Cell-Free Protein Labeling for Drug Discovery

描述(申请人提供)：人类基因组开启了药物发现的新篇章。与传统的药物发现方法不同，传统的药物发现方法针对单个蛋白质靶标筛选药库，现在可以筛选基因组的整个蛋白质补体，即蛋白质组。蛋白质组筛选可以确定先导药物化合物和整个蛋白质组之间的潜在相互作用，从而极大地加快药物发现过程。然而，这种药物-蛋白质组筛选很难使用传统技术进行，因为需要将基因组快速转换为蛋白质组，需要设计具有专门检测和亲和力标签的蛋白质，以及需要一个具有每小时检查数千种药物-蛋白质相互作用所需的高通量的平台。 在第一阶段，我们基于在体外蛋白质表达、tRNA介导的蛋白质工程(TRAMPE)和可光切割(PC)连接物(如PC-生物素)等领域的最新进展，开发了一种新的药物发现方法。这项技术允许直接表达基因中的单个蛋白质或基因组中的整个蛋白质组。这项技术的一个关键是在蛋白质的体外表达过程中将特殊设计的标记和亲和标签结合到蛋白质中，从而促进它们的快速分离和检测。一种名为PC-SNAG的中心方法允许从珠子上复杂的体外表达混合物中快速分离蛋白质，然后以纯形式光释放到溶液中或直接转移到微阵列底物的表面。 在第二阶段，这项新技术的概念验证将通过创建一个321个成员的体外表达蛋白质(Live-PRO TM)的激酶库来建立，该库将在可光释放的珠子上形成。在一项试验中，将同时筛选Live-Pro，以检测与已知的激酶抑制剂的潜在相互作用。在第二个测试中，将同时测量特定的激酶抑制剂对Live-Pro每个成员的活性的影响。这项工作将与Invitgen公司合作进行，Invitgen公司的Gateway TM重组技术和人类终极TM ORF克隆将允许创建体外可表达质粒库。千禧制药公司前首席技术官、目前在牛津生物科学公司担任常驻企业家的迈克·帕维亚博士将担任顾问。

DESCRIPTION (provided by applicant): The human genome has opened a new chapter for drug discovery. In contrast to conventional drug discovery methods, which screen a drug library against a single protein target, the entire protein complement of the genome, i.e. the proteome, is now available for screening. A proteomic screen would greatly accelerate the drug discovery process by allowing potential interactions between a lead drug compound and an entire proteome to be determined. However, such drug-proteomic screens would be difficult to perform using conventional technology because of the need to rapidly convert genome to proteome, the requirement of engineering of proteins with specialized detection and affinity tags and the need for a platform that has the high throughput necessary to examine thousands of drug-protein interactions per hour. During Phase I, we have developed a new approach for drug discovery based on recent progress in the areas of in vitro protein expression, tRNA mediated protein engineering (TRAMPE) and photocleavable (PC) linkers such as PC-biotin. This technology allows the direct expression of individual proteins from genes or entire proteomes from genomes. A key to this technology is the incorporation of specially designed markers and affinity tags into proteins during their in vitro expression, thus facilitating their rapid isolation and detection. A central approach, termed PC-SNAG, allows proteins to be rapidly isolated from complex in vitro expression mixtures on beads and then photoreleased in pure form into solution or transferred directly onto the surface of a microarray substrate. During Phase II, proof-of-concept will be established for this new technology by creating a 321 member kinase library of in vitro expressed proteins (LIVE-PRO TM) that will be formatted on photoreleasable beads. In one assay, LIVE-PRO will be simultaneously screened to detect potential interactions with known kinase inhibitors. In a second assay, the effects of specific kinase inhibitors on the activity of each member of the LIVE-PRO will be simultaneously measured. This work will be performed in collaboration with Invitrogen, whose Gateway TM recombination technology and human Ultimate TM ORF Clones will allow creation of libraries of in vitro expressible plasmids. Dr. Mike Pavia, the former Chief Technology Officer at Millennium Pharmaceuticals and currently Entrepreneur-in-Residence at Oxford Bioscience will serve as a consultant.