Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

Abstract

Whereas recent clinical studies report metastatic melanoma survival rates high as 30–50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that 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 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z’ > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

ERBB4 Drives the Proliferation of BRAF-WT Melanoma Cell Lines

“…Stably infected MEL-JUSO cells were plated in a 96-well plate (Corning) at 1×10 4 cells/well. The next day they were treated with a drug and after five days of incubation were subjected to an MTT assay as previously described [54]. The PI3K inhibitor LY294002 was obtained from Tocris Bioscience.…”

Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

“…When comparing our results to IC 50 values of phospho-blots and cell proliferation studies, we either had comparable potency values (e.g., for AG1478 on EGFR, 51 erlotinib on phospho-ERK, 52 osimertinib on ERBB4, 28 poziotinib on EGFR and ERBB2/3, 53 and TAS6417 on EGFR 48 ) or an order of magnitude higher IC 50 values, possibly due to the use of different cell lines and assay readouts (e.g., for erlotinib on EGFR, gefitinib on EGFR and ERBB4, 54 , 55 osimertinib on EGFR, 56 and pyrotinib on EGFR. 49 Furthermore, our data were comparable to results for clozapine effects with three other assays, namely the commercial DiscoveRx PathHunter assay for the HTR2A receptor, a cAMP accumulation assay, and a Fluo-4 assay to measure Ca 2+ .…”

“…Table S4 ). 49 , 52 , 54 , 55 , 56 The latter case may be due to different cell types and/or assay systems applied. Nevertheless, a substantial number of studies used phospho-blots to demonstrate the drug effect on ERBB receptors.…”

A systematic review on Drug Re-profiling/Re-Purposing

“…It can be a Sarcoma, Leukemia, lymphoma, multiple myeloma, Central nervous system cancers (begin in the tissues of the brain and spinal cord) and others 25 , 52 . Dreadful diseases like cancer necessitates the need for drug repurposing, so that an anticancer drug can be used in multiple cancers thus, with same drug we can provide new therapeutic approaches 53 . This strategy is not only cost effective but also have lifesaving tendency 54 .…”