Molecular Targets Development Program

泛素化 Meso Scale Discovery（盖瑟斯堡）的项目处于主动筛选模式。迄今为止筛选了约90，000份（共约120，000份）浸提液。初步检测的命中率为1.5- 1.8%，选择性的二次多重筛选（MDM 2、XIAP、RNF 28、Nedd 4）降低命中率约50%。所有初级和二级筛查应在10月底前完成。生物素化的VCP及其截短突变体由Li实验室提供。使用这些材料，我们目前正在筛选肽库。 IGF1R IGF 1 R的大规模磷酸化成功。IR的大规模磷酸化导致胞质构建体作为沉淀物从溶液中破碎出来。另外38 mg IR已被生产和纯化。Nissley实验室将致力于（小规模）优化IR的磷酸化以防止沉淀。我们已经收到受体结合蛋白14-3-3 sigma、14-3-3 beta、GIPC和LARG。其它结合蛋白（Shc、IRS-1、截短的IRS-1、RACK）在表达/纯化方面具有不同程度的问题。预计PEL的完整报告将在本周末公布。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Anita Roberts和MTDP项目团队举行会议，讨论最新进展。 CyclinA/CDK2 仍在等待来自Porco实验室的800个成员的螺缩酮文库，以开始小规模筛选蛋白质-蛋白质相互作用的抑制。 噬菌体展示肽库 虽然今年春天我们已经成功地构建了一个肽库，但我们发现构建第一个库的原始程序对其他库的构建不起作用。因此，在今年夏天，我们花时间开发了一种方法来简化构建过程并提高肽库的多样性。因此，我们现在有一个可重复的和简单的方法来构建一系列的噬菌体展示的组合肽库，具有不同的复杂性和独特的结构。我们还能够将连接效率提高100-1000倍。我们预计在今年年底前建成5-10个图书馆。 Kai1 我们最终能够制造顺磁性蛋白脂质体，其中Kai 1的细胞内侧位于人工脂质膜的外部，其细胞外侧位于脂质双层的内部。Tarasova博士合成了Kai 1的两个肽片段;一个代表Kai 1的第二胞外结构域的一部分，另一个代表Kai 1的C末端胞内尾。随后，她将这些肽附着在磁珠上。使用这些材料，我们目前正在筛选肽库。 STEAP Tarasova实验室为我们提供了在磁珠上代表STEAP胞外结构域的合成肽。使用这种材料，我们目前正在筛选肽库。 死亡受体 基于我们的建议，Lipkowitz和Libutti实验室分别致力于DR 4/5胞内结构域蛋白和生物素化EMAP蛋白的构建/表达/纯化。仍在等待获得高纯度的目标分子。 人scFv文库 从MRC获得人scFv文库以分离针对CCR研究者感兴趣的靶标的scFv。 核受体 对表达GFP-ER缀合物的细胞的初步实验已经阐明了核易位成像测定的问题/要求。迄今为止测试的细胞系没有足够明亮的荧光以允许基于标准筛选测定标准（例如Z因子、信号/背景比、CV等）建立可靠的、自动化的、基于图像的易位测定。另一种显然更明亮的细胞系正在戈登·哈格的实验室中扩大。预计10月份可以进行测试。如果可接受，将随后进行方法开发。 RNaseH 已完成所有纯化合物库和提取物（> 200，000个样品）的初步筛选和确认。纯化合物的二级评估已经完成，提取物优先级正在进行中。已经安排了一次会议（10月10日），与匹兹堡大学的研究小组讨论药物化学。一旦完成优先排序，将为隔离项目选择提取物。 DNA甲基转移酶 我们试图利用与甲基化DNA结合的蛋白质MeCP 2的能力，在荧光标记的DNA底物上捕获DNMT 1酶促作用的产物。虽然MeCP 2-GST融合蛋白结合甲基化的荧光标记的寡核苷酸，如通过凝胶位移测定和荧光偏振测定所测量的，但是我们已经不能在高蛋白结合或谷胱甘肽板的表面上捕获寡核苷酸。其他替代形式，如生物素捕获或毛细管电泳正在考虑中。 GPCR 等待关于收购诺拉克技术的审议结果;将电池标准化研究应用于该项目。 Suresh Arya/Geetanjali Sachdeva 完成HeLa细胞中GFP表达的评价。通过荧光显微镜和Discovery 1成像进行评估，通过掺入多重染色剂进行读数标准化，以评估细胞数量。研究已经导致获得具有明亮GFP表达的细胞作为其他成像研究的阳性对照。

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.