Molecular Targets Development Program

分子靶点开发计划

• 批准号: 6952051
• 负责人: michael lee
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6952051
• 关键词: cyclin dependent kinase; drug screening /evaluation; high throughput technology; human tissue; insulinlike growth factor; methyltransferase; nuclear receptors; phosphorylation; ribonuclease H

Ubiquitination Project in active screening mode at Meso Scale Discovery (Gaithersburg). Approximately 90,000 (out of ~120,0000) extracts so far screened. Hit rates for primary assay 1.5-1.8%, secondary multiplexed screen (MDM2, XIAP, RNF28, Nedd4) for selectivity reduces hit rate ~50%. All primary and secondary screening should be complete by the end of October. Biotinylated VCP and its truncated mutants were provided from Li lab. Using these materials, we are currently screening a peptide library. IGF1R Large-scale phosphorylation of IGF1R was successful. Large-scale phosphorylation of IR resulted in the cytosolic construct crashing out of solution as a precipitate. Additional 38 mg of IR has been produced and purified. Nissley lab will work (on small scale) to optimize phosphorylation of IR to prevent precipitation. We have received receptor binding proteins 14-3-3 sigma, 14-3-3 beta, GIPC, and LARG. The other binding proteins (Shc, IRS-1, truncated IRS-1, RACK) had varying degrees of problems with expression/purification. Full report from the PEL is expected by the end of the week. Meeting with PEL, Nissley lab and MTDP to be scheduled the following week. Smad3/4 Meeting scheduled for October 7th with Anita Roberts and MTDP project team to discuss latest developments. CyclinA/CDK2 Still waiting on the 800-member spiroketal library from Porco lab to begin small-scale screen for inhibition of protein-protein interactions. Phage-displayed peptide library Although we have successfully made one peptide library this spring, we found the original procedure to construct the first library did not work for other library construction. Therefore, during this summer we spent our time to develop a method to simplify the construction process and improve the diversity of peptide libraries. As the results, we have now reproducible and simple method to construct a series of phage-displayed combinatorial peptide libraries that possess diverse complexities and unique structures. We were also able to increase the ligation efficiency 100-1000 times. We expect to construct 5-10 libraries by the end of this year. Kai1 We finally able to make a paramagnetic proteoliposomes in which the intracellular side of Kai1 is located outside of artificial lipid membrane and the extracellular side of it is located inside of the lipid bilayer. Dr. Tarasova synthesized two peptide fragments of Kai1; one represent a part of 2nd extracellular domain of Kai1 and the other represent the C-terminal intracellular tail of Kai1. She subsequently attached the peptides on magnetic beads. Using these materials, we are currently screening a peptide library. STEAP Tarasova lab provided us the synthesized peptide that represents the extracellular domain of STEAP on magnetic beads. Using this material, we are currently screening a peptide library. Death receptor, EMAP Based on our suggestion, Lipkowitz and Libutti labs have been working on construction/expression/purification of DR4/5 intracellular domain protein and the biotinylated EMAP protein, respectively. Still waiting to obtain the highly pure target molecule. Human scFv Library Human scFv library was acquired from MRC to isolate scFv against target of interest in CCR investigators. Nuclear Receptors Preliminary experiments on cells expressing GFP-ER conjugates have clarified issues/requirements for nuclear translocation imaging assays. The cell lines tested to date have not had sufficiently bright fluorescence to allow for establishment of a reliable, automated, image-based translocation assay based on standard screening assay criteria (e.g. Z-factors, signal/background ratio, CVs, etc.). Alternative, apparently brighter, cell lines are being expanded in Gordon Hager's lab. These are expected to be available for testing in October. If acceptable, methods development will follow. RNaseH Primary screening and confirmation of all pure compound libraries and extracts (>200,000 samples) has been completed. Secondary evaluation of pure compounds is complete, and extract prioritization is well underway. A meeting has been scheduled (Oct.10) to discuss medicinal chemistry of hits with the University of Pittsburgh group. Extracts will be chosen for isolation projects once prioritization has been completed. DNA Methyl Transferase We have attempted to use the ability of a protein, MeCP2, which binds to methylated DNA, to capture the product of DNMT1 enzymatic action on a fluorescently labeled DNA substrate. While the MeCP2-GST fusion protein bound a methylated fluorescently labeled oligo, as measured by a gel shift assay and a fluorescence polarization assay, we have been unable to capture the oligo on the surface of either high protein binding or glutathione plates. Other alternative formats such as a biotin capture or capillary electrophoresis are under consideration. GPCR Awaiting resolution of deliberations on acquisition of Norak technology; application of cell standardization studies to this project. Suresh Arya/Geetanjali Sachdeva Evaluation of expression of GFP in HeLa cells completed. Assessment by fluorescent microscopy and Discovery 1 imaging with standardization of readout by incorporation of multiplexed stain to assess cell number. Studies have resulted in acquisition of cells with bright GFP expression as a positive control for other imaging studies.

泛素化 在中尺度发现号(盖瑟斯堡)的主动筛选模式下的项目。到目前为止，大约有90,000种提取物(从120,0000种提取物中筛选出来)。一次检测的命中率为1.5-1.8%，二次多重筛选(MDM2、XIAP、RNF28、Nedd4)的选择性使命中率降低约50%。所有初级和次级筛查应在10月底前完成。生物素标记的VCP及其截短突变体由LI实验室提供。利用这些材料，我们目前正在筛选多肽文库。 IGF1R IGF1R的大规模磷酸化是成功的。IR的大规模磷酸化导致胞浆结构以沉淀物的形式从溶液中崩解出来。另外还生产和提纯了38毫克的红外线。尼斯利实验室将致力于(小规模)优化IR的磷酸化，以防止沉淀。我们已经收到了受体结合蛋白14-3-3 sigma、14-3-3 beta、GIPC和LARG。其他结合蛋白(Shc、IRS-1、截短IRS-1、Rack)在表达/纯化方面存在不同程度的问题。PEL的完整报告预计将在本周末发布。与PEL、Nissley实验室和MTDP的会议安排在下周。 SMAD3/4 定于10月7日与安妮塔·罗伯茨和MTDP项目团队举行会议，讨论最新进展。 细胞周期蛋白A/CDK2 仍在等待来自波尔科实验室的800名成员的螺酮文库开始对蛋白质相互作用的抑制进行小规模筛选。 噬菌体展示多肽文库 虽然今年春天我们已经成功地制作了一个多肽文库，但我们发现原来构建第一个文库的程序不适用于其他文库的构建。因此，今年夏天，我们花时间开发了一种方法，简化了构建过程，提高了多肽文库的多样性。因此，我们现在有了一种可重复性和简单易行的方法来构建一系列具有不同复杂性和独特结构的噬菌体展示组合肽文库。我们还能将结扎效率提高100-1000倍。我们预计到今年年底将建成5-10个图书馆。 凯1 我们最终能够制造出一个顺磁性蛋白脂质体，其中Kai1的胞内侧位于人工脂膜外，胞外侧位于脂双层内。塔拉索娃博士合成了两个Kai1的多肽片段；一个代表Kai1的第二个胞外区的一部分，另一个代表Kai1的C末端胞内尾巴。随后，她将这些多肽附着在磁珠上。利用这些材料，我们目前正在筛选多肽文库。 分步前进 塔拉索娃实验室为我们提供了代表磁珠上STEAP胞外结构域的合成肽。利用这一材料，我们目前正在筛选多肽文库。 死亡受体，EMAP 基于我们的建议，Lipkowitz和Libuti实验室分别致力于构建/表达/纯化DR4/5胞内结构域蛋白和生物素标记的EMAP蛋白。仍在等待获得高纯度的目标分子。 人源单链抗体文库 从MRC获得人源ScFv文库，以分离CCR研究人员感兴趣的靶点的ScFv。 核受体 对表达GFP-ER结合物的细胞的初步实验已经阐明了核转位成像分析的问题/要求。到目前为止，测试的细胞系还没有足够明亮的荧光，无法根据标准的筛选分析标准(如Z因子、信号/背景比、CVs等)建立可靠的、自动化的、基于图像的易位分析。戈登·海格的实验室正在扩展另一种显然更明亮的细胞系。预计这些设备将在10月份进行测试。如果可以接受，方法开发将紧随其后。 RNAseH 所有纯化合物文库和提取物(&gt；200,000个样本)的初步筛选和确认已经完成。对纯化合物的二次评估已经完成，提取优先顺序正在顺利进行。已经安排了一次会议(10月10日)，与匹兹堡大学小组讨论HITS的药物化学。一旦完成优先排序，将为隔离项目选择提取部分。 DNA甲基转移酶 我们试图利用一种蛋白质MeCP2的能力，它与甲基化的DNA结合，以捕获DNMT1在荧光标记的DNA底物上的酶作用产物。虽然MeCP2-GST融合蛋白结合了甲基化的荧光标记的寡聚物，通过凝胶漂移和荧光偏振分析，我们一直无法捕获在高蛋白结合或谷胱甘肽平板上的寡聚物。其他替代形式，如生物素捕获或毛细管电泳法也在考虑之中。 GPCR型 等待有关收购Norak技术的审议结果；将细胞标准化研究应用于该项目。 Suresh Arya/Geetanjali Sachdeva 完成了绿色荧光蛋白在HeLa细胞中的表达。通过荧光显微镜和Discovery 1成像进行评估，并通过结合多重染色标准化读数来评估细胞数量。研究的结果是获得了GFP表达明亮的细胞，作为其他成像研究的阳性对照。