HIGH THROUGHPUT, HIGH CONTENT MOLECULAR LIBRARIES SCREENING

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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. Sklars 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. Sklars 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. Sklars 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. Sklars 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. Sklars 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.