In addition, the moderate inhibitor 4a and the chromen-4-one derivative 4g with potent inhibitory effect on the transcriptional synergy between GATA4 and NKX2-5 can formally be regarded as position. they represent a good target for pharmacological interventions.1 However, targeting PPIs with small molecules is challenging due to the large surface area involved in proteinCprotein binding and the lack of clear binding pouches for small molecules at many proteinCprotein interfaces. However, the number of successful examples of small molecule modulators of PPIs has been growing,1,2 and Lawson et al. recently concluded that small molecules provide significant opportunities Bivalirudin TFA for allosteric modulation of PPIs.3 Transcription factors are the fundamental regulators of gene expression, and their PPIs are of pivotal importance in the regulation of biological systems.4 The GATA family of transcription factors consists of six proteins (GATA1C6), which are involved in a variety of physiological and pathological processes.5?7 GATA1C3 are required for Rabbit polyclonal to IL20RB differentiation of mesoderm and ectoderm-derived cells, whereas GATA4C6 are implicated in the development and differentiation of endoderm- and mesoderm-derived cells such as induction of differentiation of embryonic stem cells and cardiovascular embryogenesis.6 In the developing heart, GATA4 is one of the earliest-expressed transcription factors8 and is essential for normal cardiac development.9?12 In the postnatal heart, GATA4 acts while a critical regulator of hormone response and mechanical stress13?16 Bivalirudin TFA as well as cardiac restoration and regeneration.17?20 GATA4 actions involve combinatorial interactions with a number of additional nuclear proteins, reinforcing their activity and tissue specificity.5?7 For example, functional cardiomyocytes can be directly induced from fibroblasts by a combination of three cardiac transcription factors, GATA4, MEF2C, and TBX5, in vitro and in vivo.21,22 Most of the PPIs occur through the C-terminal zinc finger, which also mediates DNA binding and is highly conserved throughout the GATA family.5 GATA4 regulates myocardial gene expression by interacting with different cardiac specific transcription factors, such as NKX2-5, NFAT, and MEF2.5?7 NKX2-5, Bivalirudin TFA a member of the evolutionary conserved NK family of homeobox proteins, is a critical GATA4 cofactor and essential for heart development.23?25 GATA4 and NKX2-5 directly interact and synergistically activate several genes including those encoding atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP).7,26 Mutational studies have shown the tandem GATA consensus sites of the proximal promoter in combination with an NKX2-5 binding element are important for the stretch-activated BNP transcription.27 As the precise balance of GATA4CNKX2-5 connection is essential for mechanical stretch-induced cardiomyocyte hypertrophy, the functional modulation of their connection could present a novel approach for cardiac restoration under pathophysiological conditions. We have previously demonstrated that solitary point mutations can interfere with GATA4 and NKX2-5 connection,28 implicating that this PPI is definitely targetable by small molecules to accomplish direct inhibition, activation, or allosteric modulation. Recently, we reported the recognition of several compounds that impact the transcriptional synergy of GATA4 and NKX2-5.29,30 In addition, we have demonstrated that a small molecule inhibitor of GATA4CNKX2-5 transcriptional synergy reduces cardiomyocyte hypertrophic response in vitro, ameliorates hypertrophic signaling in vivo, and enhances cardiac function in vivo in experimental models of myocardial infarction and hypertension.29,31,32 These studies led to the identification of compounds 1C3 (Number ?Number11), which either increase (2) or inhibit (1 or 3) the transcriptional synergy of GATA4 and NKX2-5.29 Our aim with this study was to optimize the original isoxazole hit compound 1 by means of synthesis and biological investigation of 220 structurally related compounds with modified or alternative northern, central, and southern parts (Number ?Number22). Additionally, this structureCactivity relationship (SAR) analysis was augmented by 29 commercial and 8 previously published compounds33 that were tested having a luciferase reporter assay to examine their effects within the transcriptional synergy of GATA4 and NKX2-5. The most potent compounds were also tested individually in luciferase reporter assays for NKX2-5 and GATA4 activity. Furthermore, toxicity of the selected compounds was analyzed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays in the COS-1 cell collection. Open in a separate window Number 1 Representative examples of the previously reported modulators of GATA4CNKX2-5 transcriptional synergy.29 Compounds 1 and 3 inhibit and compound 2 enhances transcriptional synergy of GATA4 and NKX2-5. Open in a separate window Number 2 Synthesis strategy for structural modifications of compound 1. Results Chemistry Synthesis of Compounds To gain info on the chemical space of the hit compound 1 in regard to the ability of such molecules to inhibit GATA4CNKX2-5 transcriptional synergy, we synthesized 220 structurally related compounds. For any simplified look at, we divided the.