Affinity chromatography reveals direct binding of the GATA4-NKX2-5 interaction inhibitor (3i-1000) with GATA4.

Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors.

Switching of hypertrophic signalling towards enhanced cardiomyocyte identity and maturity by a GATA4-targeted compound.

BACKGROUND: The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte. METHODS: A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing. RESULTS: The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch. CONCLUSIONS: There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.

TAF1 bromodomain inhibition as a candidate epigenetic driver of congenital heart disease.

Heart formation requires transcriptional regulators that underlie congenital anomalies and the fetal gene program activated during heart failure. Attributing the effects of congenital heart disease (CHD) missense variants to disruption of specific protein domains allows for a mechanistic understanding of CHDs and improved diagnostics. A combined chemical and genetic approach was employed to identify novel CHD drivers, consisting of chemical screening during pluripotent stem cell (PSC) differentiation, gene expression analyses of native tissues and primary cell culture models, and the in vitro study of damaging missense variants from CHD patients. An epigenetic inhibitor of the TATA-Box Binding Protein Associated Factor 1 (TAF1) bromodomain was uncovered in an unbiased chemical screen for activators of atrial and ventricular fetal myosins in differentiating PSCs, leading to the development of a high affinity inhibitor (5.1 nM) of the TAF1 bromodomain, a component of the TFIID complex. TAF1 bromodomain inhibitors were tested for their effects on stem cell viability and cardiomyocyte differentiation, implicating a role for TAF1 in cardiogenesis. Damaging TAF1 missense variants from CHD patients were studied by mutational analysis of the TAF1 bromodomain, demonstrating a repressive role of TAF1 that can be abrogated by the introduction of damaging bromodomain variants or chemical TAF1 bromodomain inhibition. These results indicate that targeting the TAF1/TFIID complex with chemical compounds modulates cardiac transcription and identify an epigenetically-driven CHD mechanism due to damaging variants within the TAF1 bromodomain.

Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation.

Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated α-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of α-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological α-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing α-synuclein aggregation via systemic administration.

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line.

BACKGROUND: Pharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration. METHODS: Transcription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression. RESULTS: GATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression. CONCLUSIONS: Collectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo: establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes.

Doxorubicin is a widely used anticancer drug that causes dose-related cardiotoxicity. The exact mechanisms of doxorubicin toxicity are still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose doxorubicin treatments. Here, we developed an in vitro model of long-term low-dose administration of doxorubicin utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Moreover, given that current strategies for prevention and management of doxorubicin-induced cardiotoxicity fail to prevent cancer patients developing heart failure, we also investigated whether the GATA4-targeted compound 3i-1000 has cardioprotective potential against doxorubicin toxicity both in vitro and in vivo. The final doxorubicin concentration used in the chronic toxicity model in vitro was chosen based on cell viability data evaluation. Exposure to doxorubicin at the concentrations of 1-3 µM markedly reduced (60%) hiPSC-CM viability already within 48 h, while a 14-day treatment with 100 nM doxorubicin concentration induced only a modest 26% reduction in hiPCS-CM viability. Doxorubicin treatment also decreased DNA content in hiPSC-CMs. Interestingly, the compound 3i-1000 attenuated doxorubicin-induced increase in pro-B-type natriuretic peptide (proBNP) expression and caspase-3/7 activation in hiPSC-CMs. Moreover, treatment with 3i-1000 for 2 weeks (30 mg/kg/day, i.p.) inhibited doxorubicin cardiotoxicity by restoring left ventricular ejection fraction and fractional shortening in chronic in vivo rat model. In conclusion, the results demonstrate that long-term exposure of hiPSC-CMs can be utilized as an in vitro model of delayed doxorubicin-induced toxicity and provide in vitro and in vivo evidence that targeting GATA4 may be an effective strategy to counteract doxorubicin-induced cardiotoxicity.

Targeting GATA4 for cardiac repair.

Various strategies have been applied to replace the loss of cardiomyocytes in order to restore reduced cardiac function and prevent the progression of heart disease. Intensive research efforts in the field of cellular reprogramming and cell transplantation may eventually lead to efficient in vivo applications for the treatment of cardiac injuries, representing a novel treatment strategy for regenerative medicine. Modulation of cardiac transcription factor (TF) networks by chemical entities represents another viable option for therapeutic interventions. Comprehensive screening projects have revealed a number of molecular entities acting on molecular pathways highly critical for cellular lineage commitment and differentiation, including compounds targeting Wnt- and transforming growth factor beta (TGFß)-signaling. Furthermore, previous studies have demonstrated that GATA4 and NKX2-5 are essential TFs in gene regulation of cardiac development and hypertrophy. For example, both of these TFs are required to fully activate mechanical stretch-responsive genes such as atrial natriuretic peptide and brain natriuretic peptide (BNP). We have previously reported that the compound 3i-1000 efficiently inhibited the synergy of the GATA4-NKX2-5 interaction. Cellular effects of 3i-1000 have been further characterized in a number of confirmatory in vitro bioassays, including rat cardiac myocytes and animal models of ischemic injury and angiotensin II-induced pressure overload, suggesting the potential for small molecule-induced cardioprotection.

Synthesis, Identification, and Structure-Activity Relationship Analysis of GATA4 and NKX2-5 Protein-Protein Interaction Modulators.

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.

Stem cells are the most sensitive screening tool to identify toxicity of GATA4-targeted novel small-molecule compounds.

Safety assessment of drug candidates in numerous in vitro and experimental animal models is expensive, time consuming and animal intensive. More thorough toxicity profiling already in the early drug discovery projects using human cell models, which more closely resemble the physiological cell types, would help to decrease drug development costs. In this study we aimed to compare different cardiac and stem cell models for in vitro toxicity testing and to elucidate structure-toxicity relationships of novel compounds targeting the cardiac transcription factor GATA4. By screening the effects of eight compounds at concentrations ranging from 10 nM up to 30 µM on the viability of eight different cell types, we identified significant cell type- and structure-dependent toxicity profiles. We further characterized two compounds in more detail using high-content analysis. The results highlight the importance of cell type selection for toxicity screening and indicate that stem cells represent the most sensitive screening model, which can detect toxicity that may otherwise remain unnoticed. Furthermore, our structure-toxicity analysis reveals a characteristic dihedral angle in the GATA4-targeted compounds that causes stem cell toxicity and thus helps to direct further drug development efforts towards non-toxic derivatives.

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction.

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.

Discovery of Small Molecules Targeting the Synergy of Cardiac Transcription Factors GATA4 and NKX2-5.

Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC50 = 3 µM), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.

In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart.

Myocardial infarction (MI), commonly known as a heart attack, is the irreversible necrosis of heart muscle secondary to prolonged ischemia, which is an increasing problem in terms of morbidity, mortality and healthcare costs worldwide. Along with the idea to develop nanocarriers that efficiently deliver therapeutic agents to target the heart, in this study, we aimed to test the in vivo biocompatibility of different sizes of thermally hydrocarbonized porous silicon (THCPSi) microparticles and thermally oxidized porous silicon (TOPSi) micro and nanoparticles in the heart tissue. Despite the absence or low cytotoxicity, both particle types showed good in vivo biocompatibility, with no influence on hematological parameters and no considerable changes in cardiac function before and after MI. The local injection of THCPSi microparticles into the myocardium led to significant higher activation of inflammatory cytokine and fibrosis promoting genes compared to TOPSi micro and nanoparticles; however, both particles showed no significant effect on myocardial fibrosis at one week post-injection. Our results suggest that THCPSi and TOPSi micro and nanoparticles could be applied for cardiac delivery of therapeutic agents in the future, and the PSi biomaterials might serve as a promising platform for the specific treatment of heart diseases.