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Characterization of Cellular Proteins Involved in HIV Infection

HIV 感染相关细胞蛋白的表征

基本信息

批准号：
7757981
负责人：
WILLIAM AUSTIN O'BRIEN
金额：
$ 43.09万
依托单位：
ZIRUS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-15 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7757981
关键词：
AIDS/HIV problem Adopted Adrenergic beta-Antagonists Affect Animal Model Animals Anti-Infective Agents Anti-Retroviral Agents Antisense Oligonucleotides Antiviral Agents Antiviral Therapy Binding Biological Assay Biological Models Biotechnology CCR5 gene Calcium Channel Blockers Candidate Disease Gene Cell Line Cell Survival Cells Centers for Disease Control and Prevention (U.S.)Chemokine (C-C Motif) Receptor 5 Cholinesterase Inhibitors Clinical Collaborations Collection Communicable Diseases Confocal Microscopy Coupled Cowpox virus Data Development Disease Doctor of Philosophy Down-Regulation Drug Delivery Systems Drug resistance Ebola virus Event Frankfurt-Marburg Syndrome Virus Gene Expression Gene Targeting Genes Goals HIV HIV Infections HIV vaccine HIV-1 Human Human immunodeficiency virus test Immunity Infection Insertional Mutagenesis Institution Integrase Integrase Inhibitors Knowledge Laboratories Lead Libraries Life Cycle Stages Lytic Measles virus Measures Medical Methodology Methods Molecular Biology Mus National Institute of Allergy and Infectious Disease Organ Outcome Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phase Preclinical Drug Development Principal Investigator Production Proteins Quality of life RNA Interference Reovirus Research Resistance Resolution Reverse Transcription Role Safety Screening procedure Simplexvirus Small Business Innovation Research Grant Small Interfering RNA Staging Structure System Technology Testing Texas Therapeutic Therapeutic Intervention Time Toxic effect Translating Universities Validation Viral Viral Proteins Virus Virus Diseases Virus Replication Work antiretroviral therapy base biodefense cellular targeting design drug development drug discovery expectation experience extracellular gene discovery gene function gene repression improved in vivo influenzavirus inhibitor/antagonist innovation macrophage meetings multidisciplinary non-nucleoside reverse transcriptase inhibitors novel novel therapeutics pathogen pre-clinical prevent programs public health relevance research study resistant strain small molecule small molecule libraries success therapy development vaccine development viral resistance virology virus pathogenesis

项目摘要

DESCRIPTION (provided by applicant): Important advances have been made in the treatment of viral infections over the last two decades, especially with the extensive availability of drugs to treat HIV infection, but there is a need to develop new drugs particularly with the challenges facing HIV vaccine development and the propensity to develop drug resistance. With FDA approval in 2007 of the first antiretroviral drug targeting a cellular gene, the HIV coreceptor CCR5, there is now proof of concept that a cellular target can be used to develop a highly effective, safe, nontoxic and well-tolerated antiretroviral drug. Although somewhat of a paradigm shift for infectious diseases, in fact, cellular genes are targeted routinely for non-infectious diseases. In any case this demonstrates the potential for targeting cellular genes for antiviral therapy. This proposal aims to extend the work accomplished over the last several years using a proprietary technology, gene-trap, which is a rapid and efficient method for the identification of host genes that are necessary for infection, replication or pathogenesis by viruses. Gene-trap insertional mutagenesis has identified cells with genes that are not expressed; loss of these genes allows the cell to survive during lethal virus infection, but apparently is not required for cell survival. To date, we have identified several pathways used in common by a number of viruses, suggesting that there may be key cellular mechanisms necessary for pathogenesis and that blocking such mechanisms in vivo could confer broad-spectrum therapeutic intervention. Our hypothesis is that by screening siRNAs to genes identified by gene trap and confirmed by influenza virus infection and testing them in indicator cell lines and primary marrophages for HIV infection, we will discover new host gene targets that can be used to develop antiretroviral drugs. During Phase I, we will test the effects of down regulation of specific genes indicator cell lines for effect on HIV based on well-defined criteria to select lead targets for drug discovery criteria. The most important criteria is a high resolution 3D crystal structure available for the target. The importance of crystal structure is to facilitate drug discovery efforts that will begin in phase I and that will be the primary focus in phase II of the project. Using the indicator cell line U373 MAGI, which are readily transfectable with siRNA, we will test the effect on HIV replication both by beta-GAL activity, which will identify inhibition of replication at steps prior to gene expression, including entry reverse transcription and integration, and we will also measure extracellular virus by HIV p24 production and Real-time PCR for HIV RNA, to capture inhibition events that occur in the second half of the virus life cycle. By performing these two assays in parallel we determine whether the gene is important in early viral life cycle events or later ones. To confirm the relevance of these genes for HIV replication we will assess effects of gene down regulation on primary human macrophages, which are natural targets for infection. In independent studies to perform gene-trap and develop drugs for influenza virus infection, the genes that will be assessed for effects on HIV replication will have been assessed in parallel in mouse studies. Therefore, we will have animal safety data for these genes, with down regulation maintained in the animals for a period of one month. Genes identified as important for HIV infection, and meeting our criteria for drug development, will be screened with small molecule libraries with collaborators at Emory, lead by Dennis Liotta, PhD. This proposal will be likely to lead to a large number of new antiretroviral drugs that affect novel targets for therapy. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: Tremendous advances have been made in treatment of HIV infection over the last ten years, with introduction of three new drugs in the last one to two years that are safe, well-tolerated and typically effective against virus strains resistant to older drugs. The HIV pipeline is not robust, however, and after several "me too" drugs directed to the same targets over the next several years, integrase and CCR5, there are not many other antiretroviral therapies that will be developed clinically. Thus, there is an urgent need to develop new drugs against novel targets for HIV since resistance to older drugs emerges in many patients. Our proposal will identify human proteins as targets important for HIV infection that can be used to develop new classes of antiretroviral drugs. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page

描述(申请人提供)：在过去的二十年里，在治疗病毒感染方面取得了重要进展，特别是随着治疗艾滋病毒感染的药物的广泛可用，但有必要开发新药，特别是在艾滋病毒疫苗开发面临的挑战和产生耐药性的倾向的情况下。随着FDA在2007年批准了第一种针对细胞基因的抗逆转录病毒药物-HIV共受体CCR5，现在有了概念证明，可以使用细胞靶点来开发高效、安全、无毒和耐受性良好的抗逆转录病毒药物。尽管这在某种程度上是传染病的范式转变，但事实上，细胞基因通常是非传染性疾病的靶点。无论如何，这证明了以细胞基因为靶点进行抗病毒治疗的潜力。这项提议旨在利用一种名为基因陷阱的专利技术来扩展过去几年完成的工作，基因陷阱是一种快速而有效的方法，用于识别病毒感染、复制或致病所必需的宿主基因。基因陷阱插入突变技术已经确定了带有不表达基因的细胞；这些基因的丢失使细胞能够在致命病毒感染期间存活，但显然不是细胞生存所必需的。到目前为止，我们已经确定了一些病毒共同使用的几条途径，这表明可能存在致病所需的关键细胞机制，在体内阻断这些机制可以提供广泛的治疗干预。我们的假设是，通过筛选由基因陷阱识别并经流感病毒感染确认的基因的siRNA，并在HIV感染的指示细胞系和原代噬菌体中进行测试，我们将发现新的宿主基因靶点，可用于开发抗逆转录病毒药物。在第一阶段，我们将根据明确的标准测试特定基因指示细胞系对HIV影响的下调效果，以选择药物发现标准的先导靶标。最重要的标准是目标可以获得高分辨率的3D晶体结构。晶体结构的重要性是为了促进将在第一阶段开始的药物发现工作，这将是该项目第二阶段的主要重点。使用易于与siRNA一起转基因的指示细胞系U373MAGI，我们将通过β-Gal活性来测试对HIV复制的影响，这将确定在基因表达之前的几个步骤中对复制的抑制，包括进入逆转录和整合，我们还将通过HIV p24的产生和HIV RNA的实时定量PCR来检测细胞外病毒，以捕捉在病毒生命周期的后半部分发生的抑制事件。通过同时进行这两种检测，我们确定该基因在早期病毒生命周期事件中是否重要，还是在较晚的事件中起重要作用。为了确认这些基因与HIV复制的相关性，我们将评估基因下调对原代人类巨噬细胞的影响，这些巨噬细胞是自然感染的目标。在进行基因捕捉和开发流感病毒感染药物的独立研究中，将评估对艾滋病毒复制的影响的基因将在小鼠实验中进行平行评估。因此，我们将拥有这些基因的动物安全数据，并在动物身上保持一个月的下调。由丹尼斯·利奥塔博士领导的埃默里大学的合作者将利用小分子文库对被确定为对艾滋病毒感染重要并符合我们的药物开发标准的基因进行筛选。这一提议可能会导致大量新的抗逆转录病毒药物，这些药物会影响新的治疗靶点。PHS 398/2590(Rev.11/07)第1页继续格式页公共卫生相关性：在过去十年中，艾滋病毒感染的治疗取得了巨大进步，在过去一到两年中推出了三种新药，这些新药安全、耐受性好，通常对耐老药的病毒株有效。然而，艾滋病毒的渠道并不牢固，在接下来的几年里，几种“我也一样”的药物针对相同的目标，整合酶和CCR5，没有太多其他抗逆转录病毒疗法可以在临床上开发。因此，迫切需要开发针对艾滋病毒新靶点的新药，因为许多患者出现了对旧药的抗药性。我们的提案将确定人类蛋白质是艾滋病毒感染的重要靶点，可用于开发新型抗逆转录病毒药物。PHS 398/2590(11/07版)第1页续订格式页