Abstract 10935: Identification of Small-Molecule ERBB4 Agonists to Treat Heart Failure

Abstract

Introduction: Heart failure is a complex syndrome with a poor prognosis. The neuregulin-1 (NRG1)/ERBB4 axis has been shown to be cardioprotective and is a possible target for heart failure therapy. Clinical trials with NRG1 are ongoing, but require intravenous administration. Our aim is to develop a small-molecule ERBB4 agonist with cardioprotective properties. Methods: A high-throughput diversity screening (HTS) of 10,240 compounds was performed on a cellular ERBB4/ERBB4 dimerization assay. Ligand binding of hit compounds was tested in co-administration studies with NRG1 (0.1 μM) and competition binding assays with fluorescently labeled NRG1 (0.03 μM). Selectivity, post-receptor phosphorylation, and cell toxicity of hit compounds were determined using Luminex® RTK phosphoprotein, U2OS ERBB2/ERBB3 dimerization and adenylate kinase assays. Biological effects were studied in vitro in different doses (4-32 μM) on TGF-β-induced collagen synthesis by human dermal and atrial fibroblasts, and on angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) remodeling in mice with 2 selected hit compounds (83 μg/kg/h), administrated through osmotic pumps (N=4-5/group). After 28 days, left ventricular myocardial fibrosis and cardiomyocyte cross sectional area (CSA) were analyzed. Results: The HTS resulted in 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 but not ERBB2/ERBB3 dimerization (Emax 9 to 33% relative to NRG1, and EC50 6x10 -6 M to 2x10 -7 M). Two compounds were excluded due to in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but not ERBB1, ERBB2 or ERBB3 phosphorylation in EFO-21 cells. Competition assays showed allosteric binding, and co-administration studies with NRG1 revealed that compounds potentiated NRG1-induced ERBB4 receptor dimerization up to 2.5 fold. Selected compounds showed significant dose-dependent antifibrotic effects in cultured human atrial and dermal fibroblasts and significantly attenuated AngII-induced LV myocardial fibrosis by 76%±26%, whereas cardiomyocyte CSA was not affected. Conclusions: We identified novel pyrimidine derivative small-molecule ERBB4 agonists with antifibrotic properties, both in vitro and in a mouse model of AngII-induced LV fibrosis.