Molecular Screening and Protein Expression Shared Resource

项目概要-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） 具有最先进的shRNA和小分子文库高通量筛选能力的成员， 鉴定候选治疗靶点的基因和工具抑制剂。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模式方面有希望，特别是对于 目前临床实践不理想的恶性肿瘤类型。此外，专家技术援助是 在重组DNA质粒工程中提供，在细菌和杆状病毒感染的 昆虫细胞，重组蛋白的纯化，以及高滴度逆转录病毒（例如慢病毒）的生产， 将shRNA和cDNA递送至哺乳动物细胞。CC研究人员需要高质量的重组蛋白 用于表征酶活性，用于结构分析的结晶，结构表征- 蛋白质-蛋白质、蛋白质-核酸和蛋白质-小分子相互作用的功能关系;以及 免疫兔/小鼠以产生定制抗体。通过与联合国儿童基金会的机构间协议， 海伦·F格雷厄姆癌症中心（HFGCC），MSPESR为CC成员提供CRISPR/Cas9基因 编辑服务。MSPESR通过提供生物化学和基于细胞的 试验开发。这样的测定使研究人员能够鉴定小分子化合物，然后可以将其用于治疗。 用作进一步研究细胞中靶蛋白功能和信号通路的工具，或者它们 并形成用于“命中-先导”优化以识别先导系列的基础 用于药物发现计划。目前，MSPESR提供：1）基于生物化学、细胞和高含量的 适合于在384孔微量滴定板中进行高通量筛选的测定; 2）小分子文库， 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）在二级、正交和非正交试验中表征新鉴定的化合物的效力和选择性。 测定。这些服务是通过配备机器人技术的中央实验室提供的，药物库， 比如以高密度微孔板格式排列的分子，以及高效计算的基础设施， 分析、解释和管理生物和化学数据集。MSPESR由 一位经验丰富的常务董事和专门的实验室工作人员，在提供的所有服务交叉培训。这 允许及时的项目管理、质量保证和关键数据的传播/集成， 将基本生物学观察转化为潜在的治疗策略。通过其在 在上一个预算周期，MSPESR已经能够解剖肿瘤发病的复杂信号通路， 进展、抗癌药物的验证和最终纳入的概念验证结果 进入早期临床试验作为多学科研究合作的引擎，MSPESR 为所有三个CC计划的关键出版物和赠款资金做出了贡献。

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.