Fusion assay of the HIV-induced CD4 down-modulation

HIV 诱导的 CD4 下调的融合测定

• 批准号: 7140590
• 负责人: JUAN LAMA
• 金额: $ 32.3万
• 依托单位: LA JOLLA INST FOR MOLECULAR MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140590
• 关键词: CD4 molecule; HIV envelope protein gp120; HIV infections; antiAIDS agent; cell fusion; combinatorial chemistry; human tissue; protein protein interaction; small molecule; technology /technique development; virus infection mechanism

DESCRIPTION (provided by applicant): Currently used antiretrovirals targeting the viral reverse transcriptase, protease, and envelope proteins have reduced mortality and morbidity in HIV-infected patients. However, the emergence of viral strains resistant to the inhibitors, and the appearance of side effects after prolonged drug administration pose important problems to the efficacy of HAART treatments. Characterization and evaluation of novel targets for therapeutic intervention is a priority in HIV/AIDS research. CD4 down-modulation plays an important role in HIV pathogenesis. In vitro, failure to down-modulate CD4 results in the production of HIV-1 particles with severely reduced infectivity whereas in vivo enhanced receptor down-modulation confers a replicative advantage to HIV-1 in primary lymphocytes. To date, no specific inhibitors of this function have been characterized. To facilitate the discovery of new drugs, we propose to develop a fusion-based assay to screen for specific inhibitors of the HIV-induced CD4 down-modulation activity. This assay will rely on the fact that the rate of fusion between gp120-expressing cells and CD4-positive cells can be modulated by the concentration of these proteins on the cell surface. We hypothesize that since HIV down-modulates expression of CD4, interfering with this function in infected cells will result in higher amounts of surface-CD4, and therefore fusion will occur to a higher extent than in cells in which efficient down-modulation occurs. Experiments to evaluate the sensitivity and specificity of this assay are proposed in Specific Aim 1. To validate the assay, we will screen synthetic combinatorial libraries for potential inhibitors of the virus-induced CD4 down-modulation (Specific Aim 2). We hypothesize that drugs inhibiting this function will impair HIV replication. Our goal is to identify lead compounds with defined chemical structures that could evolve into a discovery program for a new class of anti-HIV drugs. The specific aims are: Aim 1, to develop a fusion-based assay for the identification of inhibitors of the HIV-induced CD4 down-modulation, and; Aim 2, to characterize small-molecule inhibitors of the HIV-induced CD4 down-modulation activity.

描述（由申请人提供）： 目前使用的针对病毒逆转录酶、蛋白酶和包膜蛋白的抗逆转录病毒药物已降低了艾滋病毒感染患者的死亡率和发病率。然而，对抑制剂产生耐药性的病毒株的出现，以及长期给药后副作用的出现，给HAART治疗的疗效带来了重要问题。治疗干预新目标的表征和评估是艾滋病毒/艾滋病研究的首要任务。 CD4下调在HIV发病机制中发挥着重要作用。在体外，未能下调 CD4 会导致产生感染性严重降低的 HIV-1 颗粒，而体内增强的受体下调则赋予 HIV-1 在原代淋巴细胞中的复制优势。迄今为止，尚未表征该功能的特异性抑制剂。为了促进新药的发现，我们建议开发一种基于融合的测定法来筛选 HIV 诱导的 CD4 下调活性的特异性抑制剂。该测定将依赖于以下事实：表达 gp120 的细胞和 CD4 阳性细胞之间的融合率可以通过细胞表面上这些蛋白质的浓度来调节。我们假设，由于 HIV 下调 CD4 的表达，干扰受感染细胞中的这种功能将导致表面 CD4 含量更高，因此与发生有效下调的细胞相比，融合的程度会更高。具体目标 1 中提出了评估该测定的敏感性和特异性的实验。为了验证该测定，我们将筛选合成组合文库以寻找病毒诱导的 CD4 下调的潜在抑制剂（具体目标 2）。我们假设抑制这种功能的药物会损害艾滋病毒的复制。我们的目标是识别具有明确化学结构的先导化合物，这些化合物可以发展成为新型抗艾滋病毒药物的发现计划。 具体目标是： 目标 1，开发一种基于融合的检测方法，用于鉴定 HIV 诱导的 CD4 下调抑制剂；目标 2，表征 HIV 诱导的 CD4 下调活性的小分子抑制剂。