Discovery and characterization of small molecule inhibitors of the BET family bromodomains.

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.

Suppression of inflammation by a synthetic histone mimic.

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.

Design and evaluation of a novel series of 2,3-oxidosqualene cyclase inhibitors with low systemic exposure, relationship between pharmacokinetic properties and ocular toxicity.

We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of cholesterol biosynthesis at 2mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not sufficient to circumvent cataract liabilities.

Hepatocyte nuclear factor-4alpha regulates the human apolipoprotein AV gene: identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway.

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-4alpha (HNF-4alpha) as a novel regulator of human apoAV gene. Inhibition of HNF-4alpha expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4alpha directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4alpha consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha was capable of stimulating the HNF-4alpha-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-4alpha. Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4alpha gene revealed a species-distinct regulation of apoAV by HNF-4alpha, which resembles that of a subset of HNF-4alpha target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4alpha and underscore the role of HNF-4alpha in regulating triglyceride metabolism.

Thyroid hormone regulates the hypotriglyceridemic gene APOA5.

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 beta (TRbeta) 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 TRbeta-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 TRbeta may be a potential pharmacological target for the treatment of hypertriglyceridemia.

The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 1: 5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione analogues.

Starting from ethyl beta-carboline-3-carboxylate (beta-CCE), 1, a modest inhibitor of type 5 phosphodiesterase (PDE5), a series of functionalized tetrahydro-beta-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.

The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 2: 2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione analogues.

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 (IC(50) = 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 human apolipoprotein AV gene is regulated by peroxisome proliferator-activated receptor-alpha and contains a novel farnesoid X-activated receptor response element.

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-alpha (PPARalpha) 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 PPARalpha specifically enhanced apoAV promoter activity. By deletion, site-directed mutagenesis, and binding analysis, a PPARalpha response element located 271 bp upstream of the transcription start site was identified. Finally, treatment with a specific PPARalpha activator led to a significant induction of apoAV mRNA expression in hepatocytes. The identification of apoAV as a PPARalpha target gene has major implications with respect to mechanisms whereby pharmacological PPARalpha agonists may exert their beneficial hypotriglyceridemic actions.

Orphan nuclear hormone receptor Rev-erbalpha regulates the human apolipoprotein CIII promoter.

Apolipoprotein CIII (apoCIII) plays an important role in plasma triglyceride and remnant lipoprotein metabolism. Because hypertriglyceridemia is an independent risk factor in coronary artery disease and the presence in plasma of triglyceride-rich remnant lipoproteins is correlated with atherosclerosis, considerable research efforts have been focused on the identification of factors regulating apoCIII gene expression to decrease its production. Here we report that the orphan nuclear hormone receptor Rev-erbalpha regulates the human apoCIII gene promoter. In apoCIII expressing human hepatic HepG2 cells, transfection of Rev-erbalpha specifically repressed apoCIII gene promoter activity. We determined by deletion and site-directed mutagenesis experiments that Rev-erbalpha dependent repression is mainly due to an element present in the proximal promoter of the apoCIII gene. In contrast, we found no functional Rev-erbalpha response elements in the convergently transcribed human apoAI gene or the common regulatory enhancer. The identified Rev-erbalpha response element coincides with a RORalpha1 element, and in the present study we provide evidence that functional cross-talk between these orphan receptors modulates the apoCIII promoter. In vitro binding analysis showed that monomers of Rev-erbalpha bound this element but not another upstream RORalpha1 response element. In addition, we showed that the closely related nuclear orphan receptor RVR also specifically repressed the human apoCIII gene. These studies underscore a novel physiological role for members of the Rev-erb family of nuclear receptors in the regulation of genes involved in triglyceride metabolism and the pathogenesis of atherosclerosis.