Molecular Approaches To Antiviral and Vaccine Development For Viral Hepatitis and Other Viral Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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. A novel aryloxazole-based HCV entry inhibitor was identified previously through a quantitative phenotypic high-throughput screen. Structureactivity relationship (SAR) optimization yielded the compound fluoxazolevir with the improved efficacy, cytotoxicity and in vitro absorption-distribution-metabolism-excretion properties as a lead candidate for preclinical development. Here we characterize its mechanism of action, in vitro efficacy against various HCV genotypes, synergy with other HCV drugs, pharmacokinetics and in vivo efficacy in a HCV-infected humanized mouse model. We showed that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 (E1) to prevent fusion. 9 of 10 fluoxazolevir-resistance-associated substitutions are in the E1 protein and 4 are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A four-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotype 1b, 2a or 3 resulted in a 2-log reduction in viremia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3-log of viremia but is associated with emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment. 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.