JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	David D. Roberts
Title:	Principal Investigator
Address:	Building 10 Room 2A33 10 Center Drive MSC1500 Bethesda, MD 20892
Telephone:	301-496-6264
Fax:	301-402-0043
Email:	droberts@helix.nih.gov
Sponsoring Laboratory/Branch:	Laboratory of Pathology

Project Title:	A high throughput screen for CD47 antagonists
Target(s) of Interest:	CD47

Project Synopsis:
This project has 2 goals: to optimize a high throughput screening assay for peptide and small molecule agonists of CD47, and to identify novel peptide and small molecule agonists/antagonists of CD47 predicted from computational modeling studies or by screening of compound libraries. CD47, also known as integrin associated protein (IAP), is a ~50kDa transmembrane protein of the IgV superfamily [1]. The expression of CD47 is greatest in leukocytes, platelets, and erythrocytes, but it is expressed in nearly all cell types. CD47 was at first an orphan receptor that was identified as a contaminant of integrin preparations, but it is now recognized as an important counter-receptor for signal inhibitory receptor protein-alpha (SIRP-alpha or SHPS-1) and a receptor for the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1). Binding to SIRP-alpha is necessary for the innate immune system to recognize self. Engagement of CD47 by immune cells bearing SIRP-alpha inhibits phagocytosis of the target cell. Results from our lab have shown that interactions of TSP-1 with CD47 profoundly control tissue perfusion, radiosensitivity, angiogenesis, and immune functions [2]. Functionally, blocking TSP-1/CD47 interactions in animal model systems results in lowering of blood pressure [3], an inhibition of thrombosis [4], dramatic increases in tissue survival following ischemia or ischemia/reperfusion injuries [5], and nearly complete sparing of peripheral tissues following tumor radiation while simultaneously increasing tumor ablation [6]. The effects of CD47 ligation on blood pressure, thrombosis, tissue survival [7], and anti-angiogenesis [8] primarily result from inhibition of nitric oxide (NO) signaling through soluble guanylate cyclase. Radiation protection/tumor sensitization probably involves additional effecter pathways. These important pathophysiological functions of CD47 were revealed using a combination of genetic knockout mice, antisense knockdown, monoclonal antibodies, and peptides that target TSP-1 or CD47. Some CD47 antagonists have activities to improve radiation responses in tumor bearing mice, suggesting a therapeutic value of targeting TSP-1/CD47 signaling. This therapeutic potential justified an effort to develop small molecule drugs that target this pathway. This project will optimize a homogeneous cell-based assay developed in our lab that quantifies a CD47 dependent fluorescence signal for use in high throughput screening (HTS). If necessary, additional endpoints will be examined to establish a rigorous HTS. The validated HTS assay will be used to test peptide agonists synthesized based on rational design strategies, to assess candidate inhibitors identified by ongoing computational modeling of the CD47/TSP-1 binding site, and to screen small molecule libraries. Candidates identified from the HTS will be confirmed using appropriate secondary biochemical and cellular screens and, finally, tested for therapeutic activity in vivo in TSP-1/CD47-dependent mouse injury and tumor irradiation models. 1. Brown, E.J. and W.A. Frazier, Integrin-associated protein (CD47) and its ligands. Trends Cell Biol, 2001. 11(3): p. 130-5. 2. Isenberg, J.S., et al., Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. Nat Rev Cancer, 2009. 9(3): p. 182-94. 3. Isenberg, J.S., et al., Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress. Matrix Biol, 2009. 28(2): p. 110-9. 4. Isenberg, J.S., et al., Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling. Blood, 2008. 111(2): p. 613-23. 5. Isenberg, J.S., et al., Thrombospondin-1 limits ischemic tissue survival by inhibiting nitric oxide-mediated vascular smooth muscle relaxation. Blood, 2007. 109(5): p. 1945-52. 6. Isenberg, J.S., et al., Thrombospondin-1 and CD47 limit cell and tissue survival of radiation injury. Am J Pathol, 2008. 173(4): p. 1100-12. 7. Isenberg, J.S., et al., Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner. Proc Natl Acad Sci U S A, 2005. 102(37): p. 13141-6. 8. Roberts, D.D., et al., Nitric oxide and its gatekeeper thrombospondin-1 in tumor angiogenesis. Clin Cancer Res, 2007. 13(3): p. 795-8.
Fellow Research Plan:
The fellow will receive training and conduct research to identify novel CD47 signaling agonists and antagonists. They will optimize an existing fluorescence cell-based reporter assay for HTS with guidance from Dr. Barry OKeefe in the Molecular Targets Development Program. Using the optimized assay, they will establish IC50 values for existing and novel peptides and small molecules and validate their effectiveness using biochemical and cell-based assays currently used in the Roberts lab. The fellow will conduct screening assays using peptides prepared by the Roberts lab, candidate inhibitors identified by computational screening and purchased from Chem Navigator, and compound libraries available at the NCI. The fellow will be expected to gain expertise in HTS screening assay optimization and validation. The fellow will also gain expertise and provide input for the rational design of improved peptides and selection of small molecules for screening. The fellow will also receive training and participate in the functional validation of positive compounds using a variety of in vitro assays currently used in the Roberts lab such as ELISA, cell migration/proliferation/spreading/contraction assays, and animal models of tissue ischemia and radiation responses. The fellow will prepare manuscripts for publication in peer reviewed journals and attend appropriate scientific conferences to present the findings. They will also review current literature relevant to their work and present at weekly Section meetings.