Cell-Free Protein Labeling for Drug Discovery

描述（由申请人提供）：人类基因组开启了药物发现的新篇章。传统的药物发现方法是针对单个蛋白质靶点筛选药物文库，与之相反，基因组的整个蛋白质补体，即蛋白质组，现在可用于筛选。通过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，蛋白质组筛选将大大加快药物发现过程。然而，由于需要将基因组快速转化为蛋白质组，需要对具有专门检测和亲和力标签的蛋白质进行工程设计，以及需要具有高通量的平台来检查每小时数千次药物-蛋白质相互作用，因此使用传统技术很难进行此类药物-蛋白质组学筛选。

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.