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.

α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor.

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.

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.

Genome-wide profiling of miRNA-gene regulatory networks in mouse postnatal heart development-implications for cardiac regeneration.

Background: After birth, mammalian cardiomyocytes substantially lose proliferative capacity with a concomitant switch from glycolytic to oxidative mitochondrial energy metabolism. Micro-RNAs (miRNAs) regulate gene expression and thus control various cellular processes. Their roles in the postnatal loss of cardiac regeneration are however still largely unclear. Here, we aimed to identify miRNA-gene regulatory networks in the neonatal heart to uncover role of miRNAs in regulation of cell cycle and metabolism. Methods and results: We performed global miRNA expression profiling using total RNA extracted from mouse ventricular tissue samples collected on postnatal day 1 (P01), P04, P09, and P23. We used the miRWalk database to predict the potential target genes of differentially expressed miRNAs and our previously published mRNA transcriptomics data to identify verified target genes that showed a concomitant differential expression in the neonatal heart. We then analyzed the biological functions of the identified miRNA-gene regulatory networks using enriched Gene Ontology (GO) and KEGG pathway analyses. Altogether 46 miRNAs were differentially expressed in the distinct stages of neonatal heart development. For twenty miRNAs, up- or downregulation took place within the first 9 postnatal days thus correlating temporally with the loss of cardiac regeneration. Importantly, for several miRNAs, including miR-150-5p, miR-484, and miR-210-3p there are no previous reports about their role in cardiac development or disease. The miRNA-gene regulatory networks of upregulated miRNAs negatively regulated biological processes and KEGG pathways related to cell proliferation, while downregulated miRNAs positively regulated biological processes and KEGG pathways associated with activation of mitochondrial metabolism and developmental hypertrophic growth. Conclusion: This study reports miRNAs and miRNA-gene regulatory networks with no previously described role in cardiac development or disease. These findings may help in elucidating regulatory mechanism of cardiac regeneration and in the development of regenerative therapies.

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists.

Protein kinase C (PKC) modulators hold therapeutic potential for various diseases, including cancer, heart failure, and Alzheimer's disease. Targeting the C1 domain of PKC represents a promising strategy; the available protein structures warrant the design of PKC-targeted ligands via a structure-based approach. However, the PKC C1 domain penetrates the lipid membrane during binding, complicating the design of drug candidates. The standard docking-scoring approach for PKC lacks information regarding the dynamics and the membrane environment. Molecular dynamics (MD) simulations with PKC, ligands, and membranes have been used to address these shortcomings. Previously, we observed that less computationally intensive simulations of just ligand-membrane interactions may help elucidate C1 domain-binding prospects. Here, we present the design, synthesis, and biological evaluation of new pyridine-based PKC agonists implementing an enhanced workflow with ligand-membrane MD simulations. This workflow holds promise to expand the approach in drug design for ligands targeted to weakly membrane-associated proteins.

Protein Kinase A-Mediated Effects of Protein Kinase C Partial Agonist 5-(Hydroxymethyl)Isophthalate 1a3 in Colorectal Cancer Cells.

Colorectal cancer is the third most commonly occurring cancer in men and the second in women. The global burden of colorectal cancer is projected to increase to over 2 million new cases with over 1 million deaths within the next 10 years, and there is a great need for new compounds with novel mechanisms of action. Our group has developed protein kinase C (PKC)-modulating isophthalic acid derivatives that induce cytotoxicity toward human cervical and prostate cancer cell lines. In this study, we investigated the effects of 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) on colorectal cancer cell lines (Caco-2, Colo205, and HT29). HMI-1a3 inhibited cell proliferation, decreased cell viability, and induced an apoptotic response in all studied cell lines. These effects, however, were independent of PKC. Using serine/threonine kinome profiling and pharmacological kinase inhibitors, we identified activation of the cAMP/PKA pathway as a new mechanism of action for HMI-1a3-induced anticancer activity in colorectal cancer cell lines. Our current results strengthen the hypothesis for HMI-1a3 as a potential anticancer agent against various malignancies. SIGNIFICANCE STATEMENT: Colorectal cancer (CRC) is a common solid organ malignancy. This study demonstrates that the protein kinase C (PKC)-C1 domain-targeted isophthalatic acid derivative 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) has anticancer activity on CRC cell lines independently of PKC. We identified PKA activation as a mechanism of HMI-1a3-induced anticancer effects. The results reveal a new anticancer mechanism of action for the partial PKC agonist HMI-1a3 and thus provide new insights for the development of PKC and PKA modulators for cancer therapy.

Application of Human Induced Pluripotent Stem Cell Technology for Cardiovascular Regenerative Pharmacology.

Cardiovascular diseases are one of the leading causes of mortality in the western world. Myocardial infarction is among the most prevalent and results in significant cell loss within the myocardium. Similarly, numerous drugs have been identified as having cardiotoxic side effects. The adult human heart is however unable to instigate an effective repair mechanism and regenerate the myocardium in response to such damage. This is in large part due to the withdrawal of cardiomyocytes (CMs) from the cell cycle. Thus, identifying, screening, and developing agents that could enhance the proliferative capacity of CMs holds great potential in cardiac regeneration. Human induced pluripotent stem cells (hiPSCs) and their cardiovascular derivatives are excellent tools in the search for such agents. This chapter outlines state-of-the art techniques for the two-dimensional differentiation and attainment of hiPSC-derived CMs and endothelial cells (ECs). Bioreactor systems and three-dimensional spheroids derived from hiPSC-cardiovascular derivatives are explored as platforms for drug discovery before focusing on relevant assays that can be employed to assess cell proliferation and viability.

Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate-free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress-activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. KEY POINTS: Spatiotemporal contractility analysis of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only 'twitch'-like transients are observed. HiPSC-CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress-activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell-substrate adhesion underly multiphasic contractile behaviour of hiPSC-CMs.

Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoforms: Possible Implications for New Antifibrotic Therapies.

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. Although previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathologic fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-hour exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of α-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared with control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in 5-bromo-2'-deoxyuridine-positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen-encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment. SIGNIFICANCE STATEMENT: Cardiac fibrosis is a pathological process that contributes to the development of heart failure. The molecular mechanisms regulating fibrosis in the heart are, however, not fully understood, which hinders the development of new therapies. Here, we demonstrate that classical and novel protein kinase C (PKC) isoforms distinctly regulate cardiac fibroblast transdifferentiation and proliferation, the two central processes in fibrosis. Our results indicate that pharmacological PKC activation may be a promising strategy to inhibit myocardial fibrosis.

Rigorous Computational Study Reveals What Docking Overlooks: Double Trouble from Membrane Association in Protein Kinase C Modulators.

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-1gP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membrane-associated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Cell adhesion and proliferation on common 3D printing materials used in stereolithography of microfluidic devices.

Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture. In this work, we characterized four commercially available materials commonly used in stereolithography-based 3D printing with respect to long-term (2 month) cell survival on native 3D printed surfaces. Cell proliferation rates, along with material-induced effects on apoptosis and cell survival, were examined in mouse embryonic fibroblasts. Additionally, the feasibility of Dental SG (material with the most favored properties) for culturing of human hepatocytes and human-induced pluripotent stem cells was evaluated. The strength of cell adhesion to Dental SG was further examined over a shear force gradient of 1-89 dyne per cm2 by using a custom-designed microfluidic shear force assay incorporating a 3D printed, tilted and tapered microchannel sealed with a polydimethylsiloxane lid. According to our results, autoclavation of the devices prior to cell seeding played the most important role in facilitating long-term cell survival on the native 3D printed surfaces with the shear force threshold in the range of 3-8 dyne per cm2.

Missing Selectivity of Targeted 4ß-Phorbol Prodrugs Expected to be Potential Chemotherapeutics.

Targeting cytotoxic 4ß-phorbol esters toward cancer tissue was attempted by conjugating a 4ß-pborbol derivative with substrates for the proteases prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) expressed in cancer tissue. The hydrophilic peptide moiety was hypothesized to prevent penetration of the prodrugs into cells and prevent interaction with PKC. Cleavage of the peptide in cancer tumors was envisioned to release lipophilic cytotoxins, which subsequently penetrate into cancer cells. The 4ß-phorbol esters were prepared from 4ß-phorbol isolated from Croton tiglium seeds, while the peptides were prepared by solid-phase synthesis. Cellular assays revealed activation of PKC by the prodrugs and efficient killing of both peptidase positive as well as peptidase negative cells. Consequently no selectivity for enzyme expressing cells was found.

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.

Dual-peptide functionalized acetalated dextran-based nanoparticles for sequential targeting of macrophages during myocardial infarction.

The advent of nanomedicine has recently started to innovate the treatment of cardiovascular diseases, in particular myocardial infarction. Although current approaches are very promising, there is still an urgent need for advanced targeting strategies. In this work, the exploitation of macrophage recruitment is proposed as a novel and synergistic approach to improve the addressability of the infarcted myocardium achieved by current peptide-based heart targeting strategies. For this purpose, an acetalated dextran-based nanosystem is designed and successfully functionalized with two different peptides, atrial natriuretic peptide (ANP) and linTT1, which target, respectively, cardiac cells and macrophages associated with atherosclerotic plaques. The biocompatibility of the nanocarrier is screened on both macrophage cell lines and primary macrophages, showing high safety, in particular after functionalization of the nanoparticles' surface. Furthermore, the system shows higher association versus uptake ratio towards M2-like macrophages (approximately 2-fold and 6-fold increase in murine and human primary M2-like macrophages, respectively, compared to M1-like). Overall, the results demonstrate that the nanosystem has potential to exploit the "hitchhike" effect on M2-like macrophages and potentially improve, in a dual targeting strategy, the ability of the ANP peptide to target infarcted heart.

Pharmacological Protein Kinase C Modulators Reveal a Pro-hypertrophic Role for Novel Protein Kinase C Isoforms in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro. Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs. Conclusion: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer's disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.

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.

Molecular Atlas of Postnatal Mouse Heart Development.

Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA , protein, and metabolite changes in the neonatal heart for identification of metabolism-related mechanisms associated with cardiac regeneration. Methods and Results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c-means clustering were used to identify up- or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNA s and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS ( HMG -CoA [hydroxymethylglutaryl-coenzyme A] synthase)-mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU-positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems-level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration.

The heart is a complex organ consisting of various cell types, each of which plays an important role in both physiological and pathophysiological conditions. The cells communicate with each other through direct cell-cell interactions and paracrine signaling, and both homotypic and heterotypic cell interactions contribute to the organized structure and proper function of the heart. Cardiomyocytes (CMs) and endothelial cells (ECs) are two of the most abundant cardiac cell types and they also play central roles in both cardiac remodeling and regeneration. The postnatal cell cycle withdrawal of CMs, which takes place within days or weeks after birth, represents the major barrier for regeneration in adult mammalian hearts, as adult CMs exhibit a very low proliferative capacity. Recent evidence highlights the importance of ECs not only as the most abundant cell type in the heart but also as key players in post-infarction remodeling and regeneration. In this MiniReview, we focus on blood vascular ECs and CMs and their roles and interactions in cardiac physiology and pathologies, with a special emphasis on cardiac regeneration. We summarize the known mediators of the bidirectional CM-EC interactions and discuss the related recent advances in the development of therapies aiming to promote heart repair and regeneration targeting these two cell types.

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.

Anticancer activity of the protein kinase C modulator HMI-1a3 in 2D and 3D cell culture models of androgen-responsive and androgen-unresponsive prostate cancer.

Prostate cancer is one of the most common cancers in men. Although it has a relatively high 5-year survival rate, development of resistance to standard androgen-deprivation therapy is a significant clinical problem. Therefore, novel therapeutic strategies are urgently needed. The protein kinase C (PKC) family is a putative prostate cancer drug target, but so far no PKC-targeting drugs are available for clinical use. By contrast to the standard approach of developing PKC inhibitors, we have developed isophthalate derivatives as PKC agonists. In this study, we have characterized the effects of the most potent isophthalate, 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3), on three prostate cancer cell lines (LNCaP, DU145, and PC3) using both 2D and 3D cell culture models. In 2D cell culture, HMI-1a3 reduced cell viability or proliferation in all cell lines as determined by the metabolic activity of the cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay) and thymidine incorporation. However, the mechanism of action in LNCaP cells was different to that in DU145 or PC3 cells. In LNCaP cells, HMI-1a3 induced a PKC-dependent activation of caspase 3/7, indicating an apoptotic response, whereas in DU145 and PC3 cells, it induced senescence, which was independent of PKC. This was observed as typical senescent morphology, increased ß-galactosidase activity, and upregulation of the senescence marker p21 and downregulation of E2F transcription factor 1. Using a multicellular spheroid model, we further showed that HMI-1a3 affects the growth of LNCaP and DU145 cells in a 3D culture, emphasizing its potential as a lead compound for cancer drug development.