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

随着最近几种直接作用的抗病毒药物的批准，丙型肝炎病毒(丙型肝炎病毒)感染的治疗已经迅速发展。然而，大多数临床应用或临床试验中的DAA针对的是丙型肝炎病毒复制周期的同一阶段，并与快速出现耐药病毒突变有关。此外，不同的丙型肝炎病毒基因分型和临床情况也可能需要调整治疗方案。因此，仍然需要开发针对丙型肝炎病毒复制周期不同阶段的新的丙型肝炎病毒抑制剂，如进入和组装。 乙型肝炎病毒(乙肝病毒)感染肝细胞，引起免疫介导的肝损伤，使慢性感染的患者发展为肝硬化和肝细胞癌的高风险。目前治疗慢性乙肝的方法是有效的，但有许多局限性，迫切需要开发新的治疗方法。在这项研究中，我们通过高通量筛选确定了新的抗乙肝药物，验证了这些化合物，并正在确定它们的抑制机制。首先，建立了扩增发光均相联免疫吸附试验(AlphaLISA)检测乙肝e抗原(HBeAg)的方法。在高通量模式下，用14,402个小分子化合物文库感染并处理HepG2-NTCP细胞。AlphaLISA和基于ATP的细胞活力测定分别用于检测细胞的抑制和细胞毒性。从高通量筛选中，选择显示最大抑制率80%和CC50；5um的20个匹配项进行进一步验证。采用常规细胞培养方法，进一步在HepG2.215细胞、病毒感染的HepG2-NTCP和病毒感染的原代人肝细胞(PXB细胞)中滴定所选HITS的抗乙肝病毒活性和细胞毒谱。总的来说，大多数化合物在HepG2.215细胞和病毒感染的HepG2-NTCP和PXB细胞中对HBeAg和HBVDNA显示出一致的抑制作用，原始细胞模型对抗病毒治疗更敏感。经过验证，基于潜在的作用模式和抗病毒效果，人们正在研究高度感兴趣的化合物，以了解其抗乙肝病毒作用的详细分子机制。 由严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)引起的冠状病毒病2019年(新冠肺炎)已成为对全球公共卫生的严重威胁，突显了开发有效疫苗和疗法的紧迫性和高度优先事项。治疗，更具体地说，抗病毒的开发，在很大程度上仍处于初级阶段。目前还没有临床批准的治疗这种疾病的方法或疫苗，除了针对患有新冠肺炎的重病患者的瑞希韦。该项目的总体目标是通过改变现有药物的用途或开发新药来确定和开发有效的抗SARS-CoV-2药物。我们正在建立基于非传染性细胞的模型系统来研究SARS-CoV-2感染和复制周期的各个阶段，开发基于这些模型系统的高通量平台来筛选抗SARS-CoV-2化合物的大的小分子文库，并从筛选的高活性和无毒化合物中进行广泛的临床前研究，为进一步的药物开发做准备。 在这里，我们报告了我们先前研究中发现的两种丙型肝炎病毒(丙型肝炎病毒)融合抑制剂，二氯环利嗪和氟唑洛韦，它们广泛地阻止了人类冠状病毒进入各种类型的细胞。我们开发了基于水疱性口炎病毒(VSV)与不同人类冠状病毒刺突蛋白的假型和刺突介导的合胞体形成的多重进入试验，以检验这些抑制剂的有效性并确定其作用机制。两种化合物均有效，半数最大有效浓度(EC50)在个位数微摩尔范围内。在SARS-CoV-2活体感染系统中证实了其抗病毒作用。这些化合物对最近出现的具有N439K、Y453F、E484K、N501Y、D614G或P681H突变的尖峰变种同样有效。结构模拟表明，这些化合物结合在S蛋白融合肽附近的疏水口袋上，这与它们作为融合抑制剂的潜在作用机制一致。综上所述，这些融合抑制剂具有广谱的抗病毒活性，有望成为治疗SARS-CoV-2及其变异体和其他致病冠状病毒的先导药物。 自2021年底奥米克龙变种出现以来，它们迅速成为全球主导变种。与较早的武汉和其他变种相比，奥米克龙变种可能更容易传播。在这项研究中，我们旨在阐明与Omicron变体相关的感染性改变的机制。我们系统地评估了位于尖峰S2序列中的突变，并确定了导致病毒融合改变的突变。我们证明，S1/S2裂解位点附近的突变减少了S1/S2裂解，导致融合性降低。HR1和其他S2序列的突变也影响细胞-细胞融合。基于核磁共振研究和计算机模拟，这些突变可能在病毒融合的多个步骤中影响融合活性。我们的发现表明，Omicron变体积累了有助于减少合胞形成的突变，从而减弱了致病性。

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.