Discovery And Characterization Of Selective And Nonselective Inhibitors Of Erbb4 Signaling: Putative Targeted Melanoma Therapeutics.

Introduction: Gain-of-function mutations in the gene that encodes the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma samples. Melanoma cell lines that harbor some of these mutations are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent melanomas. Our drug discovery approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist/antagonist 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. ErbB4 partial agonists that function as antagonists at ErbB4 may hold promise as targeted therapeutics for ErbB4-dependent melanomas. Experimental Procedures: Automated, high-throughput phospho-ErbB4 sandwich ELISA and ErbB4-dependent proliferation assays were developed and deployed to identify 19 small-molecule compounds that stimulate ErbB4 tyrosine phosphorylation, yet inhibit ErbB4-dependent cell proliferation. These compounds were prioritized on the basis of their potency, efficacy, and specificity for inhibition of ErbB4-dependent proliferation. Traditional immunoblotting approaches specific to candidate proteins have been deployed to identify targets for hit molecules of interest. Data: The single high-priority candidate (AU-05) inhibits agonist-induced ErbB4-dependent cellular proliferation with an IC50 of 6.8 uM, but inhibits IL3-dependent proliferation with an IC50 of ~2100 uM, suggesting that AU-05 possesses specificity for ErbB4-dependent proliferation. In contrast, AU-39 potently inhibits both ErbB4-dependent cell proliferation with an IC50 of 1.05 nM and IL3-dependent cellular proliferation with an IC50 of 2.61 nM. We have hypothesized that AU-39 targets the PI3K/Akt signaling pathway, which lies downstream of both ErbB4 and the IL3 receptor. We are testing this hypothesis in part by examining the effects of AU-39 on agonist-induced ErbB4 and IL3 receptor coupling to phosphorylation of Akt and other components of the PI3K/Akt pathway. Conclusions: AU-05 is a starting point for the development of specific ErbB4 inhibitors, whereas AU-39 is a candidate inhibitor of the PI3K/Akt signaling pathway. The inhibition of ErbB4-dependent cellular proliferation by these molecules justifies further development of both molecules. Citation Format: Lauren M. Lucas, Richard L. Cullum, Jared I. Senfeld, Laura J. Cook, Mackenzie H. Harris, David Z. Kaufmann, Cristina C. Rael, Darby C. Taylor, John T. Piazza, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese II. Discovery and characterization of selective and nonselective inhibitors of ErbB4 signaling: Putative targeted melanoma therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A19.