Molecular Targets Development Program

分子靶点开发计划

• 批准号: 7055482
• 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/7055482
• 关键词: adenosinetriphosphatase; binding sites; cell line; chemical structure function; combinatorial chemistry; drug discovery /isolation; drug screening /evaluation; enzyme activity; gene induction /repression; green fluorescent proteins; high throughput technology; human tissue; liquid chromatography mass spectrometry; nuclear magnetic resonance spectroscopy; p53 gene /protein; phosphorylation; protein binding; protein structure; ribonuclease H

RNase H - LeGrice A provisional patent application was filed August 30, 2004, on a class of vinylogous ureas which were found in the ChemBridge library. Extract hit evaluation is complete, and fractionation of a select set of extracts has begun. All routine secondary evaluation of pure compounds from the various libraries is now complete. A paper is in preparation on a second lead series of tropolone natural products, and a crystallographic collaboration with Ed Arnold (Rutgers) has begun in an attempt to co-crystallize hits with the HIV-1 target. Biacore and ultracentrifugation studies have begun with Inna Goroschkova and Peter Schuck of DBEPS, ORS, NIH, while Bob Crouch (NICHD) is pursuing compounds with selectivity for the human RNase H to better understand the function of this enzyme. An automated calorimeter has been acquired with IATAP funding to conduct studies of the binding of hit compounds with the protein target.V-ATPase - Khanna/Helman In vivo work with a murine osteosarcoma model is planned with Dr. Khanna. To support this, we have been working with DTP staff (Stinson, Hollingshead, Alley) to obtain pharmacokinetic data on oximidine III with a cellular bioassay. Preliminary results have shown that therapeutic blood levels can be achieved in mice.Dr. Porco (BU) is making more oximidine III for further studies, while Yamanouchi Pharmaceutical has recently expressed interest in supplying oximidine III and lobatamide A from fermentation sources. There has been no recent progress on Australian sources from invertebrates, though much is promised. A license application has been submitted for our V-ATPase patents by Reata Pharmaceuticals, and a CRADA with that company jointly with the Pediatric Oncology Branch is under discussion.We recently correlated cellular sensitivity in the 60-cell panels with RNA expression of certain isoforms of V-ATPase subunits, especially those in the V1 domain. Among other things, this may implicate the V1 domain as the site of action of these compounds. We plan to seek similar data for the osteosarcoma cell lines. This is consistent with a recent publication from the Dallas group that shows that salicylihalamide A has a novel effect, causing redistribution of the V1 domain to the membrane. This is quite distinct from the bafilomycin class of V-ATPase inhibitors, which act on subunit c of the V0 domain. As compound becomes available, we are also actively working with the Lyon group on bone biology in osteoclasts with our compounds. We are also planning on comparing the activity of the compounds in traditional metastasis models such as wound healing and migration for highly metastatic and non-metastatic counterparts in breast and prostate cell lines.HDAC inhibitor screen - HagerStatus Low throughput screeningScreening of the structural diversity set has been completedInduction of GFP expression is the endpoint for the HDAC inhibitor screen. This assay should be a plate reader-type assay, but the cells are not bright enough for detection. Consequently, the assay is being conducted using the Discovery-1 to acquire the images in a 96-well plate format in a low-throughput manner. Hits are identified by qualitative examination of the images. The parental cell line is being used to identify false positive hits which are the result of autofluorescent compounds. Low-throughput screening using the structural diversity set has been completed on the imaging system. Fourteen of the 2,080 compounds in the structural diversity set demonstrated some activity in the screen (hit rate 0.67%). Some compounds gave strong GFP induction, while others demonstrated weaker activity. We are in the process of cherry picking the hits and re-confirming the activity found in the primary screen. Screening of additional small chemical libraries will follow. We tested the feasibility of using an anti-GFP Alexa 594 antibody to improve signal in the HDAC inhibitor screen for detection on the plate reader. An antibody titer experiment demonstrated that a 1:100 antibody dilution was necessary for justification for a high throughput screen (z'factor=0.57 and %CV=11%). The antibody is very expensive ($228/100ul), therefore, use of this antibody was not a suitable alternative for increasing screening throughput.MDM2 Ubiquitination - WeissmanProject finished with primary screening. Approximately 148,000 natural product extracts screened. Hit rates for primary assay 1.9 %, secondary multiplexed screen (MDM2, XIAP, RNF28, Nedd4) for ring finger selectivity reduces hit rate 80% (n= 472, 94 MDM2 specific). Pure natural products library screened (0.1% hit rate, n=19). All secondary screening is completed on extract hits, pure natural product hits and Weissman lab Chembridge compounds (n = 44). Cell-based p53 assay has been performed on 387 available extracts, pure natural product hits and Weissman lab Chembridge compounds. Initial results showed 51 extracts and 5 pure compounds had >25% induction of p53 (normalized to Adriamycin). Three extracts showed p53 induction greater than that of Nutlin-3. Additional multiplexed assay in development to assess up to eight cellular markers of MDM2 or p53 mediated activity. Initial chemical evaluation of active extracts has begun. Several extracts have been through three rounds of purification and retain activity. Active fractions are currently being evaluated by LC/MS and NMR.IGF1R - BarrettInitial production of IRS-1 failed. Shc production was low (3 mg realized). PEL has grown up more Shc batches and has provided samples to MTDP for evaluation. PEL is also producing 14-3-3s. Truncated IRS-1 (t-IRS1)has been re-grown and PEL has provided samples to MTDP, initial evaluations appear positive. Quantities of IR, IGF1R, IGF1Rmut, Shc, GIPC, LARG, and 14-3-3?, and ? are now in MTDP. PEL has confirmed protein identity by MS/MS. Second attempt at large-scale phosphorylation of IR by Nissley lab was more successful than first attempt (no precipitation) but level of phosphorylation was low. Additional quantities of IGF1r and t-IRS1 are being produced by PEL. Initial assay results with IR and IGFR phosphorylated by Nissley lab showed that Shc binding was quantifiable and that the phosphorylated forms bound twice the Shc the non-phosphorylated forms bound. Binding to both receptor constructs was also measurable with truncated IRS-1, 14-3-3? and GIPC using either antibodies or streptavidin detection systems (all binding partners are biotinylated). Some issues with differential binding based on phosphorylation level persist (I.e. With t-IRS1 we do not visualize significant differences). Initial QC for thermodynamic studies is ongoing (complicated by phosphorylation issues). Preliminary isothermal titration calorimetry (ITC) studies have been completed with IGF1R-PO4 and 14-3-3, GIPC and t-IRS1, sub-micromolar binding affinities were determined for all of the binding partners with t-IRS1 apparently possessing the highest affinity for IGF1R-PO4 (5 nM). Binding of IGF1R-PO4 to Shc was not detected in the first ITC experiment. Currently looking at time resolved fluorescence endpoints for primary assay with IGF1R and various binding partners to be followed by secondary assay with IR-PO4 and the same binding partners. The possible multiplexing of the assay to include Terbium-labeled binding partners is also under examination.Johns HopkinsProgram has officially started as of Fall 2004. Thirteen students met both Hopkins and CCR standards for application for NCI/CCR fellowship.Three fellowships were offered and accepted and one additional student received paid tuition through the program. Outside of NCI fellows, 11 other new students were accepted into the program by JHU. Fifteen students was the maximum class size for year one; currently, JHU has additional students seeking admission to next year's class.

2004年8月30日提交了一份临时专利申请，申请的是一类在ChemBridge文库中发现的葡萄状尿素。提取命中评估已经完成，并且已经开始对选定的提取集进行分馏。所有来自不同文库的纯化合物的常规二级评价现已完成。关于tropolone天然产物的第二个主要系列的论文正在准备中，与Ed Arnold（罗格斯大学）的结晶学合作已经开始尝试与HIV-1靶标共结晶。Biacore和超离心研究已经由美国国立卫生研究院DBEPS的Inna Goroschkova和Peter Schuck开始，而Bob Crouch （NICHD）正在研究对人类rna酶H具有选择性的化合物，以更好地了解这种酶的功能。在IATAP的资助下，已经获得了一个自动量热计，用于研究hit化合物与蛋白质靶标的结合。V-ATPase - Khanna/Helman博士计划与小鼠骨肉瘤模型进行体内研究。为了支持这一点，我们一直在与DTP工作人员（Stinson, Hollingshead, Alley）合作，通过细胞生物测定法获得肟胺III的药代动力学数据。初步结果表明，在小鼠体内可以达到治疗性的血液水平。Porco （BU）正在生产更多的肟胺III以供进一步研究，而Yamanouchi Pharmaceutical最近表示有兴趣从发酵来源供应肟胺III和洛巴他胺A。最近在澳大利亚的无脊椎动物资源方面没有任何进展，尽管有很多承诺。我们的V-ATPase专利已由Reata制药公司提交许可申请，该公司与儿科肿瘤学部门联合的CRADA正在讨论中。我们最近将60个细胞的细胞敏感性与v - atp酶亚基的某些同工异构体的RNA表达，特别是V1结构域的RNA表达联系起来。除此之外，这可能意味着V1结构域是这些化合物的作用位点。我们计划在骨肉瘤细胞系中寻找类似的数据。这与达拉斯小组最近发表的一篇文章一致，该论文表明水杨柳卤酰胺a具有一种新的作用，导致V1结构域重新分布到膜上。这与作用于V0结构域c亚基的巴霉素类v - atp酶抑制剂完全不同。随着化合物的出现，我们也在积极地与里昂小组合作，用我们的化合物在破骨细胞中进行骨生物学研究。我们还计划比较这些化合物在传统转移模型中的活性，如伤口愈合和乳腺癌和前列腺细胞系中高度转移和非转移对应物的迁移。HDAC抑制剂筛选- HagerStatus低通量筛选结构多样性集的筛选已经完成，诱导GFP表达是HDAC抑制剂筛选的终点。这个实验应该是一个平板阅读器式的实验，但是细胞不够亮，无法检测。因此，使用Discovery-1进行分析，以低通量方式获取96孔板格式的图像。命中是通过对图像的定性检查来识别的。亲本细胞系被用来鉴定假阳性命中，这是自体荧光化合物的结果。利用结构多样性集在成像系统上完成了低通量筛选。在结构多样性集中的2080个化合物中，有14个在筛选中显示出一定的活性（命中率为0.67%）。一些化合物具有很强的GFP诱导作用，而另一些则表现出较弱的活性。我们正在挑选命中并重新确认主屏幕上发现的活动。随后将筛选更多的小型化学文库。我们测试了使用抗gfp Alexa 594抗体改善HDAC抑制剂屏幕信号的可行性，以便在平板阅读器上检测。抗体效价实验表明，1:100的抗体稀释是高通量筛选的必要条件（z因子=0.57,%CV=11%）。该抗体非常昂贵（228美元/100ul），因此，使用该抗体不是提高筛选通量的合适选择。MDM2泛素化- weissman项目初步筛选完成。筛选了约148,000种天然产品提取物。初级检测的命中率为1.9%，二级多路筛选（MDM2， XIAP, RNF28, Nedd4）的环指选择性降低了80%的命中率（n= 472, 94 MDM2特异性）。筛选纯天然产物库（0.1%命中率，n=19）。所有的二次筛选都是对萃取物、纯天然产物和Weissman实验室Chembridge化合物（n = 44）进行的。基于细胞的p53检测已对387种可用提取物、纯天然产品和Weissman实验室Chembridge化合物进行了检测。初步结果显示，51种提取物和5种纯化合物对p53的诱导作用为bb0 25%（归一化为阿霉素）。3种提取物对p53的诱导作用均大于Nutlin-3。额外的多重检测正在开发中，以评估多达8种MDM2或p53介导活性的细胞标志物。活性提取物的初步化学评价已经开始。部分提取物经过三轮提纯后仍保持活性。活性组分目前正在用LC/MS和NMR进行评价。IGF1R - barrett公司IRS-1的初始生产失败。Shc产量低（实现3 mg）。PEL生产了更多的Shc批次，并提供样品给MTDP进行评估。PEL也在生产14-3-3。截断的IRS-1 （t-IRS1）已重新生长，PEL已向MTDP提供样品，初步评价呈阳性。IR、IGF1R、IGF1Rmut、Shc、GIPC、LARG、14-3-3？，和？都在MTDP中。PEL通过MS/MS确认了蛋白的同源性。Nissley实验室第二次大规模磷酸化IR的尝试比第一次更成功（没有沉淀），但磷酸化水平较低。PEL正在生产更多的IGF1r和t-IRS1。Nissley实验室磷酸化IR和IGFR的初步分析结果表明，Shc的结合是可量化的，磷酸化形式的结合是非磷酸化形式的两倍。与两种受体结构的结合也可以用截断的irs - 1,14-3-3 ？和GIPC使用抗体或链亲和素检测系统（所有结合伙伴都被生物素化）。基于磷酸化水平的差异结合的一些问题仍然存在（即，对于t-IRS1，我们没有看到显著差异）。热力学研究的初始QC正在进行中（磷酸化问题使其复杂化）。对IGF1R-PO4、14-3-3、GIPC和t-IRS1进行了初步等温滴定量热法（ITC）研究，测定了所有结合伙伴的亚微摩尔结合亲和力，其中t-IRS1对IGF1R-PO4的亲和力最高（5 nM）。在第一次ITC实验中未检测到IGF1R-PO4与Shc的结合。目前正在寻找IGF1R和各种结合伙伴进行初级分析的时间分辨荧光终点，随后进行IR-PO4和相同结合伙伴的二级分析。可能的多重分析包括铽标记的结合伙伴也在审查中。约翰霍普金斯项目已于2004年秋季正式启动。13名学生同时符合霍普金斯大学和CCR的标准，申请NCI/CCR奖学金。三名学生获得了奖学金，另外一名学生通过该项目获得了学费。除了NCI的研究员，JHU还接收了另外11名新生。十五名学生是第一年的最大班级规模；目前，JHU有更多的学生申请进入明年的班级。