High-Throughput Screening for Human Immunodeficiency Virus Fusion Inhibitors

描述（由申请方提供）：本提案描述了有效对抗人类免疫缺陷病毒1型（HIV-1）融合的小分子的高通量筛选试验的开发。防止HIV-1融合将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方案。目前有一种单一的融合抑制剂，一种肽Enfuvirtide（r），它价格昂贵，只能通过静脉注射获得。可口服的药物是低分子量化合物，其通常可以以较低的成本制造并且更广泛地分布。筛选试验将能够自动测试学术、政府和制药设施中可用的数千种低分子量化合物。通过初步筛选选择的化合物将进行进一步的测试和修改，以提高效力。所描述的测定法包括简单地将来自HIV-1 gp 41（病毒融合蛋白）的两种肽添加到平板文库化合物的威尔斯孔中。用荧光团和金属连接的染料络合物修饰肽，这使得它们的微摩尔缔合能够随后进行简单的荧光强度读出。通过来自文库的化合物竞争性抑制肽缔合的能力以及伴随的荧光增加来评估其活性。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。这是一种生化分析，使用廉价和无害的组件。我们将证明它对病毒靶点具有高度特异性，非常稳健和敏感，并且是化合物抑制细胞培养中融合能力的极好指标。在这项研究中，我们将优化最大灵敏度的测定，扩大小分子的选择范围，包括可以被束缚以产生更强大的抑制剂的片段，并探索NMR和荧光方法，以促进从库中新发现的命中的优化过程。该项目将能够系统地筛选HIV-1融合抑制剂的化合物库。筛选将特异性地鉴定以防止有效病毒融合所需的构象变化的方式结合gp 41融合蛋白的分子。新发现的小分子候选物可能被开发成有效控制HIV-1感染的进入抑制剂，作为多药物治疗策略的一部分。

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.