(512e) Development of Partial Agonists Targeted At Receptor Tyrosine Kinases for Cancer Drug Discovery

Development of Partial Agonists Targeted at Receptor Tyrosine Kinases for Cancer Drug Discovery

Richard L. Cullum*, David J. Riese, II**, and Ram B. Gupta*

*Department of Chemical Engineering, Auburn University, Auburn, AL 36849

**Department of Pharmacal Sciences, Auburn University, Auburn, AL 36849

            According to the American Cancer Society, it is estimated that about 580,350 Americans will die of cancer in 2013. This is almost 1,600 people per day. Figures as large as this indicate that there needs to be a push in the improvement of chemotherapeutics.  In recent years, receptor tyrosine kinases (RTKs) have been shown to be promising targets for cancer drug discovery.  RTKs are cell surface receptors that regulate normal cell growth and development, but also function in the growth of cancer tumors.  Targeting RTKs with ligands that function as partial agonists could potentially prohibit the proliferation of tumor cells.  The partial agonists would compete with the full agonists for receptor binding and therefore preventing the full agonists from stimulating coupling to cell proliferation.     

            ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, a family that also includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3).  Conflicting reports in the literature suggest that ErbB4 may function either as an oncogene or as a tumor suppressor in human malignancies.^1,2  Thus, our long-term goal is to develop ErbB4 ligands that can be used to elucidate ErbB4 function and ErbB4 antagonists that could be used to treat ErbB4-dependent tumors.  Our strategy is based on the observation that the Q43L mutant of the naturally-occurring ErbB4 agonist Neuregulin 2beta (NRG2β) functions as a partial agonist at ErbB4; NRG2β/Q43L stimulates ErbB4 tyrosine phosphorylation, but does not stimulate ErbB4 coupling to cell proliferation and competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation.  Therefore, we seek to develop medium- and high-throughput assays for stimulation of ErbB4 tyrosine phosphorylation and for stimulation of ErbB4 coupling to cell proliferation.  Molecules that stimulate ErbB4 tyrosine phosphorylation but do not stimulate ErbB4 coupling to cell proliferation are highly likely to function as ErbB4 antagonists.  Here we describe our efforts to develop these assays.

            The goal of the first assay will be to screen for molecules that stimulate ErbB4 tyrosine phosphorylation.  After seeding CEM/ErbB4 cells in 96-well dishes, they will be stimulated with a library of potential ErbB4 ligands, NRG2β/Q43L, or PBS.  The Q43L mutant will act as the positive control for stimulation of tyrosine phosphorylation and the PBS will act as the negative control.  After stimulation, the CEM/ErbB4 cells will be lysed and the lysates will be added to 96-well dishes that have been coated with a capture antibody that recognizes ErbB4.  An anti-phosphotyrosine antibody, an HRP-conjugated secondary antibody, and a colorimetric assay will then be used to quantify ErbB4 tyrosine phosphorylation.  Several parameters will be varied to optimize detection and quantification of ErbB4 tyrosine phosphorylation.  These parameters include the amount of ErbB4 capture antibody, the amount of CEM/ErbB4 lysate, the concentration of NRG2β/Q43L, the amount of anti-phosphotyrosine antibody, and the amount of the HRP-conjugated secondary antibody. 

            The second assay will be designed to detect molecules that do or do not stimulate ErbB4 coupling to cell proliferation.  To accomplish this, BaF3/ErbB2+ErbB4 cells that have been seeded in a 96-well dish will be stimulated with NRG2β, NRG2β/Q43L, the library of potential ErbB4 ligands, and PBS.  Cells treated with NRG2β will serve as the positive controls.  Cells treated with NRG2β/Q43L will serve as the negative controls. Cells treated with PBS will serve as the diluent controls.  After stimulation, the cells will be incubated for 48-144 hours before a tetrazolium salt and a colorimetric assay are used to analyze the ErbB4 coupling to cell proliferation.  In an effort to maximize detection of ErbB4 coupling to cell proliferation the following parameters will be varied: number of BaF3/ErbB2+ErbB4 cells, NRG2β and NRG2β/Q43L concentrations, incubation time, and tetrazolium salt dye incubation time.

            The development of these assays will be of great importance in the search for ligands that function as partial agonists for ErbB4.  Once all experimental parameters have been optimized, libraries of potential ErbB4 ligands such as a series of monoclonal antibodies could be effectively scanned in hope of discovering ligands that competitively bind with full agonists for the ErbB4 receptor.  Partial agonists at ErbB4 would be promising candidates for the establishment of a new paradigm in cancer treatment. 

(1)        Mill, C. P.; Gettinger, K. L.; Riese II, D. J. Experimental Cell Research 2011, 317, 392–404.

(2)        Koutras, A. K.; Fountzilas, G.; Kalogeras, K. T.; Starakis, I.; Iconomou, G.; Kalofonos, H. P. Critical Reviews in Oncology/Hematology 2010, 74, 73–78.