High-Throughput Screening for Human Immunodeficiency Virus Fusion Inhibitors

描述（由申请人提供）：本提案描述了一种针对人类免疫缺陷病毒1型（HIV-1）融合有效的小分子高通量筛选试验的发展。防止艾滋病毒-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现有治疗选择。目前有一种单一的融合抑制剂，一种肽恩福韦肽(r)，价格昂贵，只能通过静脉给药。可口服的药物是低分子量化合物，通常可以以较低的成本制造并更广泛地分布。筛选试验将能够自动测试学术、政府和制药机构提供的数千种低分子量化合物。通过初步筛选选出的化合物将接受进一步的测试和修饰以提高效力。所描述的实验包括简单地将来自HIV-1 gp41（病毒融合蛋白）的两个肽添加到镀库化合物的孔中。这些肽是用荧光团和金属连接的染料络合物修饰的，这使得它们的微摩尔结合之后可以简单地读出荧光强度。通过竞争性抑制肽结合的能力来评估文库中的化合物的活性，并伴随荧光增加。阳性结果表明该化合物具有抑制融合的能力。荧光信号的强度与化合物的效价直接相关。这是一种生化试验，使用廉价和无害的成分。我们将证明它对病毒靶标具有高度特异性，非常健壮和敏感，并且是化合物在细胞培养中抑制融合能力的极好指标。在这项研究中，我们将优化检测方法以获得最大的灵敏度，扩大小分子的选择范围，以包括可以连接到更强大的抑制剂的片段，并探索NMR和荧光方法，以促进从文库中新发现的hits的优化过程。该项目将使HIV-1融合抑制剂化合物文库的系统筛选成为可能。这种筛选将专门识别与gp41融合蛋白结合的分子，以防止有效病毒融合所需的构象变化。新发现的候选小分子可能会成为有效控制HIV-1感染的入口抑制剂，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.