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.
Introduction: Morbidity and mortality of heart failure remain high, mandating new therapeutic approaches. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and an attractive target for treatment. Clinical trials with recombinant NRG1 are ongoing, but require intravenous administration, limiting applicability and efficacy. Purpose: To develop selective small-molecule ERBB4 agonists with cardioprotective effects. Methods: A high-throughput screening of 10,240 compounds (cpds) was performed on a ERBB4/ERBB4 dimerization assay. Hit cpds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, toxicity and metabolic stability were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization and adenylate kinase assays or LC-MS/MS. Apoptotic and hypertrophic effects of cpds (4-32μM) on cultured cardiomyocytes were studied after exposure to 100μM H 2 O 2 and 100nM angiotensin II (AngII). Antifibrotic effects (4-32μM) were studied on TGF-β-induced collagen synthesis in cultured human fibroblasts, and in mice (n=9-10/group) treated with AngII (1000 ng/kg/min) or cpd (83 μg/kg/h) using osmotic pumps. Results: We identified 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 dimerization (Emax 9-33% relative to NRG1, EC50 6E-6 to 2E-7M). Competition assays indicated allosteric receptor binding and potentiation of NRG1-induced ERBB4 receptor dimerization, up to 2.7 fold (P<0.0001). Six were non-toxic and induced ERBB4 phosphorylation, but ERBB1, ERBB2 or ERBB3 phosphorylation remained unaffected and cpds did not induce ERBB2/ERBB3 dimerization, showing ERBB4 selectivity. Cpds showed a t1/2 of 170min in human liver microsomes. Cpds attenuated hypertrophic and apoptotic effects of AngII or H 2 O 2 (P<0.05), and decreased collagen upregulation in vitro (P<0.05). In vivo , a selected cpd attenuated AngII-induced myocardial interstitial fibrosis (-76±26%, P<0.01), and cardiac Col1a1, Col3a1 (-64±22%; -71±25%, P<0.01) and Nppa (-77±22%, P<0.01) mRNA expression. Conclusion: We identified selective novel pyrimidine derivative small-molecule ERBB4 agonists with cardioprotective effects in vitro and in vivo .
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