Molecular Screening and Protein Expression Shared Resource

项目摘要-分子筛选和蛋白质表达 分子筛选和蛋白质表达共享资源(MSPESR)提供癌症中心(CC) 拥有最先进的shRNA和小分子文库高通量筛选能力的成员 确定候选治疗靶点的基因和工具抑制物。识别类似药物的小分子 调节治疗靶点的活性在定义新的治疗范例方面有希望，特别是对 顽固性肿瘤类型，目前的临床实践并不理想。此外，专家技术援助是 重组DNA工程中提供的蛋白质在细菌和杆状病毒感染中的表达 昆虫细胞，重组蛋白的纯化，以及生产高效价逆转录病毒(如慢病毒) 将shRNA和cDNA递送到哺乳动物细胞。CC调查人员需要高质量的重组蛋白 对于酶活性的表征，用于结构分析的结晶，结构的表征- 蛋白质与蛋白质、蛋白质与核酸、蛋白质与小分子相互作用的功能关系； 免疫兔子/小鼠以产生定制抗体。通过与机构间协议 海伦·F·格雷厄姆癌症中心(HFGCC)，MSPESR为癌症中心成员提供CRISPR/Cas9基因 编辑服务。MSPESR通过提供生化和基于细胞的专业知识来促进合作 化验发展。这样的分析使研究人员能够识别小分子化合物，然后可以 用作进一步研究细胞中目标蛋白功能和信号通路的工具，或者作为替代方案 可以表示命中化合物或先导化合物，并且形成用于识别先导序列的“命中到先导”优化的基础 用于药物发现计划。目前，MSPESR提供：1)基于生化、细胞和高含量的 适合在384孔微滴定板上进行高通量筛选的分析方法；2)小分子和 ShRNA；3)小分子文库的高通量筛选；4)生物和化学分析 数据集.5)二次正交法中新鉴定的化合物的效力和选择性的表征 化验。这些服务是通过配备机器人的中央实验室、药物图书馆- 像以高密度微板格式排列的分子，以及高效的计算基础设施 生物和化学数据集的分析、解释和管理。MSPESR由以下人员操作 一位经验丰富的常务董事和敬业的实验室工作人员，接受过所有服务的交叉培训。这 允许及时进行项目管理、质量保证和关键数据的传播/集成 将基本的生物学观察转化为潜在的治疗策略。通过它在过去几年的活动 在上一个预算周期，MSPESR使人们能够剖析肿瘤发生和发展的复杂信号通路 最终纳入的抗癌药物的进展、验证(S)和概念验证结果 进入早期临床试验阶段。作为多学科研究协作的引擎，MSPESR拥有 为所有三个CC计划的关键出版物和赠款提供资金。

PROJECT SUMMARY – MOLECULAR SCREENING AND PROTEIN EXPRESSION The Molecular Screening and Protein Expression Shared Resource (MSPESR) provides Cancer Center (CC) members with state-of-the-art high-throughput screening capabilities of shRNA and small molecule libraries to identify genes and tool inhibitors of candidate therapeutic targets. Identifying drug-like, small molecules that regulate the activity of therapeutic targets holds promise in defining new treatment paradigms, especially for recalcitrant tumor types, where current clinical practice is suboptimal. In addition, expert technical assistance is provided in recombinant DNA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins, and production of high-titer retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells. CC investigators require high quality recombinant proteins for characterization of enzymatic activities, crystallization for structural analysis, characterization of structure- function relationships of protein-protein, protein-nucleic acid, and protein-small molecule interactions; and immunization of rabbits/mice to generate custom antibodies. Through an inter-institutional agreement with the Helen F. Graham Cancer Center (HFGCC), the MSPESR offers CC members access to CRISPR/Cas9 gene editing services. The MSPESR fosters collaboration by providing expertise in biochemical and cell-based assay development. Such assays enable researchers to identify small molecule compounds which can then be used as tools to further study the target protein functions and signaling pathways in cells, or alternatively they may represent hit or lead compounds and form the basis for 'hit-to-lead" optimization to identify a lead series for drug discovery initiatives. Currently, the MSPESR offers: 1) biochemical-, cell-, and high-content based assays amenable to high-throughput screening in 384-well microtiter plates; 2) libraries of small molecules and shRNA; 3) high-throughput screening of small molecule libraries; 4) analysis of biological and chemistry datasets; 5) characterization of potency and selectivity of newly identified compounds in secondary, orthogonal assays. These services are provided through a centralized laboratory equipped with robotics, libraries of drug- like molecules arrayed in high-density microplate formats, and computational infrastructure for efficient analysis, interpretation, and management of biological and chemistry datasets. The MSPESR is operated by an experienced Managing Director and dedicated laboratory staff, cross-trained in all services offered. This allows for timely project management, quality assurance, and dissemination/integration of data critical for translation of basic biological observations into potential therapeutic strategies. Through its activity over the last budget cycle, the MSPESR has enabled dissection of complex signaling pathways of tumor onset and progression, validation of anticancer agent(s), and proof of concept results that were ultimately incorporated into early phase clinical trials. As an engine for multidisciplinary research collaboration, the MSPESR has contributed to critical publications and grant funding across all three CC Programs.