral compound><anti-viral development><anti-viral drug development><anti-viral drugs><anti-viral efficacy><anti-viral medication><anti-viral therapeutic><anti-viral therapeutic development><anti-viral therapy development><anti-virals><antiviral compound><antiviral development><antiviral drug development><antiviral efficacy><antiviral medication><antiviral therapeutic><antiviral therapeutic development><antiviral therapy development><attenuate><attenuates><cell culture><cell cultures><cell type><chemical library><chronic HBV infection><chronic hepatitis B virus infection><corona virus><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 infected patient><coronavirus disease 2019 infection><coronavirus disease 2019 patient><coronavirus disease 2019 positive patient><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease infected patient><coronavirus disease patient><coronavirus disease positive patient><coronavirus disease-19><coronavirus disease-19 patient><coronavirus disease-19 virus><coronavirus infectious disease-19><coronavirus patient><cytotoxic><cytotoxicity><determine efficacy><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><developmental><drug development><drug resistant virus><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><evaluate efficacy><examine efficacy><genome mutation><hCoV19><hep C><hepatic damage><hepatic injury><hepatitis non A non B><hepatitis virus infection><high risk><high throughput screening><human CoV><human corona virus><human coronavirus><in silico><infancy><infantile><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><infection by hepatitis c virus><inhibitor><interest><liver carcinoma><liver damage><liver injury><luminescence><nCoV2><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><non A, non B hepatitis><non-A, non-B hepatitis><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><omicron variant of COVID-19><omicron variant of SARS-CoV-2><patient infected with COVID><patient infected with COVID-19><patient infected with SARS-CoV-2><patient infected with coronavirus disease><patient infected with coronavirus disease 2019><patient infected with severe acute respiratory syndrome coronavirus 2><patient with COVID><patient with COVID-19><patient with COVID19><patient with SARS-CoV-2><patient with coronavirus disease><patient with coronavirus disease 2019><patient with severe acute respiratory distress syndrome coronavirus 2><pharmaceutical><pre-clinical study><preclinical study><remdesivir><severe acute respiratory syndrome coronavirus 2 B.1.1.529><severe acute respiratory syndrome coronavirus 2 infected patient><severe acute respiratory syndrome coronavirus 2 patient><severe acute respiratory syndrome coronavirus 2 positive patient><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><small molecule><small molecule libraries><transmission process><treat COVID-19><treat COVID19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><vaccination access><vaccination availability><vaccine access><vaccine availability><validations><viral DNA><viral infection><viral inhibitor><viral multiplication><viral replication><virus DNA><virus infection><virus multiplication><virus-induced disease>