Matthew Blake Wright

Minireview: Challenges and Opportunities in Development of PPAR Agonists

The clinical impact of the fibrate and thiazolidinedione drugs on dyslipidemia and diabetes is driven mainly through activation of two transcription factors, peroxisome proliferator-activated receptors (PPAR)-α and PPAR-γ. However, substantial differences exist in the therapeutic and side-effect profiles of specific drugs. This has been attributed primarily to the complexity of drug-target complexes that involve many coregulatory proteins in the context of specific target gene promoters. Recent data have revealed that some PPAR ligands interact with other non-PPAR targets. Here we review concepts used to develop new agents that preferentially modulate transcriptional complex assembly, target more than one PPAR receptor simultaneously, or act as partial agonists. We highlight newly described on-target mechanisms of PPAR regulation including phosphorylation and nongenomic regulation. We briefly describe the recently discovered non-PPAR protein targets of thiazolidinediones, mitoNEET, and mTOT. Finally, we summarize the contributions of on- and off-target actions to select therapeutic and side effects of PPAR ligands including insulin sensitivity, cardiovascular actions, inflammation, and carcinogenicity.

Examining the safety of PPAR agonists – current trends and future prospects

Introduction: The peroxisome proliferator-activated receptor (PPAR)-α and -γ agonists, fibrates and glitazones, are effective treatments for dyslipidemia and type 2 diabetes mellitus, respectively, but exhibit class-related, as well as compound-specific safety characteristics. Areas covered: This article reviews the profiles of PPAR-α, PPAR-γ, and dual PPAR-α/γ agonists with regard to class-related and compound-specific efficacy and adverse effects. We explore how learnings from first-generation drugs are being applied to develop safer PPAR-targeted therapies. Expert opinion: The finding that rosiglitazone may increase risk for cardiovascular events has led to regulatory guidelines requiring demonstration of cardiovascular safety in appropriate outcome trials for new type 2 diabetes mellitus drugs. The emerging data on the possibly increased risk of bladder cancer with pioglitazone may prompt the need for post-approval safety studies for new drugs. Since PPAR-α and -γ affect key cardiometabolic risk factors (diabetic dyslipidemia, insulin resistance, hyperglycemia, and inflammation) in a complementary fashion, combining their benefits has emerged as a particularly attractive option. New PPAR-targeted therapies that balance the relative potency and/or activity toward PPAR-α and -γ have shown promise in retaining efficacy while reducing potential side effects.

Preclinical Evaluation of GS-9160, a Novel Inhibitor of Human Immunodeficiency Virus Type 1 Integrase

ABSTRACT GS-9160 is a novel and potent inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN) that specifically targets the process of strand transfer. It is an authentic inhibitor of HIV-1 integration, since treatment of infected cells results in an elevation of two-long terminal repeat circles and a decrease of integration junctions. GS-9160 has potent and selective antiviral activity in primary human T lymphocytes producing a 50% effective concentration (EC50) of ∼2 nM, with a selectivity index (50% cytotoxic concentration/EC50) of ∼2,000. The antiviral potency of GS-9160 decreased by 6- to 10-fold in the presence of human serum. The antiviral activity of GS-9160 is synergistic in combination with representatives from three different classes of antiviral drugs, namely HIV-1 protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Viral resistance selections performed with GS-9160 yielded a novel pattern of mutations within the catalytic core domain of IN; E92V emerged initially, followed by L74M. While E92V as a single mutant conferred 12-fold resistance against GS-9160, L74M had no effect as a single mutant. Together, these mutations conferred 67-fold resistance to GS-9160, indicating that L74M may potentiate the resistance caused by E92V. The pharmacokinetic profile of GS-9160 in healthy human volunteers revealed that once-daily dosing was not likely to achieve antiviral efficacy; hence, the clinical development of this compound was discontinued.

Comparative molecular profiling of the PPARα/γ activator aleglitazar: PPAR selectivity, activity and interaction with cofactors.

Peroxisome proliferator‐activated receptors (PPARs) are a family of nuclear hormone receptors that control the expression of genes involved in a variety of physiologic processes, through heterodimerization with retinoid X receptor and complex formation with various cofactors. Drugs or treatment regimens that combine the beneficial effects of PPARα and γ agonism present an attractive therapeutic strategy to reduce cardiovascular risk factors. Aleglitazar is a dual PPARα/γ agonist currently in phase III clinical development for the treatment of patients with type 2 diabetes mellitus who recently experienced an acute coronary event. The potency and efficacy of aleglitazar was evaluated in a head‐to‐head comparison with other PPARα, γ and δ ligands. A comprehensive, 12‐concentration dose–response analysis using a cell‐based assay showed aleglitazar to be highly potent, with EC50 values of 5 nM and 9 nM for PPARα and PPARγ, respectively. Cofactor recruitment profiles confirmed that aleglitazar is a potent and balanced activator of PPARα and γ. The efficacy and potency of aleglitazar are discussed in relation to other dual PPARα/γ agonists, in context with the published X‐ray crystal structures of both PPARα and γ.

Hematopoietically expressed homeobox is a target gene of farnesoid X receptor in chenodeoxycholic acid–induced liver hypertrophy

Farnesoid X receptor (FXR/Fxr) is a bile acid–regulated nuclear receptor that promotes hepatic bile acid metabolism, detoxification, and liver regeneration. However, the adaptive pathways under conditions of bile acid stress are not fully elucidated. We found that wild‐type but not Fxr knockout mice on diets enriched with chenodeoxycholic acid (CDCA) increase their liver/body weight ratios by 50% due to hepatocellular hypertrophy. Microarray analysis identified Hex (Hematopoietically expressed homeobox), a central transcription factor in vertebrate embryogenesis and liver development, as a novel CDCA‐ and Fxr‐regulated gene. HEX/Hex was also regulated by FXR/Fxr and CDCA in primary mouse hepatocytes and human HepG2 cells. Comparative genomic analysis identified a conserved inverted repeat‐1–like DNA sequence within a 300 base pair enhancer element of intron‐1 in the human and mouse HEX/Hex gene. A combination of chromatin immunoprecipitation, electromobility shift assay, and transcriptional reporter assays demonstrated that FXR/Fxr binds to this element and mediates HEX/Hex transcriptional activation. Conclusion: HEX/Hex is a novel bile acid–induced FXR/Fxr target gene during adaptation of hepatocytes to chronic bile acid exposure. (HEPATOLOGY 2009.)

Discovery of tetrahydro-cyclopenta[b]indole as selective LXRs modulator.

A series of tetrahydro-cyclopenta[b]indoles modulating the activity of the liver-X-receptor (LXR) were derived from a high throughput screening hit. The potency and selectivity for LXRbeta versus LXRalpha was improved. One compound, administered to wild-type mice modestly increased plasma HDL-cholesterol with no change in plasma triglycerides (TG) and reduced effects on liver TG content compared to T0901317. This novel series of LXR agonists shows promise to improve therapeutic efficacy with reduced potential to increase TG.

Candidate Genes and Mechanisms for 2-Methoxyestradiol–Mediated Vasoprotection

2-Methoxyestradiol (2-ME; estradiol metabolite) inhibits vascular smooth muscle cell (VSMC) growth and protects against atherosclerosis and vascular injury; however, the mechanisms by which 2-ME induces these actions remain obscure. To assess the impact of 2-ME on biochemical pathways regulating VSMC biology, we used high-density oligonucleotide microarrays to identify differentially expressed genes in cultured human female aortic VSMCs treated with 2-ME acutely (4 hours) or long term (30 hours). Both single gene analysis and Gene Set Enrichment Analysis revealed 2-ME–induced downregulation of genes involved in mitotic spindle assembly and function in VSMCs. Also, Gene Set Enrichment Analysis identified effects of 2-ME on genes regulating cell-cycle progression, cell migration/adhesion, vasorelaxation, inflammation, and cholesterol metabolism. Transcriptional changes were associated with changes in protein expression, including inhibition of cyclin D1, cyclin B1, cyclin-dependent kinase 6, cyclin-dependent kinase 4, tubulin polymerization, cholesterol and steroid synthesis, and upregulation of cyclooxygenase 2 and matrix metalloproteinase 1. Microarray data suggested that 2-ME may activate peroxisome proliferator-activated receptors (PPARs) in VSMCs, and 2-ME has structural similarities with rosiglitazone (PPAR&ggr; agonist). However, our finding of weak activation and lack of binding of 2-ME to PPARs suggests that 2-ME may modulate PPAR-associated genes via indirect mechanisms, potentially involving cyclooxygenase 2. Indeed, the antimitogenic effects of 2-ME at concentrations that do not inhibit tubulin polymerization were blocked by the PPAR antagonist GW9662 and the cyclooxygenase 2 inhibitor NS398. Finally, we demonstrated that 2-ME inhibited hypoxia-inducible factor 1&agr;. Identification of candidate genes that are positively or negatively regulated by 2-ME provides important leads to investigate and better understand the mechanisms by which 2-ME induces its vasoprotective actions.

The Ras Inhibitors Caveolin-1 and Docking Protein 1 Activate Peroxisome Proliferator-Activated Receptor γ through Spatial Relocalization at Helix 7 of Its Ligand-Binding Domain

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that promotes differentiation and cell survival in the stomach. PPARγ upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPARγ is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPARγ signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPARγ and enhanced nuclear translocation and ligand-independent transcription of PPARγ target genes. In contrast, Cav1 overexpression sequestered PPARγ in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPARγs ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPARγ and to inhibit cell proliferation. Ligand-activated PPARγ also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPARγ regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPARγ to ligands, limiting proliferation of gastric epithelial cells.

Comparative Transcriptional Network Modeling of Three PPAR-α/γ Co-Agonists Reveals Distinct Metabolic Gene Signatures in Primary Human Hepatocytes

Aims To compare the molecular and biologic signatures of a balanced dual peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, aleglitazar, with tesaglitazar (a dual PPAR-α/γ agonist) or a combination of pioglitazone (Pio; PPAR-γ agonist) and fenofibrate (Feno; PPAR-α agonist) in human hepatocytes. Methods and Results Gene expression microarray profiles were obtained from primary human hepatocytes treated with EC50-aligned low, medium and high concentrations of the three treatments. A systems biology approach, Causal Network Modeling, was used to model the data to infer upstream molecular mechanisms that may explain the observed changes in gene expression. Aleglitazar, tesaglitazar and Pio/Feno each induced unique transcriptional signatures, despite comparable core PPAR signaling. Although all treatments inferred qualitatively similar PPAR-α signaling, aleglitazar was inferred to have greater effects on high- and low-density lipoprotein cholesterol levels than tesaglitazar and Pio/Feno, due to a greater number of gene expression changes in pathways related to high-density and low-density lipoprotein metabolism. Distinct transcriptional and biologic signatures were also inferred for stress responses, which appeared to be less affected by aleglitazar than the comparators. In particular, Pio/Feno was inferred to increase NFE2L2 activity, a key component of the stress response pathway, while aleglitazar had no significant effect. All treatments were inferred to decrease proliferative signaling. Conclusions Aleglitazar induces transcriptional signatures related to lipid parameters and stress responses that are unique from other dual PPAR-α/γ treatments. This may underlie observed favorable changes in lipid profiles in animal and clinical studies with aleglitazar and suggests a differentiated gene profile compared with other dual PPAR-α/γ agonist treatments.

Farnesoid X receptor protects human and murine gastric epithelial cells against inflammation-induced damage

Bile acids from duodenogastric reflux promote inflammation and increase the risk for gastro-oesophageal cancers. FXR (farnesoid X receptor/NR1H4) is a transcription factor regulated by bile acids such as CDCA (chenodeoxycholic acid). FXR protects the liver and the intestinal tract against bile acid overload; however, a functional role for FXR in the stomach has not been described. We detected FXR expression in the normal human stomach and in GC (gastric cancer). FXR mRNA and protein were also present in the human GC cell lines MKN45 and SNU5, but not in the AGS cell line. Transfection of FXR into AGS cells protected against TNFα (tumour necrosis factor α)-induced cell damage. We identified K13 (keratin 13), an anti-apoptotic protein of desmosomes, as a novel CDCA-regulated FXR-target gene. FXR bound to a conserved regulatory element in the proximal human K13 promoter. Gastric expression of K13 mRNA was increased in an FXR-dependent manner by a chow diet enriched with 1% (w/w) CDCA and by indomethacin (35 mg/kg of body weight intraperitoneal) in C57BL/6 mice. FXR-deficient mice were more susceptible to indomethacin-induced gastric ulceration than their WT (wild-type) littermates. These results suggest that FXR increases the resistance of human and murine gastric epithelial cells to inflammation-mediated damage and may thus participate in the development of GC.