Identification of small-molecule ERBB4 agonists for the treatment of heart failure

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Dehousse fellowship Introduction Although progress has been made in the treatment of heart failure, morbidity and mortality remain high, requiring new therapeutic targets. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and antifibrotic when activated in the myocardium, and therefore a possible target for therapy. Phase 2 and 3 clinical trials with NRG1 are ongoing, but require intravenous administration regimens, limiting applicability and efficacy. Purpose To develop small-molecule ERBB4 agonists with cardioprotective and antifibrotic properties. Methods A high-throughput screening (HTS) of 10,240 compounds was performed on a ERBB4/ERBB4 dimerization assay. Hit compounds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, cell proliferation and toxicity were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization, WST-1 colorimetric, and adenylate kinase assays. Antifibrotic effects were studied in vitro on TGF-β-induced collagen synthesis in human dermal and atrial fibroblasts, and in a mouse model of angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) myocardial fibrosis with selected compounds (83 µg/kg/h), administrated with osmotic minipumps (N=4–5/group). mRNA expression was evaluated after 7 days; LV myocardial fibrosis area, cardiomyocyte cross sectional area (CSA), echocardiographic parameters and heart- to bodyweight ratio (HW:BW) were analyzed at 28 days. Antiapoptotic effects were studied on rat atrial cardiomyocytes (AM) after hydrogen peroxide (H2O2)-induced cardiotoxicity. Results The HTS (Z’=0.7) resulted in 8 similar pyrimidine derivatives (EF-1–8) inducing ERBB4/ERBB4 dimerization (Emax 9–33% relative to NRG1, EC50 6E-6 to 2E-7 M). Competition assays indicate allosteric binding. The compounds also significantly potentiated NRG1-induced ERBB4 receptor dimerization up to 2.7 fold. Two compounds were excluded because of in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but neither ERBB1, ERBB2 or ERBB3 phosphorylation, nor tumor growth–inducing ERBB2/ERBB3 dimerization. Selected compounds showed significant dose-dependent antiapoptotic properties on H2O2-stimulated AM, and antifibrotic effects on human atrial and dermal fibroblasts. In vivo, compound EF-1 significantly decreased myocardial fibrosis (by 76±26%) and Col1a1, Col3a1 (-70±17%; -61±20%), and Nppa (-78±32%) mRNA expression, and significantly enhanced cardiomyocyte CSA (+24±8%). No differences were observed in cardiac function or HW:BW ratio. Conclusion We identified novel pyrimidine derivative small-molecule ERBB4 agonists with cardiomyocyte protective effects and antifibrotic properties in vitro and in AngII-induced myocardial fibrosis in vivo.