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High-Throughput Screening for Human Immunodeficiency Virus Fusion Inhibitors

人类免疫缺陷病毒融合抑制剂的高通量筛选

基本信息

批准号：
7678669
负责人：
MIRIAM GOCHIN
金额：
$ 3.83万
依托单位：
TOURO UNIVERSITY OF CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-05 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7678669
关键词：
Affinity Antiviral Agents Binding Binding Sites Biochemical Biological Assay Cells Chimeric Proteins Classification Cobalt Coiled-Coil Domain Complex Cultured Cells Data Detection Development Drug Design Drug Kinetics Dyes Economics Fluorescence Glycoproteins Goals Government HIV HIV-1 Human In Vitro Infection Investigation Ions Laboratories Length Libraries Ligands Link Literature Measurement Metals Methods Modification Molecular Conformation Molecular Weight Patients Peptides Pharmaceutical Preparations Pharmacologic Substance Process Range Screening procedure Signal Transduction Site Structure T-20 Testing Variant Viral Viral Fusion Proteins Viral Proteins Virus Work base concept cost design extracellular fluorophore functional group high throughput screening improved inhibitor/antagonist intravenous administration novel peptide T20 peptidomimetics prevent protein aminoacid sequence receptor restraint scaffold small molecule small molecule libraries tool

项目摘要

DESCRIPTION (provided by applicant): This proposal describes the development of a high throughput screening assay for small molecules effective against Human Immunodeficiency Virus Type 1 (HIV-1) fusion. Preventing HIV-1 fusion would inhibit the entry of virus into human host cells, effectively protecting uninfected cells and improving the available treatment options for HIV infected patients. Currently there is a single fusion inhibitor, a peptide Enfuvirtide(r), which is expensive and available only by intravenous administration. Drugs which can be taken orally are low molecular weight compounds which usually can be manufactured at lower cost and be more widely distributed. The screening assay will be capable of automated testing of thousands of low molecular weight compounds available in academic, government and pharmaceutical facilities. Compounds selected by the initial screen would be subject to further testing and modification to improve potency. The assay described involves the simple addition of two peptides derived from HIV-1 gp41, the viral fusion protein, to wells of plated library compounds. The peptides are modified with a fluorophore and metal-ligated dye complex, which enables their micromolar association to be followed by a simple fluorescence intensity readout. Compounds from a library are assessed for activity by their ability to competitively inhibit the peptide association, with a concomitant fluorescence increase. A positive result indicates that a compound is capable of fusion inhibition. The intensity of the fluorescence signal is directly correlated to the compound's potency. This is a biochemical assay, using inexpensive and non-hazardous components. We will show that it is highly specific for the viral target, extremely robust and sensitive, and an excellent indicator of a compound's ability to inhibit fusion in cell culture. In this study, we will optimize the assay for maximum sensitivity, broaden the selection of small molecules to include fragments that could be tethered to create more powerful inhibitors, and explore NMR and fluorescence methods for facilitating the optimization process of newly discovered hits from a library. This project will enable systematic screening of compound libraries for HIV-1 fusion inhibitors. The screen will specifically identify molecules that bind to the gp41 fusion protein in such as way as to prevent the conformational change required for effective viral fusion. Newly discovered small molecule candidates may be developed into entry inhibitors effective in controlling HIV-1 infection, as part of a multi-drug treatment strategy.

描述(申请人提供)：这项建议描述了一种高通量筛选小分子有效对抗人类免疫缺陷病毒1型(HIV-1)融合的方法的发展。阻止HIV-1融合将抑制病毒进入人类宿主细胞，有效地保护未感染的细胞，并改善艾滋病毒感染患者的可用治疗选择。目前有一种单一的融合抑制剂，一种多肽enfuvitide(R)，它很昂贵，只有通过静脉注射才能获得。可以口服的药物是低分子化合物，通常可以更低的成本制造，分布更广泛。该筛选试验将能够对学术、政府和制药设施中提供的数千种低分子化合物进行自动测试。通过初始筛选选择的化合物将接受进一步的测试和修改，以提高效力。所描述的分析包括将来自HIV-1gp41(病毒融合蛋白)的两个肽简单地添加到多个平板库化合物中。这些多肽被荧光团和金属连接的染料复合体修饰，这使得它们的微摩尔结合之后可以通过简单的荧光强度读数来进行。来自文库的化合物通过竞争性抑制多肽结合的能力来评估活性，并伴随着荧光增加。阳性结果表明该化合物具有融合抑制作用。荧光信号的强度与化合物的效力直接相关。这是一种生化分析，使用廉价和无害的成分。我们将展示它对病毒靶标的高度特异性，极其强大和敏感，是化合物在细胞培养中抑制融合能力的极好指示器。在这项研究中，我们将优化检测以获得最高的灵敏度，扩大小分子的选择范围，包括可以连接以产生更强大的抑制剂的片段，并探索核磁共振和荧光方法，以促进从文库中新发现的HITS的优化过程。该项目将能够系统地筛选HIV-1融合抑制剂的化合物文库。该筛查将专门识别与gp41融合蛋白结合的分子，例如防止有效病毒融合所需的构象变化。作为多种药物治疗策略的一部分，新发现的小分子候选药物可能会被开发成有效控制HIV-1感染的进入抑制剂。