Mode of action of a new Tat HIV-1 inhibitor

新型 Tat HIV-1 抑制剂的作用方式

• 批准号: 8262971
• 负责人: Susana T Valente
• 金额: $ 66.78万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8262971
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alkaloids; Anti-Retroviral Agents; Antiviral Agents; Apoptosis; Binding; Binding Sites; Biological Assay; Bone Marrow; CD4 Positive T Lymphocytes; Capsid; Catalytic Domain; Cell Line; Cell Nucleolus; Cells; Chronic; Clinical Trials; Collaborations; Confocal Microscopy; Cultured Cells; DNA Polymerase II; DNA-dependent protein kinase; Data; Development; Drops; Drug resistance; Electrophoretic Mobility Shift Assay; Exclusion; Family; Fluorescence Resonance Energy Transfer; Gene Expression; Genetic Transcription; Genome; Glutathione S-Transferase; Goals; Growth; HIV; HIV Infections; HIV-1; In Vitro; Infection; Investigation; Kidney; Length; Life Cycle Stages; Liver; Lymphoid Tissue; Marines; Measures; Mediating; Monitor; Mus; Mutation; Non obese; Nuclear; Open Reading Frames; Output; Peripheral Blood Lymphocyte; Pharmaceutical Preparations; Phosphorylation; Plasma; Porifera; Property; Proviruses; Public Health; RNA; RNA Polymerase I; RNA Polymerase II; Repression; Role; Route; Running; SCID Mice; Small Interfering RNA; Sp1 Transcription Factor; Staging; Testing; Therapeutic Index; Thymus Gland; Time; Tissues; Toxic effect; Transcription Elongation; Transcription Initiation; Transcriptional Activation; Vagina; Viral; Viral Genes; Viral Proteins; Virus; Virus Replication; Zidovudine; analog; base; chromatin immunoprecipitation; cortistatin; cytotoxic; diabetic; drug candidate; drug efficacy; fighting; in vitro Assay; in vivo; inhibitor/antagonist; lymph nodes; mouse model; mutant; next generation; prevent; promoter; prophylactic; research study; telomere; viral RNA

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the mechanism by which Cortistatin A inhibits HIV-1 transcription. Human immunodeficiency virus type I (HIV-1) is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). HIV anti-retroviral therapy is based on the administration of drugs in combination, in order to minimize development of mutations that can confer single-drug resistance to the virus. The viral protein Tat, a potent activator of HIV gene expression, is a potential antiviral target. We have discovered that Cortistatin A is a promising anti-Tat drug candidate. Cortistatin A is a steroidal alkaloid isolated from the marine sponge Corticium simplex. We have found that didehydro-Cortistatin A (CA for short), is a very potent inhibitor of Tat- activated transcription of the HIV-1 provirus. CA is extremely efficient at reducing viral output from acutely infected cells as well as from chronically infected cultured cells or freshly isolated peripheral blood lymphocytes (PBLs). The half maximal effective concentration (EC50) of CA for inhibiting HIV in chronic infected cells is less than 0.1 nM and the half maximal cytotoxic concentration (CC50) for CA in cultured cell lines or freshly isolated PBLs is more than 20 5M, which confers to CA a very promising therapeutic index. We have identified an interaction between CA and Tat and with the DNA-dependent protein kinase (DNA-PK) catalytic subunit. Upon HIV infection, DNA-PK tightly associates with Tat to promote phosphorylation of the transcription factor Sp1, which results in increased HIV-1 transcription initiation, via three Sp1 binding sites in the core of the HIV-1 promoter. Our hypothesis is that CA might repress HIV replication in two ways: by inhibiting Tat-TAR interaction, thereby inhibiting elongation by RNA Polymerase I (Pol I) from the HIV promoter; and/or by inhibiting DNA-PK activation of the transcription factor Sp1 and initiation/elongation of transcription by Pol II. Both routes would result in repression of HIV-1 transcriptional activation. Our specific aims include: Aim 1.Determine whether CA inhibits HIV-1 Tat-TAR interaction in vitro and in vivo and if CA inhibits transcription elongation from the viral promoter. Assess whether CA inhibits Tat mediated DNA-PK phosphorylation of Sp1. Aim 2. In-depth assessment of CA toxicity in vivo. Aim 3. Determine CA effect on HIV-1 replication in humanized mice models. We will thus elucidate the mechanism by which CA inhibits HIV-1 transcription and evaluate its potential to inhibit HIV-1 replication in humanized mice. In order to generate a backup compound we will evaluate additional analogs generated during the course of this project. These analogs will be subsequently tested in the humanized mouse model. PUBLIC HEALTH RELEVANCE: Finding new drugs to fight AIDS is a significant contribution to public health. We will decipher the mechanism of action of a natural compound that we showed to have very potent anti HIV properties by inhibiting the action of a viral protein involved in viral amplification.

描述（由申请人提供）：该项目的目标是阐明 Cortistatin A 抑制 HIV-1 转录的机制。 I 型人类免疫缺陷病毒 (HIV-1) 是艾滋病（获得性免疫缺陷综合症）的病原体。 HIV 抗逆转录病毒治疗基于联合用药，以最大限度地减少可能对病毒产生单一药物耐药性的突变的发生。病毒蛋白 Tat 是 HIV 基因表达的有效激活剂，是潜在的抗病毒靶点。我们发现 Cortistatin A 是一种很有前途的抗 Tat 候选药物。 Cortistatin A 是一种从海洋海绵 Corticium simplex 中分离出来的甾体生物碱。我们发现二脱氢-Cortistatin A（简称CA）是一种非常有效的HIV-1原病毒Tat激活转录抑制剂。 CA 在减少急性感染细胞以及慢性感染培养细胞或新鲜分离的外周血淋巴细胞 (PBL) 的病毒输出方面非常有效。 CA在慢性感染细胞中抑制HIV的半数最大有效浓度（EC50）小于0.1 nM，在培养细胞系或新鲜分离的PBL中CA的半数最大细胞毒性浓度（CC50）超过20 5M，这赋予CA非常有前途的治疗指数。 我们已经确定了 CA 和 Tat 之间以及与 DNA 依赖性蛋白激酶 (DNA-PK) 催化亚基之间的相互作用。 HIV 感染后，DNA-PK 与 Tat 紧密结合，促进转录因子 Sp1 的磷酸化，从而通过 HIV-1 启动子核心中的三个 Sp1 结合位点增加 HIV-1 转录起始。我们的假设是，CA 可能通过两种方式抑制 HIV 复制：通过抑制 Tat-TAR 相互作用，从而抑制 RNA 聚合酶 I (Pol I) 从 HIV 启动子延伸；和/或通过抑制转录因子 Sp1 的 DNA-PK 激活和 Pol II 的转录起始/延长。两种途径都会导致 HIV-1 转录激活的抑制。我们的具体目标包括： 目的 1.确定CA是否在体外和体内抑制HIV-1 Tat-TAR相互作用，以及CA是否抑制病毒启动子的转录延伸。评估 CA 是否抑制 Tat 介导的 Sp1 DNA-PK 磷酸化。 目标 2. 深入评估 CA 体内毒性。 目标 3. 确定人源化小鼠模型中 CA 对 HIV-1 复制的影响。 因此，我们将阐明 CA 抑制 HIV-1 转录的机制，并评估其在人源化小鼠中抑制 HIV-1 复制的潜力。为了生成备用化合物，我们将评估在该项目过程中生成的其他类似物。这些类似物随后将在人源化小鼠模型中进行测试。 公共卫生相关性：寻找抗击艾滋病的新药是对公共卫生的重大贡献。我们将破译一种天然化合物的作用机制，通过抑制参与病毒扩增的病毒蛋白的作用，我们证明该化合物具有非常有效的抗艾滋病毒特性。