Cell Based EFC Assay for Protease Drug Discovery

描述（由申请人提供）：蛋白酶是参与将蛋白质裂解为较小肽的关键酶，并且是药物发现的重要分子靶点。很少有基于细胞的技术能够以 HTS 形式测定蛋白酶对底物的特异性裂解，这阻碍了针对蛋白酶的药物发现。 DiscoverRx 正在开发一种技术，可以测量底物选择性切割位点已知的任何蛋白酶的活性。该检测基于我们专有的 HitHunter β-半乳糖苷酶 (β-gal) 互补技术。该方法利用了大肠杆菌 beta-gal 的两个基因工程片段。较大的片段称为酶受体 (EA)，包含氨基末端附近的缺失，而较小的片段称为酶供体 (ED)，包含 EA 缺失的氨基末端序列。 EA 本身并不活跃。然而，它可以自发地与 ED 重组形成活性酶，可以催化形成可通过光度计检测的荧光或化学发光产物。我们采用了这项技术，通过合成含有单个蛋白酶特定切割位点的环状 ED 底物来测量无细胞蛋白酶活性。环状ED肽不能与EA重组，但当蛋白酶在其特定切割位点切割环状ED分子时，线性ED可以与EA结合以催化光度响应。我们建议采用这项技术来测量完整细胞中的蛋白酶活性。作为原理证明，我们将开发这项技术，用于检测 γ 分泌酶，该酶裂解淀粉样前体蛋白 (APP)，产生两种形式的 β-淀粉样肽，Abeta1-40 和 Abeta1-42，它们是参与阿尔茨海默病 (AD) 发病机制的有毒神经肽。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 药物的重要靶标。目前，还没有专门测量 γ 分泌酶活性的 HTS 测定法。然而，γ-分泌酶的特定切割位点是已知的，使其非常适合在我们的测定中进行测量。我们将开发一种新的基于细胞的测定法来测量γ-分泌酶活性，可用于发现治疗 AD 的药物。当在未来 II 期 SBIR 研究中进一步发展时，我们的技术可用作通用筛选分析，以发现针对大多数医学上重要的蛋白酶的药物。

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.