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

病毒性肝炎和其他病毒性疾病抗病毒药物开发的分子方法

• 批准号: 10919437
• 负责人: T. Jake Liang
• 金额: $ 219.81万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10919437
• 关键词: 2019-nCoV; Affect; Antiviral Agents; Attenuated; Binding; Biological Assay; Biological Models; COVID-19; COVID-19 patient; COVID-19 treatment; Cell Culture Techniques; Cell Survival; Cell fusion; Cell model; Cells; Chronic; Chronic Hepatitis; Chronic Hepatitis B; Clinical; Clinical Trials; Coronavirus; Detection; Development; Digit structure; Disease; Genotype; Giant Cells; Goals; HepG2; Hepatitis B Virus; Hepatitis B e Antigens; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; Hepatocyte; Human; Hydrophobicity; Immune; Immunosorbents; Infection; Investigation; Libraries; Link; Liver Cirrhosis; Measures; Mediating; Modeling; Molecular; Mutation; Normal Cell; Nuclear Magnetic Resonance; Pathogenicity; Patients; Peptides; Pharmacologic Substance; Population; Primary carcinoma of the liver cells; Proteins; Public Health; Reporting; SARS-CoV-2 B.1.1.529; SARS-CoV-2 infection; Site; Structural Models; System; Technology; Therapeutic; Titrations; Treatment Protocols; Vaccines; Validation; Variant; Vesicular stomatitis Indiana virus; Viral; Viral Physiology; Viral hepatitis; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; anti-hepatitis B; anti-viral efficacy; antiviral drug development; cell type; cytotoxic; cytotoxicity; drug development; drug resistant virus; efficacy evaluation; high risk; high throughput screening; human coronavirus; in silico; infancy; inhibitor; interest; liver injury; luminescence; novel; novel therapeutics; preclinical study; remdesivir; small molecule; small molecule libraries; transmission process; vaccine access; viral DNA

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.

丙型肝炎病毒（HCV）感染的治疗进展迅速，最近批准的几个直接作用的抗病毒药物。然而，大多数临床使用或临床试验中的DAA靶向HCV复制周期的同一阶段，并且与快速出现的耐药病毒突变相关。此外，不同的HCV基因型和临床状况也可能需要调整治疗方案。因此，仍然需要开发新的HCV抑制剂，其靶向HCV复制周期的不同阶段，例如进入和组装。 B型肝炎病毒（HBV）感染肝细胞并引起免疫介导的肝损伤，使慢性感染患者具有发展肝硬化和肝细胞癌的高风险。目前治疗慢性HBV感染是有效的，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HBV药物，验证了这些化合物，现在正在确定它们的抑制机制。首先，建立了用于检测乙型肝炎病毒感染标志物B e抗原（HBeAg）的放大发光接近均相免疫吸附试验（AlphaLISA）。在高通量形式中，感染HepG 2-NTCP细胞并用14，402种小分子化合物的文库处理。AlphaLISA和基于ATP的细胞活力测定分别用于测量抑制和细胞毒性。从高通量筛选中，选择显示最大抑制>80%和CC 50> 5 uM的20个命中物用于进一步验证。使用正常细胞培养形式，在HepG2.215细胞、病毒感染的HepG 2-NTCP和病毒感染的人源原代肝细胞（PXB细胞）中进一步滴定所选命中物的抗HBV活性和细胞毒性谱。总的来说，大多数化合物在HepG2.215细胞和病毒感染的HepG 2-NTCP和PXB细胞中显示出对HBeAg和HBV DNA的一致抑制，其中原代细胞模型对抗病毒治疗更敏感。经过验证后，基于潜在的作用模式和抗病毒功效，正在研究高度关注的化合物，以了解其抗HBV作用的详细分子机制。 由严重急性呼吸道综合征冠状病毒2型（SARS-CoV-2）引起的2019冠状病毒病（COVID-19）已成为对全球公共卫生的严重威胁，强调了开发有效疫苗和治疗方法的紧迫性和高度优先性。 治疗，更具体地说，抗病毒的发展，仍然处于起步阶段。目前，除了用于新冠肺炎重症患者的瑞德西韦外，尚无临床批准的治疗方法或疫苗可用于这种疾病。该项目的总体目标是通过重新利用现有药物或开发新药来确定和开发针对SARS-CoV-2的有效抗病毒药物。我们正在建立非感染性细胞模型系统，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，以筛选大型小分子库中的抗SARS-CoV-2化合物，并对筛选出的高活性和无毒化合物进行广泛的临床前研究，以进一步开发药物。 在这里，我们报告说，两个丙型肝炎病毒（HCV）的融合抑制剂，在我们以前的研究中确定，双氯环嗪和氟恶唑韦，广泛阻止人类冠状病毒进入各种细胞类型。 我们开发了基于水泡性口炎病毒（VSV）假型化的多种人类冠状病毒的刺突蛋白和刺突介导的合胞体形成的多重进入试验，以检查这些抑制剂的功效并确定其机制。 两种化合物均有效，半数最大有效浓度（EC 50）值在个位数微摩尔范围内。 在SARS-CoV-2感染系统中证实了抗病毒作用。 这些化合物对最近出现的具有N439 K、Y 453 F、E484 K、N501 Y、D 614 G或P681 H突变的刺突变体同样有效。 结构建模表明，化合物结合到S蛋白融合肽附近的疏水口袋，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抗病毒活性，可能是治疗SARS-CoV-2及其变体和其他致病性CoV的有希望的线索。 自2021年底Omicron变种出现以来，它们迅速成为全球的主导变种。 与早期的武汉和其他变种相比，Omicron变种可能更容易传播。 在这项研究中，我们的目的是阐明与Omicron变异体相关的感染性改变的机制。 我们系统地评估了位于刺突S2序列中的突变，并鉴定了导致病毒融合改变的突变。 我们证明了S1/S2切割位点附近的突变降低了S1/S2切割，导致融合性降低。 HR 1和其他S2序列的突变也影响细胞-细胞融合。 基于核磁共振（NMR）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性。 我们的研究结果表明，Omicron变异体已经积累了有助于减少合胞体形成的突变，因此致病性减弱。

项目成果

Antiviral and Immunoregulatory Effects of Indoleamine-2,3-Dioxygenase in Hepatitis C Virus Infection.

• DOI: 10.1159/000375161
• 发表时间: 2015
• 期刊: Journal of innate immunity
• 影响因子: 5.3
• 作者: Lepiller Q;Soulier E;Li Q;Lambotin M;Barths J;Fuchs D;Stoll-Keller F;Liang TJ;Barth H
• 通讯作者: Barth H

Building bridges and providing transparency to the hepatitis C virus drug approval process.

为丙型肝炎病毒药物审批流程搭建桥梁并提供透明度。

• DOI: 10.1053/j.gastro.2014.10.028
• 发表时间: 2014
• 期刊: Gastroenterology
• 影响因子: 29.4
• 作者: Ghany,MarcG;Liang,TJake
• 通讯作者: Liang,TJake

Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly.

• DOI: 10.1038/nm.3190
• 发表时间: 2013-06
• 期刊: Nature medicine
• 影响因子: 82.9

Discovery of Small Molecule Entry Inhibitors Targeting the Fusion Peptide of SARS-CoV-2 Spike Protein.

• DOI: 10.1021/acsmedchemlett.1c00263
• 发表时间: 2021-08-12
• 期刊: ACS medicinal chemistry letters
• 影响因子: 4.2
• 作者: Hu X;Chen CZ;Xu M;Hu Z;Guo H;Itkin Z;Shinn P;Ivin P;Leek M;Liang TJ;Shen M;Zheng W;Hall MD
• 通讯作者: Hall MD

Discovery and Optimization of a 4-Aminopiperidine Scaffold for Inhibition of Hepatitis C Virus Assembly.

• DOI: 10.1021/acs.jmedchem.1c00696
• 发表时间: 2021-07-08
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Rolt A;Talley DC;Park SB;Hu Z;Dulcey A;Ma C;Irvin P;Leek M;Wang AQ;Stachulski AV;Xu X;Southall N;Ferrer M;Liang TJ;Marugan JJ
• 通讯作者: Marugan JJ