MODULATION DE L'EXPRESSION GENIQUE PAR LE BUTYRATE DE SODIUM (DOCTORAT (BIOCHIMIE ET BIOLOGIE MOLECULAIRE))

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

International audienceIsoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)-and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2'-Omethyladenosine-3',5'-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling

Identification of a tetrahydroquinoline analog as a pharmacological inhibitor of the cAMP-binding protein Epac.

International audienceThe cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective β-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs

A cardiac-specific robotized cellular assay identified families of human ligands as inducers of PGC-1α expression and mitochondrial biogenesis.

Mitochondrial function is dramatically altered in heart failure (HF). This is associated with a decrease in the expression of the transcriptional coactivator PGC-1α, which plays a key role in the coordination of energy metabolism. Identification of compounds able to activate PGC-1α transcription could be of future therapeutic significance.We thus developed a robotized cellular assay to screen molecules in order to identify new activators of PGC-1α in a cardiac-like cell line. This screening assay was based on both the assessment of activity and gene expression of a secreted luciferase under the control of the human PGC-1α promoter, stably expressed in H9c2 cells. We screened part of a library of human endogenous ligands and steroid hormones, B vitamins and fatty acids were identified as activators of PGC-1α expression. The most responsive compounds of these families were then tested for PGC-1α gene expression in adult rat cardiomyocytes. These data highly confirmed the primary screening, and the increase in PGC-1α mRNA correlated with an increase in several downstream markers of mitochondrial biogenesis. Moreover, respiration rates of H9c2 cells treated with these compounds were increased evidencing their effectiveness on mitochondrial biogenesis.Using our cellular reporter assay we could identify three original families, able to activate mitochondrial biogenesis both in cell line and adult cardiomyocytes. This first screening can be extended to chemical libraries in order to increase our knowledge on PGC-1α regulation in the heart and to identify potential therapeutic compounds able to improve mitochondrial function in HF

A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

International audienceAlthough cardiac cytosolic cyclic 3',5'-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3(-)) and Ca(2+), sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca(2+) entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na(+)/Ca(2+) exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3(-) rescued the sensitization of mitochondria to Ca(2+)-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies

A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action

International audienceThe well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-proliferative activities, was chemically modified at different positions. The new EA derivatives have been tested in vitro in anti-proliferative assays on both tumor KB (epidermal carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity. Reduction of the α-β double bond of EA completely abolished anti-cancer activities, whereas introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-proliferative activities against human cancer cell lines. Several substitutions on the phenyl of these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in this study is more complex than the inhibition of glutathione S-transferase π ascribed as unique effect to EA and might help to overcome tumor resistances

Membranes prime the RapGEF EPAC1 to transduce cAMP signaling

International audienceEPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery