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1.
Bioorg Med Chem Lett ; 23(6): 1891-5, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23414840

ABSTRACT

We previously identified the methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its obligate transcriptional coactivators and prevent thyroid hormone signaling. As part of our lead optimization work we demonstrated that sulfonylnitrophenylthiazoles (SNPTs), which replace the ester linkage of MSNBs with a thiazole, also inhibited coactivator binding to TR. Here we report that replacement of the ester with an amide (methylsulfonylnitrobenzamides, MSNBA) also provides active TR antagonists.


Subject(s)
Benzamides/chemistry , Nuclear Receptor Coactivator 1/antagonists & inhibitors , Receptors, Thyroid Hormone/antagonists & inhibitors , Benzamides/chemical synthesis , Benzamides/toxicity , Cell Survival/drug effects , Hep G2 Cells , Humans , Nuclear Receptor Coactivator 1/genetics , Nuclear Receptor Coactivator 1/metabolism , Protein Interaction Maps/drug effects , Receptors, Thyroid Hormone/genetics , Receptors, Thyroid Hormone/metabolism , Structure-Activity Relationship , Thiazoles/chemistry , Transfection
2.
J Biol Chem ; 288(2): 807-18, 2013 Jan 11.
Article in English | MEDLINE | ID: mdl-23209300

ABSTRACT

Sirtuin 1 (SIRT1) is a nuclear deacetylase that modulates lipid metabolism and enhances mitochondrial activity. SIRT1 targets multiple transcription factors and coactivators. Thyroid hormone (T(3)) stimulates the expression of hepatic genes involved in mitochondrial fatty acid oxidation and gluconeogenesis. We reported that T(3) induces genes for carnitine palmitoyltransferase (cpt1a), pyruvate dehydrogenase kinase 4 (pdk4), and phosphoenolpyruvate carboxykinase (pepck). SIRT1 increases the expression of these genes via the activation of several factors, including peroxisome proliferator-activated receptor α, estrogen-related receptor α, and peroxisome proliferator-activated receptor γ coactivator (PGC-1α). Previously, we reported that PGC-1α participates in the T(3) induction of cpt1a and pdk4 in the liver. Given the overlapping targets of T(3) and SIRT1, we investigated whether SIRT1 participated in the T(3) regulation of these genes. Resveratrol is a small phenolic compound whose actions include the activation of SIRT1. Addition of resveratrol increased the T(3) induction of the pdk4 and cpt1a genes in hepatocytes. Furthermore, expression of SIRT1 in hepatocytes mimicked resveratrol in the regulation of gene expression by T(3). The deacetylase activity of SIRT1 was required and PGC-1α was deacetylated following addition of T(3). We found that SIRT1 interacted directly with T(3) receptor (TRß). Knockdown of SIRT1 decreased the T(3) induction of cpt1a and pdk4 and reduced the T(3) inhibition of sterol response element binding protein (srebp-1c) both in isolated hepatocytes and in rat liver. Our results indicate that SIRT1 contributes to the T(3) regulation of hepatic genes.


Subject(s)
Gene Expression Regulation/physiology , Liver/metabolism , Sirtuin 1/physiology , Triiodothyronine/physiology , Base Sequence , Cell Line , DNA Primers , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Humans , Real-Time Polymerase Chain Reaction , Resveratrol , Sirtuin 1/genetics , Stilbenes/pharmacology
3.
J Med Chem ; 55(5): 2301-10, 2012 Mar 08.
Article in English | MEDLINE | ID: mdl-22324546

ABSTRACT

We previously identified a series of methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its coactivators. MSNBs inhibit coactivator binding through irreversible modification of cysteine 298 of the thyroid hormone receptor (TR). Although MSNBs have better pharmacological features than our first generation inhibitors (ß-aminoketones), they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative to other nuclear hormone receptors (NRs).


Subject(s)
Nitro Compounds/chemical synthesis , Nuclear Receptor Coactivators/antagonists & inhibitors , Receptors, Thyroid Hormone/antagonists & inhibitors , Sulfones/chemical synthesis , Thiazoles/chemical synthesis , Genes, Reporter , Hep G2 Cells , Humans , Matrix Metalloproteinase 11/genetics , Matrix Metalloproteinase 11/metabolism , Models, Molecular , Nitro Compounds/chemistry , Nitro Compounds/pharmacology , Nuclear Receptor Coactivators/metabolism , Phosphoenolpyruvate Carboxykinase (ATP)/genetics , Phosphoenolpyruvate Carboxykinase (ATP)/metabolism , Receptors, Thyroid Hormone/metabolism , Response Elements , Structure-Activity Relationship , Sulfones/chemistry , Sulfones/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Transcription, Genetic/drug effects
4.
ACS Chem Biol ; 6(10): 1096-106, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21815645

ABSTRACT

Thyroid hormone (T3) mediates diverse physiological functions including growth, differentiation, and energy homeostasis through the thyroid hormone receptors (TR). The TR binds DNA at specific recognition sequences in the promoter regions of their target genes known as the thyroid hormone response elements (TREs). Gene expression at TREs regulated by TRs is mediated by coregulator recruitment to the DNA bound receptor. This TR-coregulator interaction controls transcription of target genes by multiple mechanisms including covalent histone modifications and chromatin remodeling. Our previous studies identified a ß-aminoketone as a potent inhibitor of the TR-coactivator interaction. We describe here the activity of one of these inhibitors in modulating effects of T3 signaling in comparison to an established ligand-competitive inhibitor of TR, NH-3. The ß-aminoketone was found to reverse thyroid hormone induced gene expression by inhibiting coactivator recruitment at target gene promoters, thereby regulating downstream effects of thyroid hormone. While mimicking the downstream effects of NH-3 at the molecular level, the ß-aminoketone affects only a subset of the thyroid responsive signaling network. Thus antagonists directed to the coregulator binding site have distinct pharmacological properties relative to ligand-based antagonists and may provide complementary activity in vivo.


Subject(s)
Gene Expression Regulation/drug effects , Receptors, Thyroid Hormone/antagonists & inhibitors , Receptors, Thyroid Hormone/metabolism , Triiodothyronine/metabolism , Cell Line , Hep G2 Cells , Humans , Ketones/pharmacology , Receptors, Thyroid Hormone/genetics , Signal Transduction/drug effects , Sulfones/pharmacology , Triiodothyronine/genetics
5.
J Biol Chem ; 286(27): 23799-807, 2011 Jul 08.
Article in English | MEDLINE | ID: mdl-21586575

ABSTRACT

The conversion of pyruvate to acetyl-CoA in mitochondria is catalyzed by the pyruvate dehydrogenase complex (PDC). Activity of PDC is inhibited by phosphorylation via the pyruvate dehydrogenase kinases (PDKs). Here, we examined the regulation of Pdk4 gene expression by the CCAAT/enhancer-binding protein ß (C/EBPß). C/EBPß modulates the expression of multiple hepatic genes including those involved in metabolism, development, and inflammation. We found that C/EBPß induced Pdk4 gene expression and decreased PDC activity. This transcriptional induction was mediated through two C/EBPß binding sites in the Pdk4 promoter. C/EBPß participates in the hormonal regulation of gluconeogenic genes. Previously, we reported that Pdk4 was induced by thyroid hormone (T(3)). Therefore, we investigated the role of C/EBPß in the T(3) regulation of Pdk4. T(3) increased C/EBPß abundance in primary rat hepatocytes. Knockdown of C/EBPß with siRNA diminished the T(3) induction of the Pdk4 and carnitine palmitoyltransferase (Cpt1a) genes. CPT1a is an initiating step in the mitochondrial oxidation of long chain fatty acids. Our results indicate that C/EBPß stimulates Pdk4 expression and participates in the T(3) induction of the Cpt1a and Pdk4 genes.


Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Gene Expression Regulation, Enzymologic/physiology , Hepatocytes/metabolism , Protein Serine-Threonine Kinases/biosynthesis , Response Elements/physiology , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , Carnitine O-Palmitoyltransferase/biosynthesis , Carnitine O-Palmitoyltransferase/genetics , Gluconeogenesis/physiology , Hep G2 Cells , Hepatocytes/cytology , Humans , Protein Serine-Threonine Kinases/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Pyruvate Dehydrogenase Complex/biosynthesis , Pyruvate Dehydrogenase Complex/genetics , Rats , Triiodothyronine/genetics , Triiodothyronine/metabolism
6.
J Biol Chem ; 286(14): 11895-908, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21321127

ABSTRACT

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRß with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRß antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRß to disrupt SRC2 association.


Subject(s)
Nitrobenzoates/pharmacology , Nuclear Receptor Coactivator 2/metabolism , Receptors, Thyroid Hormone/metabolism , Aniline Compounds/pharmacology , HEK293 Cells , Hep G2 Cells , Humans , Methylamines/pharmacology , Nitrobenzoates/chemistry , Piperidines/pharmacology , Protein Binding/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity Relationship
7.
Mol Cell Endocrinol ; 325(1-2): 54-63, 2010 Aug 30.
Article in English | MEDLINE | ID: mdl-20638986

ABSTRACT

Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARalpha) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARalpha ligands. Here, we characterized a binding site for PPARalpha in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARalpha binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1alpha did not enhance CPT-1A transcription through the PPARalpha binding site in the second intron. Following knockdown of PGC-1alpha with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARalpha and PGC-1alpha stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.


Subject(s)
Carnitine O-Palmitoyltransferase/genetics , PPAR alpha/physiology , RNA-Binding Proteins/physiology , Response Elements/physiology , Transcription Factors/physiology , Animals , Base Sequence , Carnitine O-Palmitoyltransferase/metabolism , Cells, Cultured , Gene Expression Regulation, Enzymologic , Humans , Male , Molecular Sequence Data , PPAR alpha/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , RNA-Binding Proteins/metabolism , Rats , Rats, Sprague-Dawley , Transcription Factors/metabolism , Transcriptional Activation , Up-Regulation
8.
J Biol Chem ; 285(4): 2375-85, 2010 Jan 22.
Article in English | MEDLINE | ID: mdl-19948729

ABSTRACT

PDK4 (pyruvate dehydrogenase kinase 4) regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). PDC catalyzes the conversion of pyruvate to acetyl-CoA and is an important control point in glucose and pyruvate metabolism. PDK4 gene expression is stimulated by thyroid hormone (T(3)), glucocorticoids, and long chain fatty acids. The effects of T(3) on gene expression in the liver are mediated via the thyroid hormone receptor. Here, we have identified two binding sites for thyroid hormone receptor beta in the promoter of the rat PDK4 (rPDK4) gene. In addition, we have investigated the role of transcriptional coactivators and found that the PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator) enhances the T(3) induction of rPDK4. Following T(3) administration, there is an increase in the association of PGC-1 alpha with the rPDK4 promoter. Interestingly, this increased association is with the proximal rPDK4 promoter rather than the distal region of the gene that contains the T(3) response elements. Administration of T(3) to hypothyroid rats elevated the abundance of PGC-1 alpha mRNA and protein in the liver. In addition, we observed greater association of PGC-1 alpha not only with the rPDK4 gene but also with phosphoenolpyruvate carboxykinase and CPT-1a (carnitine palmitoyltransferase 1a) genes. Knockdown of PGC-1 alpha in rat hepatocytes reduced the T(3) induction of PDK4, PEPCK, and CPT-1a genes. Our results indicate that T(3) regulates PGC-1 alpha abundance and association with hepatic genes, and in turn PGC-1 alpha is an important participant in the T(3) induction of selected genes.


Subject(s)
Hepatocytes/enzymology , Hyperthyroidism/physiopathology , Hypothyroidism/physiopathology , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Triiodothyronine/metabolism , Animals , Base Sequence , Carcinoma, Hepatocellular , Cell Line, Tumor , Hepatocytes/cytology , Humans , Hyperthyroidism/metabolism , Hypophysectomy , Hypothyroidism/metabolism , Liver Neoplasms , Male , Molecular Sequence Data , PPAR gamma/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Promoter Regions, Genetic/physiology , Protein Serine-Threonine Kinases/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Pyruvate Dehydrogenase Complex/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-Dawley , Thyroid Hormone Receptors beta/metabolism , Transcription Factors/genetics , Transcription, Genetic/physiology , Transfection
9.
Mol Cell Endocrinol ; 315(1-2): 159-67, 2010 Feb 05.
Article in English | MEDLINE | ID: mdl-19703515

ABSTRACT

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRalpha) stimulates the expression of PDK4. Here, we determined that one of the ERRalpha binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRalpha binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.


Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Glucocorticoids/pharmacology , Insulin/pharmacology , Isoenzymes/metabolism , Protein Kinases/metabolism , Animals , Cells, Cultured , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Genes, Reporter , Glucocorticoids/metabolism , Humans , Insulin/metabolism , Isoenzymes/genetics , Mutagenesis, Site-Directed , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Protein Kinases/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Rats , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Receptors, Glucocorticoid/metabolism , Response Elements , Transcription Factors/genetics , Transcription Factors/metabolism , ERRalpha Estrogen-Related Receptor
10.
J Biol Chem ; 283(45): 30788-95, 2008 Nov 07.
Article in English | MEDLINE | ID: mdl-18753136

ABSTRACT

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.


Subject(s)
Apoptosis , Gene Expression Regulation, Leukemic , Gene Expression Regulation , Leukemia/enzymology , Methionine Adenosyltransferase/biosynthesis , RNA Interference , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Apoptosis/genetics , Cell Survival/genetics , Disease Models, Animal , Gene Expression Regulation/genetics , Gene Expression Regulation, Leukemic/genetics , Humans , Jurkat Cells , Leukemia/genetics , Leukemia/therapy , Methionine/genetics , Methionine/metabolism , Methionine Adenosyltransferase/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , S-Adenosylmethionine/biosynthesis , S-Adenosylmethionine/genetics
11.
PLoS Pathog ; 4(4): e1000042, 2008 Apr 18.
Article in English | MEDLINE | ID: mdl-18421376

ABSTRACT

Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%-30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.


Subject(s)
Chromosome Mapping/methods , Genetic Predisposition to Disease , Sepsis/genetics , Streptococcal Infections/genetics , Streptococcus pyogenes/pathogenicity , Animals , Bacteremia , Female , Gene Expression Regulation, Bacterial , Genomics , Genotype , Longevity , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , Quantitative Trait Loci , Recombination, Genetic , Sepsis/microbiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/physiology
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