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Automated Screening for Small Molecule Regulators of Cardiogenesis Cardiomyocyte production from hESCs is currently too inefficient to permit large-scale clinical application. New technology is required to enhance differentiation and, possibly, replication of committed precursors. Thus a major initiative in the lab is to identify small molecules that regulate these and other critical aspects of stem cell biology through the automated screening of chemical, siRNA and microRNA libraries. The goal of our chemical library screening program is to generate small, drug like molecules that will direct differentiation of hESCs to cardiomyocytes and stimulate cell-cycle entry and proliferation of differentiated cardiomyocytes. This project, sponsored by the California Institute for Regenerative Medicine and National Institutes of Health, has led to the discovery of a number of hit classes that stimulate cardiomyocyte differentiation from ESCs. Structure activity relationship studies in collaboration with Dr. John Cashman at the Human Biomolecular Research Institute (http://www.hbri.org) have led to optimized compounds and biochemical affinity reagents. In addition, screening of combinatorial chemistry libraries in collaboration with Clemencia Pinilla and Richard Houghten at the Torrey Pines Institute for Molecular Studies (http://www.tpims.org) has yielded a number of active compounds optimized through structure activity relationship studies. The small-molecule leads are being used as probes to characterize the cellular signaling pathways and proteins that direct the multi-step process of stem cells to cardiomyocyte differentiation. Once identified, the cellular targets of the small molecules can be used in structure-based approaches to guide lead optimization. A long-term goal is to exploit the the druggable targets in biochemical screens to discover novel and more effective classes of hits. In addition, since many of the small molecules might also target processes important for cardiomyocyte regeneration from endogenous stem cells; certain of the hits have potential as therapeutic stimulators of endogenous regeneration from a patient’s own cells. Critical to the success of using small molecules identified through phenotypic screens as probes of complex biological processes, such as differentiation and functional maturation of cardiomyocytes, is developing robust methods for the identification of the cellular targets. Affinity versions of optimized small molecules are being generated for use as biochemical probes in collaboration with Professor Marcia Dawson at BIMR. In addition, the NHLBI Exploratory Program in Systems Biology http://www.nhlbi.nih.gov/resources/sys-bio/exploratory.htm is sponsoring a program in collaboration with Professor Shankar Subramaniam at UCSD and Professor Juan Carlos Izpisua Belmonte at the Salk Institute to develop informatics tools to deduce target pathways through phospho-proteomics and genomics. The screens are being designed and implemented and medicinal chemistry follow up of the hits is being conducted in partnership with the Burnham Center for Chemical Genomics [http://sdccg.burnham.org/metadot/index.pl], which is a multidisciplinary group of cell biologists, chemists and engineers funded by the NIH Multiple Libraries Probe Production Centers Network (MLPCN) [https://mli.nih.gov/mli/mlpcn/access-to-mlscn-screening]. The MLPCN was established by the NIH Roadmap for Medical Research Initiative to accelerate medical discoveries. The network allows researchers with promising assays to use the expertise and resources of the BCCG to identify novel chemical probes and make the results available to the research community through PubChem, a shared database.
People Vipul Bhargava, PhD graduate student, UCSD Bioengineering Paul Bushway PhD student, UCSD Biomedical Sciences-Molecular Pathology Wenqing Cai PhD student, BIMR PhD program Alexandre Colas, PhD postdoctoral fellow Serena Grimaldi, PhD, postdoctoral fellow Frederick Lo PhD student, UCSD Bioengineering Sean Spiering, Research Associate Joaquim Teixeira PhD student, UCSD Biomedical Sciences-Molecular Pathology Masanao Tsuda, PhD, postdoctoral fellow Erik Willems, PhD, postdoctoral fellow
Collaborators: Pedro Aza-Blanc, PhD, Director of Functional Genomics, BCCG Susanne Heynen, PhD, Director of High Content Screening, BCCG Eduard Sergienko, PhD, Director of Assay Development, BCCG John Cashman, PhD, Professor and Director, Human Biomolecular Research Institute, La Jolla Marcia Dawson, PhD, Professor, BIMR Jeff Price, MD, PhD, Associate Professor, BIMR Clemencia Pinilla, Ph.D., Associate Member, Torrey Pines Institute for Molecular Studies Richard Houghten, Ph.D., President, Torrey Pines Institute for Molecular Studies
Recent publications
Willems, E., Bushway, P., Mercola, M. (2008). Natural and Synthetic Regulators of Embryonic Stem Cell Cardiogenesis, Pediatric Cardiology, In press. Charlot D., Campa, V., Azimi, B., Mercola, M., Ingermanson, R., McDonough, P.M., and Price, J.H. (2008). Automated Calcium Measurements in Live Cardiomyocytes. IEEE Symposium Biomedical Imaging, 2008: 316-319. Bushway, P.J., Mercola, M., and Price, J.H. (2008). A Comparative Analysis of Standard Microtiter Plate Reading versus Imaging in Cellular Assays. Assay and Drug Development Technologies, 6, 557-667. [PubMed] Bushway, P.J., Azimi, B., Heynen-Genel, S., Price, J.H., and Mercola, M. (2008). Hybrid Median Filter Background Estimator for Correcting Distortions in Microtiter Plate Data. Assay and Drug Development Technologies, In press. Kita-Matsuo, H., Barcova, M., Prighozhina, N., Salomonis, N., Wei, K., Jacot, J.G., Nelson, B., Spiering, S., Haverslag, R., Kim, C., Talantova, M., McCulloch, A.D., Conklin, B.R., Price, J.H., Chen, H.S.V., and Mercola, M. (2008). Stable Human Embryonic Stem Cell Lines for Tracking and Purification of Cardiomyocytes by Genetic Selection. Submitted. Prigozhina, N., Russo, J.A., Pekurovsky, A., Kita-Matsuo, H., Clancy, J.W., Mercola, M. and Price, J.H. (2007). Toward automated analyses of migration and differentiation in cultured human embryonic stem cells. IEEE Symposium Biomedical Imaging, 1104-1107. Wei, Z. L., Petukhov, P. A., Bizik, F., Teixeira, J. C., Mercola, M., Volpe, E. A., Glazer, R. I., Willson, T. M. and Kozikowski, A. P. (2004). Isoxazolyl-serine-based agonists of peroxisome proliferator-activated receptor: design, synthesis, and effects on cardiomyocyte differentiation. J Am Chem Soc 126, 16714-5. [PubMed]
Funding California Institute for Regenerative Medicine NHLBI Exploratory Program in Systems Biology http://www.nhlbi.nih.gov/resources/sys-bio/exploratory.htm NIH Multiple Libraries Probe Production Centers Network (MLPCN) https://mli.nih.gov/mli/mlpcn/access-to-mlscn-screening
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