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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（由申请人提供）：在过去的二十年中，在治疗病毒感染方面取得了重要进展，特别是随着治疗HIV感染的药物的广泛可用性，但需要开发新药，特别是HIV疫苗开发面临的挑战和产生耐药性的倾向。随着FDA在2007年批准了第一种靶向细胞基因（HIV辅助受体CCR 5）的抗逆转录病毒药物，现在已经证明了细胞靶点可以用于开发高效，安全，无毒和耐受性良好的抗逆转录病毒药物的概念。尽管这在某种程度上是传染病的一种范式转变，但事实上，细胞基因通常是非传染性疾病的靶点。在任何情况下，这都证明了靶向细胞基因用于抗病毒治疗的潜力。该提案旨在扩大过去几年中使用专利技术-基因陷阱-所完成的工作，基因陷阱是一种快速有效的方法，用于鉴定病毒感染、复制或致病所必需的宿主基因。基因陷阱插入突变已经鉴定出具有不表达的基因的细胞;这些基因的丢失允许细胞在致命病毒感染期间存活，但显然不是细胞存活所必需的。到目前为止，我们已经确定了一些病毒共同使用的几种途径，这表明可能存在致病所必需的关键细胞机制，并且在体内阻断这些机制可以提供广谱治疗干预。我们的假设是，通过筛选基因陷阱识别的基因和流感病毒感染证实的siRNA，并在指示细胞系和HIV感染的原代骨髓中测试它们，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，我们将根据明确定义的标准测试特定基因指示细胞系下调对HIV的影响，以选择药物发现标准的主要靶点。最重要的标准是可用于目标的高分辨率3D晶体结构。晶体结构的重要性是促进药物发现的努力，这将开始在第一阶段，这将是主要的重点，在第二阶段的项目。使用指示细胞系U373 MAGI，其易于用siRNA转染，我们将通过β-GAL活性测试对HIV复制的影响，这将鉴定在基因表达之前的步骤中的复制抑制，包括进入逆转录和整合，我们还将通过HIV p24产生和HIV RNA的实时PCR测量细胞外病毒，以捕获病毒生命周期后半期发生的抑制事件。通过平行进行这两种检测，我们确定该基因在早期病毒生命周期事件或后期事件中是否重要。为了证实这些基因与HIV复制的相关性，我们将评估基因下调对原代人巨噬细胞的影响，巨噬细胞是感染的天然靶点。在进行基因捕获和开发流感病毒感染药物的独立研究中，将在小鼠研究中平行评估对HIV复制影响的基因。因此，我们将获得这些基因的动物安全性数据，并在动物中维持一个月的下调。被确定为对HIV感染重要的基因，并符合我们的药物开发标准，将与埃默里大学的合作者一起用小分子文库进行筛选，由Dennis Liotta博士领导。这一提议将可能导致大量新的抗逆转录病毒药物的产生，从而影响新的治疗目标。PHS 398/2590（Rev. 11/07）Page 1 Continuation Format Page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的进步，在过去的一到两年中，三种新药被引入，这些新药安全、耐受性良好，并且通常对对耐旧药物的病毒株有效。然而，艾滋病毒的管道并不健全，在未来几年内，几种针对相同目标的“我也是”药物，整合酶和CCR 5之后，临床上将开发的其他抗逆转录病毒疗法并不多。因此，迫切需要开发针对HIV新靶点的新药，因为许多患者出现了对旧药物的耐药性。我们的建议将确定人类蛋白质作为艾滋病毒感染的重要靶点，可用于开发新的抗逆转录病毒药物。PHS 398/2590（Rev. 11/07）第1页