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（由申请人提供）：该提案描述了针对人类免疫缺陷病毒1型（HIV-1）融合有效的小分子的高吞吐量筛选测定的开发。防止HIV-1融合会抑制病毒进入人类宿主细胞，有效保护未感染的细胞并改善HIV感染患者的可用治疗选择。当前有一个单一的融合抑制剂，一个肽enfuvirtide（R），它很昂贵，仅通过静脉内给药才能获得。可以口服的药物是低分子量化合物，通常以较低的成本制造并分布得更广泛。筛选测定法将能够自动测试学术，政府和药品设施中可用的数千种低分子量化合物。初始屏幕选择的化合物将经过进一步的测试和修改以提高效力。所描述的测定涉及简单地添加来自HIV-1 GP41病毒融合蛋白的两种肽，并将其添加到镀板库化合物的井中。用荧光团和金属绑扎的染料复合物对肽进行了修饰，这使其微摩尔缔合之后进行了简单的荧光强度读数。从文库中的化合物通过竞争性抑制肽缔合的能力来评估活性，并随着荧光的伴随量增加。阳性结果表明化合物能够抑制融合。荧光信号的强度与化合物的效力直接相关。这是一种使用廉价和非危害成分的生化测定法。我们将证明它对病毒靶标的高度特异性，非常稳健和敏感，并且是化合物抑制细胞培养融合的能力的极好指标。在这项研究中，我们将优化测定法以最大程度的敏感性，扩大小分子的选择，以包括可以束缚以创建更强大抑制剂的片段，并探索NMR和荧光方法，以促进图书馆中新发现的hits的优化过程。该项目将对HIV-1融合抑制剂的化合物库进行系统的筛选。屏幕将特别识别与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。作为多药治疗策略的一部分，新发现的小分子候选物可能被发展为有效控制HIV-1感染的进入抑制剂。