Ranjan Mukherjee

Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-activated Receptor (PPAR) Isoforms PPARγ2 versus PPARγ1 and Activation with Retinoid X Receptor Agonists and Antagonists

We describe the cloning, characterization, and tissue distribution of the two human peroxisome proliferator activated receptor isoforms hPPARγ2 and hPPARγ1. In cotransfection assays the two isoforms were activated to approximately the same extent by known PPARγ activators. Human PPARγ binds to DNA as a heterodimer with the retinoid X receptor (RXR). This heterodimer was activated by both RXR agonists and antagonists and the addition of PPARγ ligands with retinoids resulted in greater than additive activation. Such heterodimer-selective modulators may have a role in the treatment of PPARγ/RXR-modulated diseases like diabetes. Northern blot analysis indicated the presence of PPARγ in skeletal muscle, and a sensitive RNase protection assay confirmed the presence of only PPARγ1 in muscle that was not solely due to fat contamination. However, both PPARγ1 and PPARγ2 RNA were detected in fat, and the ratio of PPARγ1 to PPARγ2 RNA varied in different individuals. The presence of tissue-specific distribution of isoforms and the variable ratio of PPARγ1 to PPARγ2 raised the possibility that isoform expression may be modulated in disease states like non-insulin-dependent diabetes mellitus. Interestingly, a third protected band was detected with fat RNA indicating the possible existence of a third human PPARγ isoform.

Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters

Bile acid biosynthesis is regulated by both feed-forward and feedback mechanisms involving a cascade of nuclear hormone receptors. Feed-forward regulation of the rate limiting enzyme in bile acid biosynthesis is provided by oxysterols through liver-X-receptor alpha (NR1H3), while feedback regulation is provided by bile acids through farnesoid-X-receptor (FXR) (NR1H4). The Syrian golden hamster provides a useful model for studying lipid metabolism. The hamster metabolizes and transports dietary cholesterol in a similar manner to humans, with the resulting lipid profile being more similar to the human profile than that of other rodent models. Cloning of Fxr from Syrian golden hamster revealed four hamster Fxr splice variants that altered the N-terminal activation domain or the hinge region between the DNA and ligand binding domains. Human genomic sequence and data from hamster Fxr were used to identify and clone a novel human FXR isoform resulting from the use of an alternative promoter. RNA expression analysis indicates that the two human FXR isoforms are differentially expressed in developmental and tissue-specific patterns and are likely to provide a mechanism for cell-specific FXR-dependent transcriptional activity.

Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor α

The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331<WY14643=ciprofibrate<L165041<gemfibrozil. Similarly, for all ligands tested, the rank order of EC(50) for peptide recruitment was CBP<PGC-1<SRC-1. These data suggest that for these LXXLL coactivator peptides, the ligands do not substantially differ in their preferences. Partial agonism was observed with ciprofibrate and PGC-1 and gemfibrozil and CBP giving a lower FRET at saturation than with the other ligands. This suggests that ciprofibrate and gemfibrozil induce a different conformation to the receptor-PGC-1 and receptor-CBP complex, respectively. In cotransfection assays, unexpected differences in potencies and efficacies were observed and the rank order of EC(50)s for activation differed from that predicted by FRET assays. In most cases, the presence of a coactivator peptide led to decrease in the EC(50)s seen in FRET assays compared to the K(i)s observed in binding to receptor only, consistent with the lower EC(50)s obtained in the transfection assays. Our data demonstrate that ligand induced coactivator preferences of PPARalpha contribute to transcription potency and efficacy.

PPARs as targets for metabolic and cardiovascular diseases.

Peroxisome proliferator-activated receptors (PPARs) alpha, gamma and delta (beta) are ligand-activated transcription factors of the nuclear hormone receptor superfamily which have been shown to play key roles in maintaining glucose and lipid homeostasis. The physiological effects of several marketed drugs for the treatment of dyslipidemia (fenofibrate and gemfibrozil) and diabetes (rosiglitazone and pioglitazone) have now been shown to be mediated through PPARalpha and PPARgamma respectively. Over the past few years our understanding of how PPAR ligands and receptors modulate gene expression has greatly increased; this knowledge is being used to design even more potent and efficacious PPAR ligands for the treatment of diabetes, dyslipidemia, atherosclerosis and obesity. This review is a brief survey of the PPAR field which highlights recent progress, with an emphasis on new ligands with novel PPAR profiles, particularly compounds which are co-agonists of PPAalpha, gamma and beta (delta).

Novel Peroxisome Proliferator-Activated Receptor α Agonists Lower Low-Density Lipoprotein and Triglycerides, Raise High-Density Lipoprotein, and Synergistically Increase Cholesterol Excretion with a Liver X Receptor Agonist

The first generation peroxisome proliferator-activated receptor (PPAR) α agonist gemfibrozil reduces the risk of major cardiovascular events; therefore, more potent PPARα agonists for the treatment of cardiovascular diseases have been actively sought. We describe two novel, potent oxybenzylglycine PPARα-selective agonists, BMS-687453 [N-[[3-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]phenyl]methyl]-N-(methoxycarbonyl)-glycine] and BMS-711939 N-[[5-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]-2-fluorophenyl]methyl]-N-(methoxycarbonyl)-glycine], that robustly increase apolipoprotein (Apo) A1 and high-density lipoprotein cholesterol in human ApoA1 transgenic mice and lower low-density lipoprotein-cholesterol and triglycerides in fat-fed hamsters. These compounds have much lower potency against mouse PPARα than human PPARα; therefore, they were tested in PPARα-humanized mice that do not express murine PPARα but express human PPARα selectively in the liver. We developed hepatic gene induction as a novel biomarker for efficacy and demonstrate hepatic gene induction at very low doses of these compounds. BMS-711939 induces fecal cholesterol excretion, which is further increased upon cotreatment with a liver X receptor (LXR) agonist. It is surprising that this synergistic increase upon coadministration is also observed in mice that express PPARα in the liver only. BMS-711939 also prevented the LXR agonist-induced elevation of serum triglycerides. Such PPARα agonists could be attractive candidates to explore for the treatment of cardiovascular diseases, especially in combination with a suitable LXR agonist.

Discovery of an oxybenzylglycine based peroxisome proliferator activated receptor alpha selective agonist 2-((3-((2-(4-chlorophenyl)-5-methyloxazol-4-yl)methoxy)benzyl)(methoxycarbonyl)amino)acetic acid (BMS-687453).

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) alpha agonist, with an EC(50) of 10 nM for human PPARalpha and approximately 410-fold selectivity vs human PPARgamma in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPARdelta. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPARalpha ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPARalpha in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.

Induction of endogenous genes by peroxisome proliferator activated receptor alpha ligands in a human kidney cell line and in vivo

The peroxisome proliferator activated receptor alpha (PPARalpha) plays a key role in regulating fatty acid metabolism by regulating expression of genes involved in fatty acid oxidation. To identify endogenous transcripts that could be used as surrogate markers for on-target activity of PPARalpha agonists, we employed a global profiling approach using DNA microarrays. The HK-2 cell line derived from proximal tubules of the human kidney, showed induction of several genes, including pyruvate dehydrogenase kinase 4 (PDK-4) and adipocyte differentiation related protein (ADRP) by PPARalpha ligands. HK-2 cells express detectable levels of PPARalpha and its dimerization partner the retinoid X receptor (RXRalpha) proteins. Induction of PDK-4 in these cells correlates with induction of PDK-4 in the liver of fat-fed hamsters. The magnitude of fibrate induction of PDK-4 in the liver also mirrors the decrease in serum triglyceride levels. Thus, induction of PDK-4 by PPARalpha agonists in the HK-2 cell model closely correlates with its induction in vivo and may represent an early marker for PPARalpha agonist action.

Synthesis and structure–activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel, potent PPARα selective activators- PPARα and PPARγ selectivity modulation

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.

Ligand-induced coactivator recruitment to peroxisome proliferator-activated receptorα characterized by fluorescence resonance energy transfer

Publisher Summary This chapter describes the way the affinity of ligands can be measured for peroxisome proliferator-activated receptorα (PPARα) using the fluorescence resonance energy transfer (FRET) assay utilizing a small peptide derived from coactivators containing the LXXLL receptor interaction site. It also compares the results with more traditional methods, such as scintillation proximity assay. The FRET assay has several advantages over conventional assays for determining affinities of PPARα ligands. It is a nonradioactive assay and is more sensitive than the scintillation proximity assay (SPA) for certain ligands, such as gemfibrozil and fenofibric acid. A known, high-affinity (labeled) ligand is not required as in SPA and is therefore ideal for identifying agonist ligands, especially for orphan receptors. The FRET assay can also be performed in the presence of a known agonist to detect receptor antagonists. However, the reagents are relatively expensive. Because the peptide is only a small part of the whole coactivator and the coactivators have multiple receptor interacting domains, the affinities determined with a peptide may be different from that using the whole coactivator protein.

Nuclear receptors in metabolic diseases

Nuclear receptors (NR) are transcription factors that are regulated by ligand binding. Several members of this family regulate glucose and lipid metabolism in vivo and consequently play a major role in the overall energy balance of the host. Serendipitously, some of these NRs were found to bind to known drugs for the treatment of dyslipidaemia, insulin resistance and diabetes, proving that these receptors may also play a role in these diseases. Because of this historical success as a target for clinically acceptable drugs, there is now renewed effort at discovering further receptor modulators for the metabolic diseases. This review will summarise the current understanding and treatment of the metabolic diseases, describe the role of specific NRs and evaluate their potential as therapeutic targets.