Abstract 1245: Screening methodologies for the discovery of small molecule melanoma therapeutics targeted at the ErbB4 receptor tyrosine kinase
Authors: Cullum Richard L. , Piazza John T. , Senfeld Jared I. , Neel Logan T. , Gupta Ram B. , David Allan E. , Riese David J. 
Published: July 2017

Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Consequently, we have developed high-throughput screening assays to identify small molecule ErbB4 antagonists that may hold promise as targeted melanoma therapeutics. Our approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation.

Experimental procedures: Therefore, we have developed three high-throughput screening assays to identify ErbB4 partial agonists that function as antagonists. The primary screen identifies molecules that stimulate ErbB4 tyrosine phosphorylation. The secondary screen identifies molecules that stimulate or fail to stimulate ErbB4-dependent cell proliferation. The tertiary screen identifies molecules that antagonize agonist stimulation of ErbB4-dependent cell proliferation.

Results: A phospho-ErbB4 sandwich ELISA assay identifies molecules that stimulate ErbB4 tyrosine phosphorylation with high sensitivity and fidelity (Z’ &gt;0.5). IL3-independence assays in conjunction with MTT assays using a cell line that displays ErbB4-dependent cell proliferation distinguish between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation (Z’&gt;0.5) and identify molecules that antagonize agonist stimulation of ErbB4 dependent proliferation. These assays have been used to identify small molecules that stimulate ErbB4 tyrosine phosphorylation. Efforts to determine whether these hits function as ErbB4 full agonists or partial agonists (antagonists) are underway and will be reported. Structures of these small molecule ErbB4 full and partial agonists may be reported, pending submission of a provisional patent application.

Conclusions: We have validated an HTS strategy for identifying ErbB4 partial agonists that function as ErbB4 antagonists and deployment of that strategy has led to the identification of several hits (~20 from the primary screen). Such molecules may hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors.

Citation Format: Richard L. Cullum, John T. Piazza, Jared I. Senfeld, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese. Screening methodologies for the discovery of small molecule melanoma therapeutics targeted at the ErbB4 receptor tyrosine kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2017-1245