Cell Based EFC Assay for Protease Drug Discovery

描述（由申请人提供）：蛋白酶是参与蛋白质裂解成更小肽的关键酶，是药物发现的重要分子靶点。很少有基于细胞的技术可以检测HTS格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，可以测量任何已知底物选择性切割位点的蛋白酶的活性。该检测基于我们专有的HitHunter β -半乳糖苷酶（β -gal）互补技术。这种方法利用了大肠杆菌β -gal的两个基因工程片段。较大的片段，被称为酶受体（EA），包含氨基末端附近的缺失，而较小的片段，被称为酶供体（ED），包含EA缺失的氨基末端序列。单独，EA是无活性的。然而，它可以自发地与ED重组形成一种活性酶，这种酶可以催化形成荧光或化学发光产物，可以通过光度法检测到。我们已经采用这种技术，通过合成含有单个蛋白酶特定切割位点的环状ED底物来测量细胞游离蛋白酶的活性。环状ED肽不能与EA重组，但当蛋白酶在其特定的裂解位点切割环状ED分子时，线性ED可以与EA结合，催化光度反应。我们建议将该技术应用于完整细胞中蛋白酶活性的测量。作为原理证明，我们将开发该技术作为γ -分泌酶的检测，该酶可切割淀粉样前体蛋白（APP），产生两种形式的β -淀粉样肽Abeta1-40和Abeta1-42，这两种形式的β -淀粉样肽是参与阿尔茨海默病（AD）发病机制的有毒神经肽。由于β是γ -分泌酶的产物，该蛋白酶已成为发现治疗AD药物的重要靶点。目前，还没有专门测量γ -分泌酶活性的HTS检测方法。然而，γ分泌酶的特定切割位点是已知的，这使得它非常适合我们的测定。我们将开发一种新的基于细胞的检测方法来测量γ -分泌酶的活性，该方法可用于发现治疗AD的药物。当在未来的II期SBIR研究中进一步发展时，我们的技术可以作为一种通用的筛选试验来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.