Fig 2: Five compounds are potent and selective inhibitors of ErbB4-dependent cellular proliferation.(a-e) In three independent trials and using a modified version of our semi-automated processes, BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of each candidate inhibitor in the presence of 0.1 nM IL3 or 0.3 nM NRG1β. A semi-automated MTT assay was used to analyze cellular proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC50 value for each candidate against 0.1 nM IL3 and 0.3 nM NRG1β. IC50 values are also shown in Table 3.

Fig 3: A high-priority small molecule compound selectively and potently inhibits ErbB4-dependent cellular proliferation.(a-b) In three independent trials and using a modified version of our semi-automated processes, BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of a candidate inhibitor or gefitinib (positive control inhibitor of NRG1β-induced proliferation) in the presence of 0.1 nM IL3 or 0.3 nM NRG1β. A semi-automated MTT assay was used to analyze cellular proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC50 value for each inhibitor against 0.1 nM IL3 and 0.3 nM NRG1β. IC50 values are also shown in Table 3. *Extrapolated value.

Fig 4: Representative candidates that inhibit agonist-induced ErbB4-dependent cellular proliferation.(a-d) In three independent trials and using semi-automated processes, BaF3/EGFR+ErbB4 cells were stimulated with increasing concentrations of NRG1β in the presence or absence of gefitinib at 300 nM or candidates at 30 uM. A semi-automated MTT assay was used to analyze cell proliferation at 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the EC50 and Emax of NRG1β in the presence and absence of gefitinib or the candidates. EC50 and Emax values are also shown in Table 2.

Fig 5: Deployment strategy of screening methodologies for the identification of partial agonists at the ErbB4 receptor tyrosine kinase that function as ErbB4 antagonists.Southern Research (SR) Phosphorylation Screen: SR screened a library of small molecule compounds (~100k) for stimulation of ErbB4 tyrosine phosphorylation using an ultra-high throughput assay developed by DiscoverX. Auburn University (AU) Phosphorylation Screens: The 43 most promising compounds identified from the high-throughput SRI screen were then tested for concentration-dependent stimulation of ErbB4 tyrosine phosphorylation. AU Agonist Screen: The compounds that exhibited dose-dependent stimulation of ErbB4 tyrosine phosphorylation were tested to determine whether they stimulated ErbB4-dependent cellular proliferation. AU Inhibition Screens: The compounds that failed to stimulate ErbB4-dependent cellular proliferation were tested to determine whether they inhibited agonist-induced ErbB4-dependent cellular proliferation. Selectivity of these inhibitors was determined by comparing inhibition of ErbB4-dependent cellular proliferation against inhibition of Interleukin 3-dependent (IL3-dependent) cellular proliferation.