HIGH THROUGHPUT, HIGH CONTENT MOLECULAR LIBRARIES SCREENING

高通量、高内涵分子库筛选

• 批准号: 8169400
• 负责人: LARRY A. SKLAR
• 金额: $ 3.34万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169400
• 关键词: Acoustics; Address; Area; Binding; Biological; Biological Assay; Cell Line; Cells; Collaborations; Communities; Computer Retrieval of Information on Scientific Projects Database; Computer software; Consult; Coupling; Custom; Cytometry; Data Analyses; Data Display; Detection; Development; Firefly Luciferases; Flow Cytometry; Fluorescence; Funding; Goals; Grant; Heat-Shock Proteins 70; Heating; Human; Individual; Institution; Libraries; Ligand Binding; Ligands; Luciferases; Luminescent Measurements; Manuals; Mass Spectrum Analysis; Measurable; Measurement; Measures; Molecular; Molecular Bank; Molecular Chaperones; Neomycin; New Mexico; Optics; Peptide Library; Phase; Positioning Attribute; Qualifying; Recommendation; Recording of previous events; Research; Research Personnel; Resistance; Resolution; Resources; Role; Sampling; Screening procedure; Signal Transduction; Sorting - Cell Movement; Source; Speed; Surface; System; Techniques; Technology; Testing; Time; United States National Institutes of Health; Universities; Visualization software; Walkers; Work; abstracting; anticancer research; base; combinatorial; data acquisition; design; digital; experience; fluorophore; high throughput screening; hygromycin A; improved; inhibitor/antagonist; instrument; instrumentation; luminescence; new technology; novel; particle; programs; receptor; research and development; small molecule; tool; treatment center

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract The application of flow cytometry as a high-throughput, high information content screening platform is just beginning and has not reached its full potential. The NFCR and the University of New Mexico cytometry group will extend their long and productive history of collaboration into the technical advancement and improved application of flow-based molecular screening assays. This collaboration will extend across each of the three R&D Projects, and will evolve with the instrumental advances developed as these projects progress. In Project 1, we will use the new technical capabilities of the acoustic-focusing instrument to extend the range of flow-based screening assays into the realm of luminescence measurements. In Project 2, we will use the large-particle sorter to develop improved combinatorial library screening. In Project 3, we will develop improved multiplexed and molecular interaction screening assays based on the ability to simultaneously measure complete emission spectra and fluorescence lifetime. We will also continue our ongoing collaboration to apply the advantages of the NFCR-developed Open Reconfigurable Cytometry Acquisition System (ORCAS) digital data acquisition hardware/software to flow screening instruments developed by the UNM group. This collaboration has the potential to considerably extend the throughput, range and information content of a wide variety of cell-based and particle-based flow screening assays, directly addressing major goals of the NIH Roadmap Initiative. Background In the past five years, the potential of flow cytometry as a platform for high-throughput screening assays has begun to be realized (1-3). Dr. Larry Sklar is the Director of the New Mexico Molecular Libraries Screening Center, funded as an NIH Roadmap Initiative (4). As a part of this flow cytometry screening effort, Dr. Sklar's group has developed a novel automated, high-throughput flow cytometry platform termed HyperCyt¿ (5). The major advance thus far has been an enormous increase in sample throughput: this technology has even greater potential as a screening tool by further advances in the information content of the screening assays themselves. The National Flow Cytometry Resource competitive renewal includes the development of new instrumentation that would be of great value to these molecular libraries screening efforts, as well as high throughput, high content screening efforts in general. The details of how the NFCR will contribute (in coordination with Dr. Sklar's group) are outlined in the specific sections below, but it is of value here to outline how this collaboration will be extremely synergistic and result in significant progress toward NIH Roadmap goals in the area of Molecular Libraries. In guiding the New Mexico Molecular Libraries Screening Center, Dr. Sklar is in a unique position to implement and evaluate our new technologies and assays for their use in high throughput and high content screening assays. Importantly Dr. Sklar will be exceptionally qualified to assist in pairing the new technology from the NFCR to real world screening issues. This will enable the NFCR to apply its technology to biological problems quickly by either working directly with Dr. Sklar or by recommendation of Dr. Sklar to users of the screening centers. Approach Project 1 The major technical goal of NFCR project 1 is to enable the development of new optical analysis techniques that were either impossible or impractical previously by flow cytometry. Of particular importance for molecular screening is the possibility to use luminescence in a flow cytometry format. Luminescence is not readily measurable currently using flow cytometry due to the long emission times, but Project 1 will implement acoustically-focused cytometers that can measure luminescence at high particle analysis rates. This will allow high throughput flow cytometry screening to use the advantages of luminescence as a detection assay, including high signal and increased spectral resolution. The collaboration with Dr. Sklar will consist of the NFCR developing an acoustic flow cytometer for use in luminescence assays, in a project suggested by Claire Verschraegen and Walker Wharton from the UNM Cancer Research and Treatment Center. The assay under consideration is designed to identify HSP70 inhibitors by high throughput flow cytometry. A cell line has been stably transfected with pRC/luciferase (neomycin resistant) and pcDNA3.p70 (hygromycin resistant) to have cells that express consistent and elevated levels of both firefly luciferase and human HSP70. The assay has been performed in such a way that luciferase activity is observed following heat treatment and renaturation dependent upon the molecular chaperone HSP 70. The screen involves identifying small molecules that regulate HSP 70 activity. This collaboration directly supports the Molecular Libraries Roadmap initiative to develop new tools for high throughput high content screening. Project 2 The major technical goal of NFCR project 2 is the development of a high-speed, large particle sorter. A considerable number of 'one bead-one compound' libraries have been synthesized on large polymeric particles, but current technology requires manual selection to obtain beads that have positive interactions. Flow cytometry screening of the interactions of biological targets on the particle surface followed by sorting to determine the identity of the interacting compound via traditional technologies (such as mass spectrometry) will be a major advance in throughput for this powerful screening technology. Since the libraries now available through the screening center are not 'one bead-one compound' libraries, we will focus on developing new screening approaches using more diverse library sets. In this collaboration, Dr. Sklar will consult on new initiatives and assays to convey this technology to the research community at large. His role as PI of the NIH New Mexico Molecular Libraries Screening Center will enable excellent visibility of this technology to the research community. One of the current collaborators of the NMMLSC is Richard Houghten, the president of TPIMS, who developed the teabag approach for synthesis of peptide libraries on beads. As a part of Project 2, the NFCR will develop a large particle sorter for use at UNM, where it will serve as a point of collaboration for the NFCR, the UNM Screening Center, and the UNM Screening center users. Project 3 The goal of Project 3 is to develop an integrated phase-spectral flow cytometer, allowing simultaneous measurement of fluorescence lifetimes of many fluorophores across a wide spectral range. This instrument will have several applications to improve the information content of both cell-based and particle-based flow screening assays. The ability to measure fluorescence lifetime of fluorophores provides a means to probe molecular interactions and improve the resolution of free versus bound ligands. The ability collect complete spectral emission information on individual particles also provides an avenue to increased multiplexing of bead-based screening assays: coupling this with fluorescence lifetime information should allow better resolution of molecular interactions in screening assays. Dr. Sklar's group has considerable experience in developing assays for receptor-ligand interactions, and this collaboration will be used to develop improved molecular screening assays for a variety of applications. As detailed in Project 3, the R&D approach will result initially in separate phase-resolved and spectral instruments: these will be used by Dr. Sklar's group to test and validate improved molecular screening assays. Once the integrated instrument is operational, we will investigate applying the resultant increased multiplexing potential and improved molecular interaction determination to both cell-based and bead-based screening assays, using both the expertise of the NM screening center and the collaborators that it has attracted. ORCAS Digital Data Acquisition System We have already initiated an effort to supply the NM Molecular Libraries Screening center with the NFCR-developed ORCAS digital data acquisition system (6). This system has been used to operate the HyperCyt¿ high-throughput screening instrument for data acquisition. Several of the features of ORCA system provide distinct advantages for this instrument, notably significantly increased time resolution and range. We will provide a dedicated ORCA system for use on this instrument, along with the NFCR-developed Tailorable Rapid Acquisition and Visualization Software (TRAViS) program for data display and analysis. We will continue to collaborate with Dr. Sklar's group to enhance the ORCA system for high-throughput applications, specifically developing instrument control systems and increased analysis sensitivity. We will also use the custom data analysis routines developed in Project 3 for spectral/lifetime measurement to increase the information content and throughput of screening assays.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 中心，不一定是研究者的机构。 抽象的 流式细胞术作为高通量、高信息含量筛选平台的应用才刚刚开始，尚未充分发挥其潜力。 NFCR 和新墨西哥大学细胞计数小组将把他们长期而富有成效的合作历史扩展到技术进步。这种合作将扩展到三个研发项目中的每一个，并将随着这些项目的进展而发展，我们将利用声学的新技术能力。 - 聚焦仪器可延长在项目 2 中，我们将使用大颗粒分选仪来开发改进的组合库筛选。在项目 3 中，我们将开发基于流式筛选分析的改进的多重和分子相互作用筛选分析。我们还将继续持续合作，将 NFCR 开发的开放式可重构细胞计数采集系统 (ORCAS) 数字数据采集硬件/软件的优势应用到流式细胞术中。 UNM 小组开发的筛选仪器有可能大大扩展各种基于细胞和基于颗粒的流式筛选测定的通量、范围和信息内容，直接实现 NIH 路线图计划的主要目标。 背景 在过去的五年中，流式细胞术作为高通量筛选分析平台的潜力已开始显现 (1-3)。Larry Sklar 博士是新墨西哥州分子图书馆筛选中心的主任，该中心由一个机构资助。 NIH 路线图计划 (4) 作为流式细胞术筛选工作的一部分，Sklar 博士的团队开发了一种新型自动化、高通量流式细胞术平台，称为 HyperCyt¿ (5) 迄今为止的主要进步是样品通量的巨大增加：通过筛选测定本身的信息内容的进一步进步，该技术作为筛选工具具有更大的潜力。国家流式细胞术资源竞争性更新包括：开发新仪器对于这些分子库筛选工作以及一般的高通量、高内涵筛选工作具有重要价值。NFCR 将如何做出贡献的详细信息（与 Sklar 博士的小组合作）概述于。下面的具体部分，但在此概述这种合作将如何发挥极大的协同作用并在分子图书馆领域实现 NIH 路线图目标方面取得重大进展是有价值的。在指导新墨西哥州分子图书馆筛选中心时，Sklar 博士处于独特的地位。重要的是，Sklar 博士将非常有资格协助将 NFCR 的新技术与现实世界的筛选问题相结合。通过直接与 Sklar 博士合作或通过 Sklar 博士向筛查中心的用户推荐，使 NFCR 能够快速将其技术应用于生物问题。 方法 项目1 NFCR 项目 1 的主要技术目标是开发以前通过流式细胞术不可能或不切实际的新光学分析技术，对于分子筛选特别重要的是能够以流式细胞术形式使用发光。由于发射时间长，目前可以使用流式细胞术轻松测量，但项目 1 将实施能够以高颗粒分析速率测量发光的声学聚焦细胞仪，这将使高通量流式细胞术筛选能够利用其优势。与 Sklar 博士的合作将包括在新墨西哥大学癌症中心的 Claire Verschraegen 和 Walker Wharton 建议的项目中开发用于发光检测的声学流式细胞仪。研究和治疗中心。正在考虑的测定旨在通过高通量流式细胞术鉴定 HSP70 抑制剂，该细胞系已用 pRC/荧光素酶（新霉素抗性）稳定转染。和 pcDNA3.p70（潮霉素抗性），使细胞表达萤火虫荧光素酶和人 HSP70 的水平一致升高。该测定的进行方式是在热处理和复性后观察到依赖于分子伴侣 HSP 70 的荧光素酶活性。该筛选涉及识别调节 HSP 70 活性的小分子。这项合作直接支持分子图书馆路线图计划，以开发高通量高含量的新工具。筛选。 项目2 NFCR项目2的主要技术目标是开发高速、大型颗粒分选机，已经在大型聚合物颗粒上合成了相当数量的“一珠一化合物”库，但目前的技术需要手动选择来获得珠子。流式细胞术筛选颗粒表面生物靶标的相互作用，然后通过传统技术（例如质谱法）进行分选以确定相互作用化合物的身份，这将是这种强大筛选技术通量的重大进步。自从图书馆现在通过筛选中心提供的不是“一珠一化合物”文库，我们将专注于使用更多样化的文库集开发新的筛选方法。在这次合作中，Sklar 博士将就新的举措和测定进行咨询，以将该技术传达给其他人。他作为 NIH 新墨西哥州分子图书馆筛选中心的 PI 的角色将使研究界能够很好地了解这项技术，NMMLSC 的当前合作者之一是 TPIMS 的总裁 Richard Houghten。这用于合成珠上肽库的茶袋方法作为项目 2 的一部分，NFCR 将开发一种供 UNM 使用的大型颗粒分选机，它将作为 NFCR、UNM 筛选中心和新墨西哥州筛查中心用户。 项目3 项目 3 的目标是开发一种集成相位光谱流式细胞仪，允许在宽光谱范围内同时测量许多荧光团的荧光寿命。该仪器将具有多种应用，以提高基于细胞和颗粒的信息内容。测量荧光团荧光寿命的能力提供了一种探测分子相互作用并提高游离配体与结合配体的分辨率的方法，收集单个颗粒的完整光谱发射信息的能力也提供了提高的途径。基于微珠的筛选分析的多重分析：将其与荧光寿命信息相结合，应该可以更好地解析筛选分析中的分子相互作用。Sklar 博士的团队在开发受体-配体相互作用的分析方法方面拥有丰富的经验，这种合作将用于开发改进的方法。正如项目 3 中详细介绍的，研发方法最初将产生单独的相位分辨和光谱仪器：Sklar 博士的团队将使用这些仪器来测试和验证改进的分子筛选。一旦集成仪器投入运行，我们将利用 NM 筛选中心及其合作者的专业知识，将由此产生的增加的多重潜力和改进的分子相互作用测定应用于基于细胞和基于微珠的筛选分析研究。吸引了。 ORCAS 数字数据采集系统 我们已经开始努力为 NM 分子库筛选中心提供 NFCR 开发的 ORCAS 数字数据采集系统 (6) 该系统已用于操作 HyperCyt¿用于数据采集的高通量筛选仪器。ORCA 系统的一些功能为该仪器提供了明显的优势，特别是显着提高的时间分辨率和范围。我们将提供专用于该仪器的 ORCA 系统以及 NFCR 开发的系统。用于数据显示和分析的可定制快速采集和可视化软件 (TRAViS) 程序我们将继续与 Sklar 博士的团队合作，增强 ORCA 系统的高通量应用，特别是开发仪器控制系统和提高分析灵敏度。还使用项目 3 中开发的自定义数据分析例程进行光谱/寿命测量，以增加筛选分析的信息内容和吞吐量。