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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

随着几种直接作用抗病毒药的批准，丙型肝炎病毒（HCV）感染的治疗已迅速发展。但是，临床使用或临床试验中的大多数DAA针对HCV复制周期的同一阶段，并且与药物耐药性病毒突变的快速出现有关。另外，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复制周期不同阶段的新的HCV抑制剂，例如进入和组装。丙型肝炎病毒（HBV）感染肝细胞并引起免疫介导的肝损伤，使慢性感染的患者患有肝硬化和肝细胞癌的高风险。当前对慢性HBV感染的治疗方法有效，但有许多局限性，迫切需要开发新疗法。在这项研究中，我们通过高通量筛选确定了新型的抗HBV剂，验证了这些化合物，现在正在确定其抑制作用机制。首先，建立了放大的发光近端均质测定与免疫吸附测定法（Alphalisa），以检测HBV感染的标志物（HBEAG）丙型肝炎抗原（HBEAG）。以高通量格式，将HEPG2-NTCP细胞感染并用14,402个小分子化合物的库处理。 α和基于ATP的细胞活力测定法分别用于测量抑制和细胞毒性。从高吞吐量屏幕中，选择了二十个命中率显示最大抑制> 80％和CC50> 5UM以进行进一步验证。使用正常的细胞培养格式，在HEPG2.215细胞，病毒感染的HEPG2-NTCP和病毒感染的人类原始原始原始原发性肝细胞（PXB细胞）中进一步滴定了所选命中的抗HBV活性和细胞毒性谱。总的来说，大多数化合物在HEPG2.215细胞中表现出对HBEAG和HBV DNA的一致性抑制作用，并且感染了病毒的HEPG2-NTCP和PXB细胞，主要细胞模型对抗病毒治疗更为敏感。验证后，基于潜在的作用方式和抗病毒功效，对其抗HBV效应的详细分子机制进行了高关注的化合物。 2019年冠状病毒病（COVID-19）是由严重的急性呼吸综合症冠状病毒2（SARS-COV-2）引起的，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疗法的紧迫性和优先级。治疗性，更具体地说是抗病毒发育，仍处于起步阶段。目前，除Remdesivir针对患有COVID-19的严重患者外，尚无临床批准的疗法或疫苗可用于该疾病。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。我们正在建立非感染的基于细胞的模型系统，以研究SARS-COV-2感染和复制周期的各个阶段，以基于这些模型系统开发高通量平台，以筛选大型小分子库，以用于抗SARS-COV-COV-2化合物，并对筛查的高度活性和非氧化型化合物进行更广泛的抗议研究。在这里，我们报告说，在我们先前的研究中鉴定出的两种丙型肝炎病毒（HCV）融合抑制剂，即二氯环嗪和氟氧唑维尔，它们广泛地阻止了人冠状病毒进入各种细胞类型。我们根据各种人类COV的尖峰蛋白和尖峰介导的合成症形成基于囊泡口腔炎病毒（VSV）开发了多个入口测定，以检查这些抑制剂的机制。两种化合物在单位微摩尔范围内的最大有效浓度（EC50）值有效。在实时SARS-COV-2感染系统中证实了抗病毒作用。这些化合物同样有效地对新兴的尖峰变体具有N439K，Y453F，E484K，N501Y，D614G或P681H突变。结构建模表明，该化合物与S蛋白融合肽附近的疏水口袋结合，与它们作为融合抑制剂的潜在作用机理一致。总而言之，这些融合抑制剂具有广谱抗病毒活性，并且可能有望治疗SARS-COV-2，其变异和其他致病性COV。自2021年底Omicron变体的出现以来，它们迅速成为全球占主导地位的变体。与较早的武汉和其他变体相比，Omicron变体可能更容易传输。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的S2序列中的突变，并确定了导致病毒融合改变的突变。我们证明了S1/S2裂解位点附近的突变会降低S1/S2裂解，从而降低融合性。 HR1和其他S2序列中的突变也影响了细胞 - 细胞融合。基于核磁共振（NMR）研究和计算机建模，这些突变可能会在病毒融合的多个步骤下影响融合。我们的发现表明，Omicron变体积累了突变，这些突变有助于降低合胞体形成，从而导致致病性减弱。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.