Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host ...due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which ...are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein–protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.
The newly identified apolipoprotein AV (apoAV) gene is a key player in determining plasma triglyceride concentrations. Because hypertriglyceridemia is a major independent risk factor in coronary ...artery disease, the understanding of the regulation of the expression of this gene is of considerable importance. We presently characterize the structure, the transcription start site, and the promoter of the human apoAV gene. Since the peroxisome proliferator-activated receptor-α (PPARα) and the farnesoid X-activated receptor (FXR) have been shown to modulate the expression of genes involved in triglyceride metabolism, we evaluated the potential role of these nuclear receptors in the regulation of apoAV transcription. Bile acids and FXR induced the apoAV gene promoter activity. 5′-Deletion, mutagenesis, and gel shift analysis identified a heretofore unknown element at positions –103/–84 consisting of an inverted repeat of two consensus receptor-binding hexads separated by 8 nucleotides (IR8), which was required for the response to bile acid-activated FXR. The isolated IR8 element conferred FXR responsiveness on a heterologous promoter. On the other hand, in apoAV-expressing human hepatic Hep3B cells, transfection of PPARα specifically enhanced apoAV promoter activity. By deletion, site-directed mutagenesis, and binding analysis, a PPARα response element located 271 bp upstream of the transcription start site was identified. Finally, treatment with a specific PPARα activator led to a significant induction of apoAV mRNA expression in hepatocytes. The identification of apoAV as a PPARα target gene has major implications with respect to mechanisms whereby pharmacological PPARα agonists may exert their beneficial hypotriglyceridemic actions.
Guanosine cyclic 3′:5′-monophosphate (cGMP) plays a crucial role in regulating vascular smooth muscle contractile state. In rat aortic smooth muscle cells (RSMC) three isozymes of phosphodiesterase ...(PDE) may be involved in the degradation of cGMP, namely PDE I, PDE III, and PDE V. To study the effective contribution of PDE V to the control of intracellular cGMP levels, a specific and potent PDE V inhibitor 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo3,4d-pyrimidin-4-(5H)-one (DMPPO) was synthesized. DMPPO is a competitive inhibitor with respect to cGMP (
K
i
= 3 nM) and displayed high selectivity for PDE V as compared to other PDE isozymes. DMPPO strongly potentiated the cGMP response of atrial natriuretic peptide- or sodium nitroprusside-treated RSMC (
EC
50 = 0.5
μM). In addition, similar intracellular cGMP levels were obtained in the presence of a saturating concentration of DMPPO or 3-isobutyl-1-methylxanthine, a nonspecific PDE inhibitor, suggesting that cGMP is almost exclusively hydrolyzed by PDE V in RSMC. Stimulation of RSMC with atrial natriuretic factor resulted in accumulation of cGMP in the extracellular media. This egression was shown to be proportional to the intracellular level of cGMP and a first-order rate constant of 0.04 min
−1 was determined for the egression process. DMPPO did not interfere with the efflux and allowed us to show that intracellular cGMP levels are mainly controlled by PDE V, rather than by egression in RSMC. DMPPO is, therefore, a useful tool for determining the role of PDE V in the control of cGMP levels in living cells and tissues.
The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4α (HNF-4α) as a ...novel regulator of human apoAV gene. Inhibition of HNF-4α expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4α directly regulates human apoAV promoter through DR1 a direct repeat separated by one nucleotide (nt), and via a novel element for HNF-4α consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor-γ coactivator-1α was capable of stimulating the HNF-4α-dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4α. Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4α gene revealed a species-distinct regulation of apoAV by HNF-4α, which resembles that of a subset of HNF-4α target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4α and underscore the role of HNF-4α in regulating triglyceride metabolism.
1
In rat aortic rings precontracted with phenylephrine, the β‐adrenoceptor agonist isoprenaline (10 nM to 30 μm) produces greater relaxant effects in preparations with endothelium than in ...endothelium‐denuded preparations. The aim of this study was to determine the mechanisms involved in this effect and in particular investigate the possibility of a synergistic action between adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and guanosine 3′:5′‐cyclic monophosphate (cyclic GMP).
2
Isoprenaline‐induced relaxation of rat aortic rings precontracted with phenylephrine was greatly reduced by the nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μm) or the soluble guanylate cyclase inhibitors methylene blue (10 μm) or 1H‐1,2,4oxadiazolo4,3‐aquinoxalin‐1‐one (ODQ, 10 μm) but unaffected by indomethacin (10 μm), a cyclo‐oxygenase inhibitor. Similarly, in intact rings, the concentration‐response curve of forskolin (10 nM to 1 μm) was shifted to the right upon endothelium removal or treatment with methylene blue.
3
In endothelium‐denuded rat aortic rings, isoprenaline‐induced relaxation was potentiated by the guanylate cyclase activators atrial natriuretic factor (ANF, 1 to 10 nM) and sodium nitroprusside (SNP, 1 to 10 nM), and to a greater extent in the presence of the cyclic GMP‐specific phosphodiesterase (PDE 5) inhibitor, 1,3dimethyl‐6‐(2‐propoxy‐5‐methane sulphonylamidophenyl) pyrazolo 3,4‐d pyrimidin‐4‐(5H)‐one (DMPPO, 30 nM). Relaxation induced by isoprenaline was also potentiated by the cyclic GMP‐inhibited PDE (PDE 3) inhibitor cilostamide (100 nM).
4
Intracellular cyclic nucleotide levels were measured either in rat cultured aortic smooth muscle cells or in de‐endothelialized aortic rings. In both types of preparation, isoprenaline (5 nM and 10 μm) increased cyclic AMP levels and this effect was potentiated by cilostamide (10 μm), by rolipram, a cyclic AMP‐specific PDE (PDE 4) inhibitor (10 μm) and by cyclic GMP‐elevating agents (50 nM ANF or 30 nM SNP plus 100 nM DMPPO). In isoprenaline‐stimulated conditions, the increase in cyclic AMP induced by rolipram was further potentiated by cilostamide and by cyclic GMP‐elevating agents. Cilostamide and cyclic GMP‐elevating agents did not potentiate each other, suggesting a similar mechanism of action.
5
We conclude that in vascular smooth muscle (VSM) cells an increase in cyclic GMP levels may inhibit PDE 3 and, thereby, cyclic AMP catabolism. Under physiological conditions of constitutive NO release, and to a greater extent in the presence of the PDE 5 inhibitor DMPPO, cyclic GMP should act synergistically with adenylate cyclase activators to relax VSM.
Modification of the hydantoin ring in the previously described lead compound 2a has led to the discovery of compound 12a, tadalafil, a highly potent and highly selective PDE5 inhibitor. The ...replacement of the hydantoin in compound 2a by a piperazinedione ring led to compound cis- 11a which showed similar PDE5 inhibitory potency. Introduction of a 3,4-methylenedioxy substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor cis- 11c with increased cellular potency. Optimization of the chain on the piperazinedione ring led to the identification of the racemic cis- N-methyl derivative 11i. High diastereospecificity for PDE5 inhibition was observed in the piperazinedione series with the cis-(6R,12aR) enantiomer displaying the highest PDE5 inhibitory activity. The piperazinedione 12a, tadalafil (GF196960), has been identified as a highly potent PDE5 inhibitor (IC50 = 5 nM) with high selectivity for PDE5 vs PDE1−4 and PDE6. Compound 12a displays 85-fold greater selectivity vs PDE6 than sildenafil 1. 12a showed profound and long-lasting blood pressure lowering activity (30 mmHg/>7 h) in the spontaneously hypertensive rat model after oral administration (5 mg/kg).
The apolipoprotein AV gene (APOA5) is a key determinant of plasma triglyceride levels, a major risk factor for coronary artery disease and a biomarker for the metabolic syndrome. Since thyroid ...hormones influence very low density lipoprotein triglyceride metabolism and clinical studies have demonstrated an inverse correlation between thyroid status and plasma triglyceride levels, we examined whether APOA5 is regulated by thyroid hormone. Here we report that 3,5,3′-triiodo-l-thyronine (T3) and a synthetic thyroid receptor β (TRβ) ligand increase APOA5 mRNA and protein levels in hepatocytes. Our data revealed that T3-activated TR directly regulates APOA5 promoter through a functional direct repeat separated by four nucleotides (DR4). Interestingly, we show that upstream stimulatory factor 1, a transcription factor associated with familial combined hyperlipidemia and elevated triglyceride levels in humans, and upstream stimulatory factor 2 cooperate with TR, resulting in a synergistic activation of APOA5 promoter in a ligand-dependent manner via an adjacent E-box motif. In rats, we observed that apoAV levels declines with thyroid hormone depletion but returned to normal levels upon T3 administration. In addition, treatments with a TRβ-selective agonist increased apoAV and diminished triglyceride levels. The identification of APOA5 as a T3 target gene provides a new potential mechanism whereby thyroid hormones can influence triglyceride homeostasis. Additionally, these data suggest that TRβ may be a potential pharmacological target for the treatment of hypertriglyceridemia.
Starting from ethyl β-carboline-3-carboxylate (β-CCE), 1, a modest inhibitor of type 5 phosphodiesterase (PDE5), a series of functionalized tetrahydro-β-carboline derivatives has been identified as a ...novel chemical class of potent and selective PDE5 inhibitors. Optimization of the side chain on the hydantoin ring of initial lead compound 2 and of the aromatic ring on position 5 led to the identification of compound 6e, a highly potent and selective PDE5 inhibitor, with greater selectivity for PDE5 vs PDE1−4 than sildenafil. Compound 6e demonstrated a long-lasting and significant blood pressure lowering effect after iv administration in the spontaneously hypertensive rat model but showed only moderate oral in vivo efficacy.
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