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.

随着最近几种直接作用抗病毒药物的批准，丙型肝炎病毒（HCV）感染的治疗取得了迅速进展。然而，大多数临床使用或临床试验中的DAA都针对HCV复制周期的同一阶段，并且与耐药病毒突变的快速出现有关。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，仍然需要开发针对HCV复制周期的不同阶段（例如进入和组装）的新HCV抑制剂。 乙型肝炎病毒（HBV）感染肝细胞并引起免疫介导的肝损伤，使长期感染的患者罹患肝硬化和肝细胞癌的风险很高。目前治疗慢性乙型肝炎病毒感染的方法有效，但存在许多局限性，迫切需要开发新疗法。在这项研究中，我们通过高通量筛选鉴定了新型抗乙肝病毒药物，验证了这些化合物，现在正在确定它们的抑制机制。首先，建立了放大发光邻近均相分析联免疫吸附分析（AlphaLISA）来检测乙型肝炎e抗原（HBeAg），这是乙型肝炎病毒感染的标志物。在高通量格式中，HepG2-NTCP 细胞被感染并用包含 14,402 种小分子化合物的文库进行处理。 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%且CC50>5uM的20个命中用于进一步验证。使用正常细胞培养形式，在 HepG2.215 细胞、病毒感染的 HepG2-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的抗 HBV 活性和细胞毒性特征。总的来说，大多数化合物对 HepG2.215 细胞以及病毒感染的 HepG2-NTCP 和 PXB 细胞中的 HBeAg 和 HBV DNA 表现出一致的抑制作用，并且原代细胞模型对抗病毒治疗更敏感。经过验证后，基于潜在的作用方式和抗病毒功效，人们正在研究高度关注的化合物，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 (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，凸显了开发有效疫苗和疗法的紧迫性和高度优先性。 治疗，更具体地说，抗病毒药物的开发仍处于起步阶段。目前，除了用于治疗 Covid-19 重症患者的瑞德西韦外，尚无临床批准的疗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识别和开发针对 SARS-CoV-2 的有效抗病毒药物。我们正在建立基于非感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，并基于这些模型系统开发高通量平台来筛选抗SARS-CoV的大型小分子库-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，以用于进一步的药物开发。 在这里，我们报告了我们之前的研究中发现的两种丙型肝炎病毒（HCV）融合抑制剂，二氯环利嗪和氟恶唑韦，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）的多重进入测定法，该病毒使用各种人类冠状病毒的刺突蛋白和刺突介导的合胞体形成进行假型化，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，半最大有效浓度 (EC50) 值均在个位数微摩尔范围内。 抗病毒作用在活的 SARS-CoV-2 感染系统中得到证实。 这些化合物对最近出现的带有 N439K、Y453F、E484K、N501Y、D614G 或 P681H 突变的刺突变体同样有效。 结构模型表明，这些化合物与 S 蛋白融合肽附近的疏水口袋结合，这与它们作为融合抑制剂的潜在作用机制一致。 总之，这些融合抑制剂具有广谱抗病毒活性，可能是治疗 SARS-CoV-2、其变体和其他致病性 CoV 的有希望的先导药物。 自 2021 年底 Omicron 变体出现以来，它们迅速成为全球的主导变体。 与早期的武汉变种和其他变种相比，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刺突 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会减少 S1/S2 裂解，从而导致融合性降低。 HR1 和其他 S2 序列的突变也会影响细胞与细胞的融合。 根据核磁共振（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 变体积累了突变，导致合胞体形成减少，从而减弱了致病性。

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

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

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 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