Abstract B17: A high-throughput screening process for the discovery of melanoma chemotherapeutics targeted at the ErbB4 receptor tyrosine kinase
Authors: Cullum Richard L. , Gupta Ram B. , Riese David J. 
Published: July 2015

Introduction. 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). Gain-of-function ErbB4 mutants are found in a significant fraction of melanoma cell lines, suggesting that ErbB4 is a melanoma oncogene. However, there is a paucity of specific inhibitors for probing ErbB4 function or for treating ErbB4-dependent tumors. Consequently, we are developing high-throughput screening (HTS) approaches to address this void.

Experimental Strategy. The Q43L mutant of the naturally-occurring ErbB4 agonist Neuregulin 2beta (NRG2beta) functions as a partial agonist at ErbB4; NRG2beta/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 are developing three high-throughput assays to identify ErbB4 partial agonists that function as antagonists. (1) The primary screen will utilize a sandwich enzyme-linked immunosorbent assay (ELISA) to identify molecules that stimulate ErbB4 tyrosine phosphorylation. (2) The secondary screen will use an MTT proliferation assay to identify molecules that fail to stimulate ErbB4 coupling to cell proliferation. (3) An MTT proliferation assay will be used in the tertiary screen to identify molecules that inhibit agonist-induced ErbB4 coupling to cell proliferation. Molecules that stimulate ErbB4 tyrosine phosphorylation, do not stimulate ErbB4 coupling to cell proliferation, and inhibit agonist-induced ErbB4 coupling to proliferation are highly likely to function as ErbB4 antagonists.

Results. (1) A high-throughput ELISA assay reproducibly detects stimulation of ErbB4 tyrosine phosphorylation by the ErbB4 full agonist NRG2beta (Z' = 0.730) and the ErbB4 antagonist (partial agonist) NRG2beta/Q43L (Z' = 0.664). (2) A high-throughput MTT assay reproducibly detects stimulation of ErbB4 coupling to cell proliferation by the ErbB4 full agonist NRG1beta (Z' = 0.236; 10 nM NRG1beta versus unstimulated). (3) A high-throughput MTT assay detects the difference between stimulation of ErbB4 coupling to cell proliferation by 2 nM NRG1beta and 10 nM NRG1beta (Z' = 0.246), suggesting that 80% inhibition of the activity of 10 nM NRG1beta at ErbB4 can be detected using this assay. (4) Initial screening of small molecule libraries using the primary screen has yielded more than 20 hits (potential novel small-molecule ErbB4 ligands). These hits are being validated and subjected to secondary and tertiary screens.

Conclusions. We have validated HTS approaches for the identification of small molecule ErbB4 partial agonists and antagonists. The initial implementation of these approaches has yielded promising results.

Citation Format: Richard L. Cullum, Ram B. Gupta, David J. Riese, II. A high-throughput screening process for the discovery of melanoma chemotherapeutics targeted at the ErbB4 receptor tyrosine kinase. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B17.