Cell Based EFC Assay for Protease Drug Discovery

用于蛋白酶药物发现的基于细胞的 EFC 测定

• 批准号: 6688470
• 负责人: PYARE L KHANNA
• 金额: $ 12.2万
• 依托单位: DISCOVERX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6688470
• 关键词: Alzheimer's disease; active sites; amyloid proteins; bioassay; biotechnology; chimeric proteins; drug delivery systems; endopeptidases; enzyme activity; high throughput technology; radioimmunoassay; technology /technique development

DESCRIPTION (provided by applicant): Proteases are critical enzymes involved in the cleavage of proteins to smaller peptides and are important molecular targets for drug discovery. Few cell-based technologies exist to assay specific cleavage of substrates by proteases in a HTS format, which has hindered drug discovery against proteases. DiscoveRx is developing a technology that can measure activity of any protease whose substrate selective cleavage site is known. The assay is based on our proprietary HitHunter beta-galactosidase (beta-gal) complementation technology. This method utilizes two genetically engineered fragments of E. coli beta-gal. The larger fragment, termed Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, termed Enzyme Donor (ED), contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, it can spontaneously recombine with ED to form an active enzyme that can catalyze the formation of a fluorescent or chemiluminescent product that can be detected photometrically. We have adapted this technology to measure cell free protease activity by synthesizing cyclic ED substrates that contain specific cleavage sites for individual proteases. The cyclic ED peptide can't recombine with EA, but when the protease cleaves the cyclic ED molecule at its specific cleavage site, the linear ED can combine with EA to catalyze a photometric response. We propose to adapt this technology to measure protease activity in intact cells. As proof of principle, we will develop this technology as an assay for gamma -secretases which cleave amyloid precursor protein (APP) to produce two forms of beta-amyloid peptide, Abeta1-40 and Abeta1-42, that are toxic neuropeptides involved in the pathogenesis of Alzheimer's disease (AD). Since Abetas are products of gamma-secretase, this protease has become an important target for the discovery of drugs to treat AD. Presently, there is no HTS assay that specifically measures gamma-secretase activity. However, the specific cleavage sites of gamma-secretase are known, making it ideally suited to e measured in our assay. We will develop a novel cell based assay to measure gamma-secretase activity that can be used to discover drugs to treat AD. When further developed in future phase II SBIR studies, our technology can be used as a general screening assay to discover drugs against most medically important proteases.

描述（由申请人提供）：蛋白酶是参与蛋白质裂解为较小肽的关键酶，是药物发现的重要分子靶标。几乎没有基于细胞的技术存在以测定HTS形式的蛋白酶对底物的特异性切割，这阻碍了针对蛋白酶的药物发现。DiscoveRx正在开发一种技术，可以测量任何蛋白酶的活性，其底物选择性切割位点是已知的。该检测基于我们专有的HitHunter β-半乳糖苷酶（β-gal）互补技术。该方法利用了两个基因工程的E. coli beta-gal.较大的片段称为酶受体（EA），在氨基末端附近含有缺失，而较小的片段称为酶供体（艾德），含有EA缺失的氨基末端序列。单独，EA是不活动的。然而，它可以自发地与艾德重组形成活性酶，该活性酶可以催化形成可以通过光度法检测的荧光或荧光发光产物。我们已经采用了这种技术，通过合成环状艾德底物来测量无细胞蛋白酶活性，所述环状底物含有针对单个蛋白酶的特异性切割位点。环状艾德肽不能与EA重组，但当蛋白酶在其特异性切割位点切割环状艾德分子时，线性艾德可以与EA联合收割机结合以催化光度响应。我们建议采用这种技术来测量完整细胞中的蛋白酶活性。作为原理的证明，我们将开发这种技术作为γ-分泌酶的测定，所述γ-分泌酶切割淀粉样前体蛋白（APP）以产生两种形式的β-淀粉样肽，A β 1 -40和A β 1 -42，其是参与阿尔茨海默病（AD）发病机制的毒性神经肽。由于Abeta是γ-分泌酶的产物，因此这种蛋白酶已成为发现治疗AD的药物的重要靶标。目前，没有专门测量γ-分泌酶活性的HTS测定。然而，γ-分泌酶的特异性切割位点是已知的，使其非常适合在我们的测定中测量。我们将开发一种新的基于细胞的检测方法来测量γ-分泌酶活性，可用于发现治疗AD的药物。当在未来的II期SBIR研究中进一步开发时，我们的技术可用作一般筛选测定，以发现针对大多数医学上重要的蛋白酶的药物。