Selective modulators of PPAR activity as new therapeutic tools in metabolic diseases

Endocrine, Metabolic & Immune, Disorders - Drug Targets, 2006, 6, 000-000 1

1871-5303/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

Selective Modulators of PPAR Activity as New Therapeutic Tools inMetabolic Diseases

Balint, B. L. and Nagy, L.*

Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen,

Medical and Health Science Center, Nagyerdei krt. 98., Debrecen, H-4012, Hungary

Abstract: Peroxisome Proliferator Activated Receptors (PPARs) are regulators of metabolic pathways mainly of lipidmetabolism and energy balance. Their medical importance is given by the fact that they have been implicated indevelopment of insulin resistance, obesity and atherosclerosis. In recent years, major progress has been made inunderstanding the molecular basis of the function of these receptors. As a result of structural studies and identification ofputative natural as well as synthetic ligands and activators of PPARs a new concept emerged and new drugs are on theirways to the clinic. The concept of Selective PPAR Modulators (SPPARM) was suggested by analogy to SelectiveEstrogen Receptor Modulators (SERM). SPPARMs activate the receptors in distinct ways leading to differential geneexpression and biological response. The key features in understanding their action is most likely at the molecular detailsof ligand binding and the subsequently induced conformational changes as well as cofactor binding. A key aspect of thisis that unlike classical steroid hormone receptors such as the retinoic acid receptor, the PPAR receptors have a rather largeligand-binding pocket which is not filled with the ligand entirely and the ligand stabilizes the receptor‘s structure. Theligand receptor can have distinct conformations and this leads to different binding affinities for the various cofactors(coactivators and corepressors). In this review, we will introduce this concept, review the literature that supports it andpresent an overview of the receptor selective ligands including data about their mechanism of action and biologicaleffects.

Key Words: PPARs, SPPARMs, diabetes, dyslipidemia, ligands.

INTRODUCTION

PPARs AND their Functions as Metabolic Sensors

PPARs are members of the nuclear receptor superfamilythat act as metabolic sensors and are involved in thedevelopment of obesity and type 2 diabetes. In this reviewwe attempt to give an introduction to the molecular basis oftheir action and to present how a new group of PPARactivating ligands termed, the SPPARMs are modulatingtheir action. To better appreciate the benefits of this newclass of drugs we will describe PPARs as metabolic sensorsand will try to give a summary on the structural basis ofPPAR introduction. It is of particular importance to contrastthe molecular details of receptor activation via distinctligands with the in vivo activity of receptor activation.

PPARs since their discovery [1] have been associatedwith the drug discovery processes. The fact that they areligand dependent transcription factors, and might beinvolved in major pathological processes brought them in thefront line of the drug discovery process. This became clearearly on in spite of the fact that the first reports linked themto carcinogenesis on the basis that they are activated byrodent carcinogens and produce massive peroxisomeproliferation in this model organism (hence their names:Peroxisome Proliferator Activated Receptors, PPAR).However in this first report Issemann and Green already

*Address correspondence to this author at the Department of Biochemistryand Molecular Biology, Research Center for Molecular Medicine,University of Debrecen, Medical and Health Science Center, Nagyerdei krt.98., Debrecen, H-4012, Hungary; Tel: +36-52-416-432; Fax:+36-52-314-989; E-mail: [email protected]

pointed to a role in lipid lowering [1]. The receptorsinvolvement in human carcinogenesis was not confirmed butthere are at least two major diseases they were shown to beinvolved in, atherosclerosis and non-insulin dependentdiabetes. Interestingly, but not surprisingly two classes ofcompounds fibrates and thiazolidinediones (TZDs) wereindependently identified and tested for their possiblebeneficial effect in metabolic diseases without priorknowledge about their pharmacophores [2-4]. These studiespreceded the cloning of PPARs and the identification ofthese compounds as ligand for the receptors. The mainpharmacologic effect of fibrates was proposed to be theirability to reduce serum lipid levels and increase HDL levels[2]. Thiazolidinediones were shown to be beneficial inpatients with non-insulin dependent diabetes by reducingperipheral insulin resistance [5].

After cloning of the three different PPAR isoforms fromXenopus laevis, Wahli and his colleagues demonstrated thatseveral polyunsaturated fatty acids were able to activatePPAR� and synthetic Wy14 643 had a similar effect [6].Shortly after, Capone el al [7] showed that a similar effectwas produced by fibrates and that fibrates were able toactivate only PPAR� . Following the demonstration thatTZDs had transcriptional effects on adipogenesis andadipocyte related gene expression [8, 9], in a search forreceptor selective ligands PPAR� was shown to be activatedby thiazolidinediones [10]. Finding molecules that canactivate these receptors was not the end but the start of anextremely interesting scientific journey towards the possiblecontrol and cure of the plague of modern societies, themetabolic X syndrome, a combination of insulin resistance,hyperinsulinaemia, glucose intolerance, hypertension and

Selective Modulators of PPAR Activity Endocrine, Metabolic & Immune, Disorders - Drug Targets, 2006, Vol. 6, No. 1 2

dyslipidaemia (increased TG and reduced HDL levels) [11] acluster of conditions with an increased risk for coronaryheart disease. To better understand the pitfalls and sidetracksof these discoveries we briefly summarize the tissuedistribution and metabolic effects of PPARs.

PPARs have been identified as ligand activated trans-cription factors belonging to the nuclear hormone receptorsuperfamily [12], for a recent update about members of thisfamily implicated directly in lipid metabolism see Chawla etal. [13]. There are three isoforms of PPARs: PPAR�(NR1C1), PPAR� (NR1C3) and PPAR� (NR1C2), calledalso �. The difference in the nomenclature of � and � comesfrom their cloning. PPAR� was cloned from Xenopus by theWahli group while � from mouse by the Evans group. Thislatter was shown to be highly similar to the human receptorand became widely used in receptor studies with the namePPAR�. For mere details we refer to the several in depthreviews published on PPARs and their roles in lipidmetabolism [14] [15].

In humans, liver is the main expression site of PPAR�but heart, kidney, skeletal muscle, and large intestine alsoexpress this receptor. As a general rule, the expression ofPPAR� correlates with high mitochondrial and peroxisomalbeta-oxidation activities. In humans, PPAR� is abundant inadipose tissue and present at low levels in skeletal muscleand placenta. The intestinal mucosa also expresses highlevels of PPAR� in the colon but lower amounts in the smallintestine. Human PPAR� is expressed in several myeloid celllines, as well as in primary bone marrow stromal cells inculture as well as in macrophages and dendritic cells [16][17]. PPAR� has been shown to be involved in adipocytedifferentiation, fatty acid (FA) storage in adipose tissues andin foam cells (lipid filled macrophages) during athero-sclerosis development in the arterial wall [18-21]. PPAR� isinvolved in regulation of lipid uptake and metabolism inliver. Some of the enzymes of both peroxisomal andmitochondrial oxidation are under the control of PPAR�.The basal expression of these enzymes is not under thecontrol of PPAR� , but the adaptation to different stresssignals, especially fasting is driven by PPAR�. As a resultPPAR� KO mice develop severe hepatic steatosis duringfasting [22]. On the other hand, PPAR� KO mice die duringembryogenesis possibly because of placental insufficiency[23].

In the mid-nineties, PPARs became intensively studiedmolecules for their proposed roles in at least two majordiseases: diabetes and hyperlipidaemia. An importantdiscovery was the identification that not only PPAR�activation is involved in the process of atherosclerosisthrough modulation of serum lipid levels but also PPAR� inthe development of atherosclerosis through its expression inmacrophages and other cell types in the wall of bloodvessels. PPAR� is activated by fatty acids derived fromoxidized LDL and their induction is increasing the level ofCD36, a scavenger receptor for oxidized LDL [19] [18].Moreover, PPAR� ligands activate a downstream nuclearreceptor LXR that will induce the expression of ABCA1 amajor component of cholesterol efflux from macrophages[21]. These studies opened up multiple lines of investigationinto the role of these receptors in macrophage lipi[24-26]d

metabolism and atherosThe identification of the moleculartarget of fibrates and thiazolidinediones eased the applicationof the simplifying concept of one disease- one gene- onedrug. Introduction of synthetic high affinity PPAR� agonistsin the clinical practice for the treatment of type 2 diabetes(non insulin dependent diabetes - NIDDM) was a directresult of the application of this concept. As more and moreclinical and biological data are becoming available, thecaveats of this concept are now obvious. There are unwantedside effects of the drugs such as weight gain [27], waterretention [28] or liver toxicity [29]. There is also a wellfounded expectation that combination of PPAR� and PPAR�ligands or better dual agonists could provide a better controlof serum lipid levels and insulin sensitivity.

The first clinical trials with PPAR ligands were carriedout with highly selective ligands. Either PPAR� withfibrates or PPAR� with thiazolidinediones were studied asthe most potent activators available. The concept behind thisis an observation that comes from classical pharmacologicalstudies. According to this concept, highly selective ligands inlow concentrations will bring about only their selective“beneficial” effect. If a ligand has a lower affinity, in orderto achieve the specific effect, a higher concentration isneeded. However, at this higher concentration, several non-specific effects will be seen and in part, these are responsiblefor the unwanted side effects. From this point of view, thesearch for high affinity, specific ligands was easy torationalise. It is important to take into consideration that nohigh affinity endogenous/natural ligands have been identifiedfor PPARs so far and therefore PPARs may act differently,more like metabolic sensors that can be activated by highconcentrations (micromolar) of naturally occurring lipids orlipid soluble compounds. These molecules and probably alsoendogenous retinoids, that are the activators of theheterodimeric partners of PPARs, the RXRs have differentconcentrations from tissue to tissue, organ to organ. As aresult, the physiological activation of PPARs is probably aresult of several convergent signals such as different fattyacids and even the availability of retinoids. In other wordsthe PPAR signaling is the readout of the combined effects ofmultiple ligands with different affinities. If the receptor hasonly two distinct states: an active and an inactive then itshould not matter, but if binding of the different ligands putthe receptor into slightly different conformations/activationstates than the biological response might be substantiallydifferent. In this latter case the availability and relativeamount of each ligand should be taken into consideration(i.e. the receptor works as a sensor reading out a givenmetabolic state). It is also conceivable that activation of sucha sensor with highly selective ligands may result in asuperphysiological activation i.e. inducing a level ofactivation normally not achieved by endogenous ligands.There is evidence to suggest that highly selective PPAR�agonists although are beneficial for the amelioration ofperipheral insulin resistance their main side effect is that theyproduce peripheral obesity by activation of the geneticprogram of adipocyte differentiation in subcutaneous tissues[30] [27] probably by altering the concentrations of certainadipocyte-derived hormones, such as leptin, TNF� ,adiponectin, or resistin [31]. Although recent developmentsshow that these effects are less common than predicted based


on animal studies [32] these side effects need to beminimized. A new concept that allows drug design to benefitfrom the clinical findings and also from the molecular detailsof receptor action that has been accumulated in the past 13years on the action of PPARs is the SPPARM concept.

The Two Groups of PPAR Modulators or SPPARM-s

The idea that ligands change the conformation of thenuclear receptors in a complete spectrum of states leading todifferent biological consequences is supported by structuralevidence (see below) and this concept opens a door towardsthe understanding of the physiological role of these receptorsin regulation of metabolic pathways.

SPPARMs can be divided into two groups: PPAR�/�dual partial agonists and PPAR� modulators. PPAR�/� dualpartial agonists have a lower affinity for both PPARisoforms than the specific agonists and they have beneficialeffects on both the serum lipid levels and insulin sensitivity.Since 2001 several compounds with PPAR�/� dual agonistactivity have been identified and reported on. PPAR� modu-lators are the other group of ligands that activate PPAR� inan alternative way if compared to the classical PPAR�activators. They act specifically on PPAR� but produceconformational changes that favor selective cofactorinteractions and by this result in tissue specific PPAR effects(e.g. FMOC-L-Leucine, PAT-5A). Currently a whole arrayof ligands is under development by pharmaceuticalcompanies and they might completely change the way wethink about PPAR� and diabetes.

What are the Molecular Determinants of SPPARMs?

Ligand Binding Pockets

Although it has been believed that PPARs in theirunliganded state do not bind corepressors with high affinitythere is increasing evidence that they do at least undercertain circumstances (Sakatomto et al. presents the releaseof NCoR after binding of a SPPARM, namely TAK559, see[33]). After ligand binding, the receptor undergoesconformational changes that result in coactivator binding andpotentially also DNA binding. This initiates a protein-proteininteraction cascade initiated that ultimately results in thetranscription of the regulated gene. PPARs similar to othernuclear receptors are made of 12 alpha helixes packed inthree layers the so-called three-layered alpha helicalsandwich form. PPARs have a rather large ligand-bindingpocket. For example: RXR� has a ligand-binding pocket of700 cubic angstrom (Å3), Vitamin D receptor 800Å3, PPAR�1400Å3, PPAR� 1500Å3 , PPAR� 1300Å3[34]. It has beenproposed that some orphan receptors such as ROR� have noopen pocket at all but bind a “structural lipid” presumablyconstitutively a cholesterol molecule [35-37] and anotherorphan receptor, Nurr1 does not have ligand binding cavityat all, based on structural evidence [38].

The concept of metabolic sensors is consistent with thesedata. Not only the Km of their naturally occurring ligands isby orders of magnitude higher than the classical nuclearreceptors‘ (like retinoid receptors‘ or Vitamin D receptor‘s)but also the huge ligand-binding pocket can bind structurallyvery different ligands. The highly selective ligand

Rosiglitazone fills only 25% of this large pocket whileGI262570 a tyrosine-based molecule that binds to thePPAR�/RXR� heterodimer occupies approximately 40% ofit [39]. In contrast, all-trans retinoic acid fills 60% of a441Å3 pocket in the retinoic acid receptor RAR [40] [41].

To further understand the way metabolic sensors workwe can illustrate with another example from the crystalstructure of PPAR� in complex with the naturally occurringeicosapentaenoic acid (EPA). EPA occupies the ligand-binding pocket of PPAR� in two distinct conformations. Ineach docking mode, EPA occupied a volume of approxi-mately 300 Å3, which is roughly 30% of the pocket. Thesetwo different conformations were the result of the flexibilityof the FA hydrocarbon tail and the large size of the pocket[42].

The naturally occurring ligands fill only a small part ofthe ligand-binding pocket. Ligands were suggested to serveas a chemical skeleton of the relatively loose unligandedprotein. Ligand binding is stabilizing the structure of theprotein and helix 12 is bent towards the core of the ligand-binding domain. In the new conformational state, thereceptor will be able to bind DNA response elements in thepromoters of regulated genes and coactivators. Coactivatorson the other hand will recruit proteins with enzymaticactivity that will switch the chromatin into an activated stateand through phosphorylation of PolII will produce geneactivation and mRNA transcription. This concept of theligand induced switch operating along the full spectra ofconformational states produced by different ligands has beenreviewed in [43].

Co-Activators Co-Repressors and PPARs

In order to understand the molecular details of PPAR actionwe have also to take into consideration the mediators of theiractivation termed the cofactors. PPARs, similarly to othertranscription factors, bind with their DNA binding domain tothe response elements in the promoters of their target genesand with their trans-activation domains to cofactors. Thesecofactors are responsible ultimately of the activation orrepression of the target gene. As PPARs are proteins with arather loose structure and ligands are likely to act asmolecular skeletons of this structure, through ligand bindingPPAR can adopt a variety of forms that ultimately can betranslated into selective binding of one or the other cofactor.Through this mechanism different ligands can activatedifferent subsets of genes and have different side effectprofile even from one tissue to the other (as illustrated in Fig.1). One can illustrate this idea by saying that PPARs do notrepresent molecular switches that can be either on or off butrather signal integrators with a variety of input signals (e.g.natural ligands and retinoids, full agonists, partial agonists orinverse agonists) and have a broad spectra of output signalsalso (e.g. specific modulation of TIF2 or SRC1 in the case ofF-Moc-Leu [44]).

Since members of the SPPARM family, modulate theinteraction of PPAR� with the transcriptional activators orrepressors as a consequence a large variety of modulators(from full agonist through partial agonist to inverse agonist)will hopefully find their place in the clinical therapy of


obesity and type2 diabetes. As a good example of theirtherapeutic usefulness let us consider the effect of a PPAR�modulator (farglitazar) on the binding and activationcharacteristics of the mutated forms of PPAR�. Patientshaving mutated PPAR� suffer of severe insulin resistanceand early onset of diabetes mellitus (PPAR� ligand resistancesyndrome). In these patients rosiglitazone or other TZDbased full agonists do not bind to the mutated form ofPPAR� and these drugs do not activate the mutated PPAR�.On the other hand, farglitazar, a bona fide member of theSPPARM family that was suggested to interact in a differentway with the ligand binding pocket of the receptor comparedto rosiglitazone was shown to bind to the mutated form ofthe receptor and also activates the mutant form of the

receptor. Treating these patients with SPPARMs is likely toimprove their insulin resistance and ameliorate their type 2diabetes. These data suggest that even in severe insulinresistance with a genetic background where classical TZD-sare ineffective a SPPARM might the right choice for therapy[45].

Dual Agonists and Metabolic Sensors

Some of the fatty acids activate PPARs under physio-logical conditions. It has been shown by Evans and colleaguesthat unsaturated fatty acids activate PPAR� and � but not �[46] and also by Wahli et al. [47] that some natural fattyacids like eicosapentanoic acid or linoleic acid and 8(S)HETE show similar values for the activation of all the threeisoforms PPAR� , � and � in a cofactor binding assay [46][47]. Similarly, a fibrate GW2331 was shown in the earlyreports that can activate both PPAR� and PPAR� [48] andthe authors of this report suggested, based on their finding, arole for PPARs as metabolic sensors. These data if puttogether with the previously proven beneficial effects ofPPAR� ligands on serum lipid levels raised the questionwhether the activation of both receptors, the PPAR� and �will bring advantages in the treatment of metabolic diseasesand/or would eliminate some of the unwanted side effects ofPPAR� activators. A search for dual �-� agonists started.

Dual Agonists

Liu et al. published on compounds [49-51], with multiple�/� /� agonist activity. In the same year, several other com-pounds with PPAR�/� dual agonist activity were publishedsuch as AZ242 [52], LY465608 [53] and Ragaglitazar (orDRF 2725) [54] and later: TAK-559 [55], Muraglitazar [56].All of them had beneficial effects in animal models, rangingfrom increasing insulin sensitivity without being adipogenicto protective effect against atherosclerosis. Some of thesehave been tested in humans (Ragaglitazar) as well [57] [58].

Tesaglitazar or AZ 242 [59] [52]

The only ligand for that structural data with both PPAR�and PPAR� is available is Tesaglitazar, previously known asAZ242. AZ242 was co-crystallized with PPAR� and themolecular determinants of dual PPAR� and PPAR� weredetermined [52]. Moreover NMR studies presented in thispaper showed that indeed the ligand AZ242 is functioning asa molecular backbone of the PPAR� molecule by stabilizingits mobility and shifting it towards a more rigid and compactstructure similarly to the effect of Rosiglitazone on thePPAR� molecule [60]. In animal studies (obese Ob/Obdiabetic mice) this compound reduced hypertriglyceridaemiabelow the level of control lean mice. Insulin level showed amoderate decrease, glucose level was reduced to the level ofcontrol lean animals, whilst body weight showed no changeif compared to obese mice. All these responses were dosedependent with an efficacy at 10% of the Rosiglitazone dose.On obese Zucker rats in euglycemic hyperinsulinemic clamps,insulin sensitivity index and free fatty acid suppressionreached the level of lean rats whilst C-peptide and basalinsulin secretion was reduced significantly compared toobese rats. The authors used an interesting proteomicsapproach (2Dgel elfo) to compare the protein levels of L-FABP induction in HepG2 cells treated with AZ242 or

Fig. (1). Proposed model of PPAR modulation through selectivebinding of different cofactors. PPARs form heterodimers with RXRand act as metabolic sensors. They have a lose ligand bindingdomain that contains a large ligand binding pocket. Different typeof ligands generate conformational changes that will result inselective binding of different cofactors represented by A, B and C.These PPAR-cofactor complexes activate only partialy overlapinggenes. Genes that are activated by all of these complexes arerepresented in the overlaping group „I“, genes activated by multiplecofactors but not all of them are represented in the subgroup „II“,genes specific for a single PPAR-cofactor complex are representedin the subgroup „III“. These later groups are larger in the case offull agonists, like TZD-s (represented as III.A) and smaler in thecase of a SPPARM (represented as III.C). Genes from these groupsare in part responsible for the unwanted effects of full agonists likeweight gain and edema.


Table 1. The PPAR�/� Agonist Group of SPPARM-s

COMMON NAME CHEMICAL STRUCTURE EC50 (nM)

PPAR� / PPAR�

REFERENCE NOTES

TESAGLITAZAR

(AZ242)

1200/1300 Cronet 2001, [47] Structural data available for both

PPAR� and �.

RAGAGLITAZAR 270/324 Chakrabarti

2003, [56]

Already tested in pilot clinical

studies.

TZD 18 26/14 Guo 2004, [59] Transactivation in COS-1 cells

transiently transfected with PPAR-

GAL4 fusion proteins and

pUAS(5X)-tk-luciferase or pCMV-

lacZ reporter plasmids.

O-ARYL-MANDELIC

ACID

n.a./n.a. Adams 2003, [60] A series of PPAR�/� agonist

developed based on this structure.

GW2331 50/31 Kliewer 1997,

[43]

EC50 values were determined

using the GAL4-PPAR chimeras.

CV-1 cells were transfected with

the various GAL4-PPAR

expression plasmids and the cells

were treated with GW2331 The

overall levels of activation of

PPAR� and PPAR� were

comparable to those obtained with

saturating concentrations of Wy

14,643 and BRL 49653,

respectively.

TAK 559

(COMPOUND27)

67/31 Sakamoto 2004,

[33]

Authors present data about release

of NCOR and recruitment of SRC1

upon ligand activation.

PPAR� was activated at 10�M

concentration.

MURAGLITAZAR 320/110 Devasthale 2005

[56]

Muraglitazar is currently under

clinical development.

n.a.= not available, n.d.= not detected

O

S

O

O

O

OH

O

O

O

OH

O

ON

O

S

NH

O

O

O O

O O

O

OH

O

N

H

F

F

N

O

O COOH

O

OO

O

N

COOH

N

O

O O

N COOH

O

OCH3


Bezafibrate. They found the protein expression profilesimilar using this method. The EC50 of AZ242 was inmicromolar range in reporter studies.

In a recent paper [61], published already by the newname Tesaglitazar, this compound was shown to increase theclearance of non-esterified fatty acids into white adiposetissue. The increased level of non-esterified fatty acidscharacteristic to fating or diabetes was achieved by aninfusion of heparin/ triglyceride infusion into male Wistarrats.

Ragaglitazar

Ragaglitazar was first reported in 2001 with the name (-)DRF 2725 (6) or Compound 6 [54]. It is a phenoxazineanalogue of phenyl propanoic acid and was reported as beinga dual PPAR�/� activator. In the initial animal studies oninsulin resistant db/db mice, Ragaglitazar showed betterreduction of plasma glucose and triglyceride levels whencompared to Rosiglitazone. Its good oral bioavailability putthis compound in the line of potential drugs for diabetes anddyslipidemia [54]. In early 2003 a report was published thatdocumented effects of Ragaglitazar on hyperglycemia andwhole body insulin sensitivity in early and late stages ofdiabetes in Zucker diabetic fatty (ZDF) rats. Ragaglitazarhad a similar effect as the PPAR� agonist Rosiglitazone witha slight increase in Hb A(1c) [62]. Later that year detailedbiochemical and animal studies using this compound wherepublished [63]. Cotransfection studies showed ligand(Ragaglitazar) activation of both PPAR� and � isoforms.Compared to the � selective ligand WY14 643, Ragaglitazarhad a similar activation profile at a concentration level thatwas lower by three orders of magnitude. On the other handcompared to the � selective ligand Rosiglitazone, Ragaglitazaractivated PPAR� in a similar range of concentration.Ragaglitazar did not show any PPAR� activity in the studiedconcentration range. In ob/ob mice, Ragaglitazar showed areduction in plasma glucose, triglycerides, FFA, and glucoselevels. In high fat diet fed SD rats Ragaglitazar reducedplasma total and LDL-cholesterol, and also plasmatriglyceride levels, with a concomitant increase in HDL-cholesterol levels without affecting food consumption. In aninteresting model of hyperlipidaemia, fat-fed rats, treatedwith Ragaglitazar showed a significant reduction in triton-induced hepatic triglyceride secretion compared to controlmice, and an improvement in lipid clearance when challengedintravenously with a lipid emulsion [63]. Rosiglitazone orPioglitazone, on the other hand did not show any significantactivity in this model of hyperlipidaemia. On the mRNAlevels, FABP4 (also called aP2) and ACO were induced byboth Ragaglitazar and Rosiglitazone but not by fenofibrate.In animals treated with Ragaglitazar (10mg/kg) LPL activityin both white adipose tissue and liver was increased to higherlevels compared to animals treated with Rosiglitazone(30mg/kg) (Rosiglitazone had no effect on liver LPL activityat all) [63]. In 2003 the first report has been published thatused Ragaglitazar in humans [58]. As a start single-dosepharmacokinetics and tolerability of Ragaglitazar in healthysubjects was studied. In a second step multiple-dosepharmacokinetics, pharmacodynamics, and tolerability ofRagaglitazar in healthy subjects and in patients with type 2

diabetes was investigated. Healthy subjects received a singleoral dose (1-120 mg), and healthy subjects and type 2diabetic patients received a loading dose and thereafter once-daily doses (0.5-16 mg) of Ragaglitazar for 6 and 20 days,respectively. The active ingredient was rapidly absorbed(tmax: 1.5-1.7 h). The T1/2 was 80 hours following a singledose and 122 hours in patients after multiple dosing.Administration of 4 mg Ragaglitazar to patients (n= 4) for 21days resulted in mean decreases from baseline in fastinglevels of plasma glucose (18%), C-peptide (18%), trigly-cerides (36%), free fatty acids (49%), total cholesterol(11%), low-density lipoprotein (LDL) cholesterol (21%), andvery low-density lipoprotein (VLDL) cholesterol (15%), aswell as an increase in high-density lipoprotein (HDL) choles-terol (33%). Overall, Ragaglitazar was well tolerated, asadverse effects peripheral edema and anemia were reported[58]. In a recent study with Ragaglitazar 177 hypertrigly-ceridaemia type 2 diabetic subjects were involved [57]. Inthis 12-week, double blind, parallel, randomized, placebo-controlled dose-ranging study (open Pioglitazone arm)subjects received Ragaglitazar (0.1, 1, 4, or 10 mg), placebo,or Pioglitazone (45 mg). Ragaglitazar provided glycemiccontrol that was comparable with that of Pioglitazone and,compared with placebo, provided significant improvementon the lipid profile. Changes in triglycerides for Pioglitazonetreatment were similar to 1 mg ragaglitazar. [57]. Otheranimal studies that were using Ragaglitazar reported: in ratswith adiponectin was involved in the mediation of the effectsof Ragaglitazar [64] and also in rats PPAR� componentcounteracted the orexigenic component of PPAR� activators[65]. On the contrary, Pioglitasone produced increase in thefood intake of the animals.

TZD18

Recently another TZD based PPAR�/� agonist namedTZD18 [66] was reported. The uniqueness of this report isthat it is analyzing in depth the lipid lowering level of themolecule in db/db mice, dogs and hamsters and compares itseffects to Simvastatin and fenofibrate. The authors reportthat both fenofibrate and TZD18 inhibited the HMG-CoAreductase activity of hepatocytes and the hepatic cholesterolsynthesis similar to Simvastatin and these effects appeared tobe additive.

TAK-559

TAK 559 was identified in the search for potent non-TZD based PPAR� activators [55]. A series ofoxyiminoacetic and oxyiminoalkaloic acid derivatives weretested in vitro and on KKAy mice. The compound laternamed TAK-559 was chosen for further evaluation based onits pharmacokinetic activities and strong glucose and lipidlowering activity accompanied with no increase in bodyweight. Later this compound was shown to be a partialagonist of PPAR�, and to activate both PPAR� and PPAR�in transfection studies [33]. In competition binding assaysTAK559 was also shown to bind all three subtypes of PPARand to activate the transcription of ap2 (FABP4) mRNA[33]. In a model of atherosclerosis in hypercholesterolemicnon-diabetic rabbits TAK-559 was shown to inhibit intimalhyperplasia due to inhibition of macrophage recruitment and


smooth muscle cells [67]. No other data were publishedabout this compound.

Muraglitazar [56]

Muraglitazar was tested on db/db mice with a dose of 10mpk/day for 14 days and was found to be highly efficaciousin reducing glucose, TG levels to normal levels and FFAwith 62% and insulin with 48%. In rats had a goodbioavailability (88%) with t1/2 of 7.3 hour, beagle dogs andcynomolgus monkeys were also tested for bioavailability.The performed studies suggested that muraglitazar is apotent PPAR a/g agonist. Muraglitazar is currently underclinical development as a novel drug for the treatment oftype 2 diabetes

Non-TZD Based �/� Agonists

As previously mentioned in the case of TAK559 severalgroups are working on the development of non-TZD basedPPAR agonists. TAK559 is an oxyiminoalkaloic acid deri-vative, but other chemically different structures were tested.

Non-TZD based activators promise to eliminate the sideeffects of TZDs such as edema, fluid retention, fatty changesin bone marrow and significant increases in heart weight inrodents. A PPAR� agonist-mandelic acid- was shown tohave micromolar affinity for PPAR� with nanomolar EC50 onPPAR� [68]. Based on this finding a list of compoundsderived from O-Arylmandelic acid as novel PPAR�/� dualagonists were developed and tested in different experimentalsystems. In the animal system used (db/db mouse) thecorrection of fasting plasma glucose (FPG) levels of thesenew ligands were compared to the highly specific PPAR�agonist Rosiglitazone and the highly potent PPAR� agonistpirinixic acid (Wy-14643). Interestingly, in the case of twoof these ligands (out of 13 tested new ligands) the FPG wassimilar to the effect of the PPAR� agonist Rosiglitazonewhilst Wy14643 had no effect. The paper reports onadditional changes in the chemical structure of the compoundin order to improve bioavailability and other optimizations ofthese ligands. The overall data presented by the authors areencouraging especially if taken into consideration that theseO-arylmandelic acids showed decrease in total cholesteroland triglycerides, with little or no increase in heart weight orbody weight compared to control animals. Although theseresults are promising, further studies are needed to establishthese ligands as potential drugs.

PPAR� PARTIAL AGONISTS

In recent years, some excellent demonstrations of thevarious ways of PPAR� activation were published [44, 69,70] [71] and the number of tested molecules belonging tothis group of PPAR� activators is increasing exponentially.

FMOC-L-Leucine

In a paper published in 2001 [44] Auwerx and colleaguesreported a ligand with potent PPAR� activity, FMOC-L-Leucine. The thiasolidine ring of this substance is replacedby a carboxylic acid. After analyzing a large number of L-Tyr based ligands they found that FMOC-L-Leucine has aunique PPAR� binding and activating profile. RXR, PPAR�or LXRs were not activated by FMOC-L-Leucine.

In several cell lines optimal PPAR� activation wasachieved by FMOC-L-Leucine at a concentration of 10-5 Mwhereas Rosiglitazone achieved its maximal efficacy between10-8 and 10-7 M. This concentration 10-5 was similar to thatof 15-deoxy-delta 12, 14-prostaglandin J2 one of the proposednatural ligand of PPAR� . Moreover, FMOC-L-Leucineshows a tendency to synergize Rosiglitazone activation in10-8M concentration of Rosiglitazone. In protease protectionassay PPAR� showed a conformational change after FMOC-L-Leucine binding, and this conformation digestion patternseemed to be different from the one produced byRosiglitazone.

Interestingly, PPAR� during activation binds twomolecules of FMOC-L-Leucine as documented by ESI-MS.The most interesting feature of this activation is that itchanges the receptor‘s structure in a way that it changes itscofactor specificity. FMOC-L-Leucine activation enablesbinding of p300 and SRC-1 but not of TIF2. Rosiglitazoneactivated PPAR� preferentially binds TIF2. This cofactorselectivity suggests that different ligands induce docking ofdifferent cofactors and as a result, different pharmacologicalprofiles can be achieved. This finding, which has itscounterparts in the estrogen receptor biology [72], also hasbroad biological consequences and can guide thedevelopment of new drugs with favourable side effectsprofiles. In a subsequent paper by the same group TIF2 -/-animals were shown to be protected against obesity and theratio between TIF2/SRC-1 was shown to control the energymetabolism and the white/brown fat energy balance [70]. Inadipocyte differentiation study, FMOC-L-Leucine has beenshown to stimulate the expression of ap2 (FABP4) and LPL,two bona fide PPAR� target genes but had a much loweradipogenic effect if compared to Rosiglitazone. FMOC-L-Leucine has also been shown in animal studies to improveglucose tolerance while Rosiglitazone did not in normal C57BL/6J mice. Furthermore, in db/db diabetic mice, FMOC-L-Leucine improved glucose tolerance without inducing weightgain.

NTZDpa [69]

A non TZD partial agonist was identifed recently [69]. Itis named nTZDpa, binds potently to PPAR� with highselectivity if compared to PPAR� or PPAR�. In cell-basedassays for transcriptional activation, nTZDpa proved to be aselective, potent PPAR� partial agonist and was able toantagonize the activity of PPAR� full agonists. Thismolecule also displayed partial agonist effects when itsability to promote adipogenesis in 3T3-L1 cells wasevaluated. The most elegant way to show its distinct way ofaction compared to classical TZDs was shown at thestructural level. Assessment of protein conformation usingprotease protection assay of the nTZDpa bound PPAR�showed a strikingly distinct digestion pattern compared toeither unliganded PPAR� or to TZD bound PPAR�. Assessedwith nuclear magnetic resonance spectroscopy nTZDpaproduced different, less compact conformation of PPAR�.DNA microarray analysis of RNA from 3T3-L1 adipocytestreated with nTZDpa or several structurally diverse PPAR�full agonists showed qualitative differences in the affectedgene expression profile for nTZDpa. Non-TZDpa activated adistinct set of genes than four other TZDs. Chronic treatment


of fat-fed, C57BL/6J mice with nTZDpa or a TZD fullagonist ameliorated hyperglycemia and hyperinsulinaemia.However, unlike the TZD, nTZDpa caused reductions inweight gain and adipose depot size. Unlike TZDs, nTZDpadid not cause cardiac hypertrophy in mice. When a panel ofPPAR� target genes were examined in white adipose tissue,nTZDpa produced a different in vivo expression pattern vs.

the full agonist. These findings establish that novel selectivePPAR� modulators can produce altered receptor activationstate leading to distinctive gene expression profiles, reducedadipogenic cellular effects, and potentially improved in vivobiological responses. Such compounds are candidates tobecome efficient therapeutic tools for diabetes, obesity, ormetabolic syndrome.

Table 2. The Group of SPPARM-s that Act Through Modulation of PPAR� -Cofactor Bindings

COMMON NAME CHEMICAL STRUCTURE EC50 (nM) PPAR

�/ PPAR �

REFERENCE NOTES

FMOC-L-LEU n.a./n.a. Rocchi 2001,[63]

Different cofactor binding withhigher affinity for TIF2.

Two molecules of FMOC Leubind the PPAR � ligand binding

pocket.

FARGLITAZAR n.a./n.a. Agostini 2004,[45]

Potential benefic effect on patientswith mutation in PPAR� ligand

binding domain

nTZDpa n.d./57 Berger 2003,[61]

Transactivation in COS-1 cellstransiently transfected with

PPAR-GAL4 fusion proteins aGAL4-responsive reportersystem.

PAT 5A n.a./33% ofrosiglitazone

Mishra 2003,[65]

HEK 293 cells transfected withfull length PPAR � PPARRE

upstream of a luciferase reportersystem.

M-BENZYL INDOLE n.d/10 Acton 2005,

[77]

A large series of benzyl methylindoles are presented in this

reference.

L 764406 n.d./69 Elbrecht 1999,[68]

Transactivation in COS-1 cellstransfected with PPAR-GAL4

fusion proteins a GAL4-responsive reporter system.PPAR � agonist by covalent

modification of cystein Cys 331residue on helix 3

n.a.= not available, n.d.= not detected

O

H

OH

O

N

O

O

N

O

O

COOH

NH

O

N

Cl

S

O

OH

Cl

N

N

O

NHS

O

O

COOH

COOH

CH3O

O

N

O

S

COOH

F3CO

N

N

Cl

S

O

O


PAT5A [73, 74]

A novel unsaturated thiazolidinedione analog has beenfound to be effective in reducing plasma glucose levels andimproving insulin sensitivity of db/db mice. The authorsfound that PAT5A binds to PPAR� with an affinity of 10% ifcompared to Rosiglitazone but is efficient in biologicalassays at only of a 3 fold higher concentration. The authorsreport that they have found a different protease digestionpattern of ligand bound recombinant PPAR� when comparedto Rosiglitazone. In an extensive search for the molecularmechanism of these differences, the authors comparedcofactor binding and structural data available with molecularmodels. As presented in their article, the authors suggest thatPAT5A activates PPAR� through a different conformationthan Rosiglitazone. This lower affinity is sufficient for thetherapeutic effect but has weaker adipogenic effect and bythis a better side effect profile. An interesting observation isthe differential regulation of adipsin and resistin genes byPAT5A and rosiglitazone in the adipose tissue. Similarly tothe SERM concept in cases of SPPARMs the receptor-cofactor complexes also may be different with differentligands leading to variations in the covalent modification oftranscriptional machinery resulting in alteration of trans-criptional regulation [75]. The differential gene regulation ofadipocyte-specific target genes may explain the mechanismof the weak adipogenic potential of PAT5A.

L-764406

In a paper published by Moller and his colleagues theauthors report on a ligand that has partial agonist effect, L-764406 [76]. Besides different binding assays, the authorsshow, that this ligand is able to activate a chimericPPARreceptor and also to induce the ap2 (FABP4) gene.They do not compare this activation to other known PPAR�ligands though. The most important finding presented intheir paper is that they claim this ligand binds covalently tothe PPAR� molecule and this binding is dependent on theCys313 residue. They do not show however by othermethods whether this ligand could become a pharma-cologically relevant molecule.

FK-614

Is a newly published potent selective non TZD PPAR�agonist [71]. The efficacy of this ligand was similar torosiglitazone. FK614 also reduced hematocrit, hemoglobin,and red blood cells and increased cardiac mass similar torosiglitazone although the minimum dose of FK614 at whichthese effects were significant was 10-fold higher than ofrosiglitazone.

Benzoyl 2 Methyl Indoles

Benzoyl 2 methyl indoles [77] were already developedbased on the SPPARM concept. The indole core is present ina large number of compounds that have been reported asbeing activators of PPAR� (like indomethacin, nTZDpa,etc.). Based on this observation, an array of compounds weregenerated and tested for activation of PPAR isoforms.Majority of the compounds were selective for PPAR�. As anexample, compound identified as compound 24 had an ECvalue of 3nM compared to 23 nM of Rosiglitazone, but the

maximal luciferase activity achieved with this compoundwas only 20% compared to rosiglitazone. This observationfits perfectly in the SPPARM concept. When tested in db/dbmouse and Sprague-Dawley rats it showed a glucosecorrection comparable with rosiglitazone with a lowerweight gain, less heart weight increase and brown adiposetissue weight increase. According to these observations thiscompound possesses pharmacological advantages comparedto classical full agonists, like rosiglitazone.

Farglitazar

Farglitazar, previously known as GI262570 is currentlyevaluated in clinical trials. According to the crystallographicmodel of interactions between PPAR� and the ligand,additional hydrogen bonds are formed between the ligandand its binding pocket compared to rosiglitazone. As aconsequence Farglitazar is activating the receptor using adifferent mechanism. As described previously this ligand is apotent activator of even the mutated dominant negative formof PPAR� , the one that is found some of the patientssuffering of severe insulin resistance and early onset of type2 diabetes [45]. Animal studies conducted with this ligandshowed multifocal fatty infiltrations around the vasculaturein brown adipose tissue of treated rats but not in other organs[78]. In renal system increased NO production was observedthat might explain the blood pressure lowering effect of thisagent but also signs of volume expansion (decreasedhaematocrit, serum hemoglobin and albumin levels) weredetected. These changes were not a result of decreasedglomerular filtration [79]. It is also possible that these effectsare the result of a false hyperaldosteronism caused by ligandtreatment with normal aldosteron levels but electrolytechanges characteristic to hyperaldosteronism such a asdecreased potassium and increased sodium and chloride inthe plasma. These changes are probably a result of PPAR�ligand dependent change in the level of ion transporters inthe renal tissue [80]. The side effect profile needs to becarefully taken into consideration during the clinicalevaluation of farglitazar.

SUMMARY AND PERSPECTIVES

Several pharmaceutical companies are putting seriousefforts in finding new drugs that would be useful in thetreatment of type 2 diabetes and insulin resistance. PPARa/gagonists and PPAR� modulators according to the publishedliterature seem to provide substantial benefits compared tothe classical agonists in order to become useful tools in thetreatment of metabolic X syndrome.

Taken together the reviewed literature the classical view ofPPAR activation has definitely changed over the last coupleof years. PPARs seem to be rather flexible molecules andthey provide a ligand binding surface for a variety ofmolecules. This is in contrast to classical steroid hormonereceptors. The adopted conformation may be loose orcompact and a whole variety on this spectra. By this some ofthe ligands are able to provide cofactor selectivity as it wasshown in the case of PPAR� modulators. On the other hand,the relatively large binding pockets of PPARs make possiblethe development of dual PPAR�/� agonists. These moleculessynthesized in a growing number. The leading force in their


development is the desire to combine the beneficial effectson lipid levels of the PPAR� agonists and the insulinsensitizing effects of PPAR� agonists. These ligands seem tofulfill the original goal and avoid the commonly seen sideeffects of TZDs like liquid retention, edema, adipogenity andincrease of cardiac weight in rodents. We assume some ofthese ligands will fail on their way towards the clinic, butnothing seems to stop at least some of them to becomeclinically used drugs in the treatment of metabolic Xsyndrome.

ACKNOWLEDGEMENTS

The work in the authors’ laboratory is supported bygrants from the EU FP5– RTN (to L.N.). HFSP and theHungarian Scientific Research Fund (OTKA) T034434 (toL.N.). L.N is an International Scholar of the Howard HughesMedical Institute and holds a Wellcome Trust SeniorResearch Fellowship in Biomedical Sciences in CentralEurope. B.L.B. is a Young Researcher of the EU NUC RECNET (an EU FP5 training network).

REFERENCES

[1] Issemann, I. and Green, S. (1990) Nature, 347, 645-50.[2] Watts, G. F. and Dimmitt, S. B. (1999) Curr. Opin. Lipidol., 10,

561-74.[3] Chang, A. Y.; Wyse, B. M. and Gilchrist, B. J. (1983) Diabetes, 32,

839-45.[4] Chang, A. Y.; Wyse, B. M.; Gilchrist, B. J.; Peterson, T.; Diani, A.

R. (1983) Diabetes, 32, 830-8.[5] Barnett, A. H. (2002) Curr. Med. Res. Opin., 18(Suppl. 1), s31-9.[6] Keller, H.; Dreyer, C.; Medin, J.; Mahfoudi, A.; Ozato, K. and

Wahli, W. (1993) Proc. Natl. Acad. Sci. USA, 90, 2160-4.[7] Marcus, S. L.; Miyata, K. S.; Zhang, B.; Subramani, S.;

Rachubinski, R. A. and Capone, J. P. (1993) Proc. Natl. Acad. Sci.

USA, 90, 5723-7.[8] Kletzien, R. F.; Clarke, S. D. and Ulrich, R. G. (1992) Mol.

Pharmacol., 41, 393-8.[9] Kletzien, R. F.; Foellmi, L. A.; Harris, P. K.; Wyse, B. M. and

Clarke, S. D. (1992) Mol. Pharmacol., 42, 558-62.[10] Lehmann, J. M.; Moore, L. B.; Smith-Oliver, T. A.; Wilkison, W.

O.; Willson, T. M. and Kliewer, S. A. (1995) J. Biol. Chem., 270,12953-6.

[11] Reaven, G. M. (2000) Curr. Atheroscler. Rep., 2, 503-7.[12] Kliewer, S. A.; Lehmann, J. M. and Willson, T. M. (1999) Science,

284, 757-60.[13] Chawla, A.; Repa, J. J.; Evans, R. M. and Mangelsdorf, D. J.

(2001) Science, 294, 1866-70.[14] Evans, R. M.; Barish, G. D. and Wang, Y. X. (2004) Nat. Med., 10,

355-61.[15] Willson, T. M.; Lambert, M. H. and Kliewer, S. A. (2001) Annu.

Rev. Biochem., 70, 341-67.[16] Greene, M. E.; Blumberg, B.; McBride, O. W.; Yi, H. F.;

Kronquist, K.; Kwan, K.; Hsieh, L.; Greene, G. and Nimer, S. D.(1995) Gene Expr., 4, 281-99.

[17] Szatmari, I.; Gogolak, P.; Im, J. S.; Dezso, B.; Rajnavolgyi, E. andNagy, L. (2004) Immunity, 21, 95-106.

[18] Nagy, L.; Tontonoz, P.; Alvarez, J. G.; Chen, H. and Evans, R. M.(1998) Cell, 93, 229-40.

[19] Tontonoz, P.; Nagy, L.; Alvarez, J. G.; Thomazy, V. A. and Evans,R. M. (1998) Cell, 93, 241-52.

[20] Chawla, A.; Barak, Y.; Nagy, L.; Liao, D.; Tontonoz, P. and Evans,R. M. (2001) Nat. Med., 7, 48-52.

[21] Chawla, A.; Boisvert, W. A.; Lee, C. H.; Laffitte, B. A.; Barak, Y.;Joseph, S. B.; Liao, D.; Nagy, L.; Edwards, P. A.; Curtiss, L. K.;Evans, R. M. and Tontonoz, P. (2001) Mol. Cell, 7, 161-71.

[22] Kersten, S.; Seydoux, J.; Peters, J. M.; Gonzalez, F. J.; Desvergne,B. and Wahli, W. (1999) J. Clin. Invest., 103, 1489-98.

[23] Barak, Y.; Nelson, M. C.; Ong, E. S.; Jones, Y. Z.; Ruiz-Lozano,P.; Chien, K. R.; Koder, A. and Evans, R. M. (1999) Mol. Cell, 4,585-95.

[24] Szanto, A.; Benko, S.; Szatmari, I.; Balint, B. L.; Furtos, I.; Ruhl,R.; Molnar, S.; Csiba, L.; Garuti, R.; Calandra, S.; Larsson, H.;Diczfalusy, U. and Nagy, L. (2004) Mol. Cell Biol., 24, 8154-66.

[25] Laffitte, B. A. and Tontonoz, P. (2002) Curr. Atheroscler. Rep., 4,213-21.

[26] Lazar, M. A. (2001) Nat. Med., 7, 23-4.[27] Akazawa, S.; Sun, F.; Ito, M.; Kawasaki, E. and Eguchi, K. (2000)

Diabetes Care, 23, 1067-71.[28] Mudaliar, S.; Chang, A. R. and Henry, R. R. (2003) Endocr. Pract.,

9, 406-16.[29] Tolman, K. G. and Chandramouli, J. (2003) Clin. Liver Dis., 7,

369-79, vi.[30] Chaput, E.; Saladin, R.; Silvestre, M. and Edgar, A. D. (2000)

Biochem. Biophys. Res. Commun., 271, 445-50.[31] Mayerson, A. B.; Hundal, R. S.; Dufour, S.; Lebon, V.; Befroy, D.;

Cline, G. W.; Enocksson, S.; Inzucchi, S. E.; Shulman, G. I. andPetersen, K. F. (2002) Diabetes, 51, 797-802.

[32] Stumvoll, M. (2003) Expert Opin. Investig. Drugs, 12, 1179-87.[33] Sakamoto, J.; Kimura, H.; Moriyama, S.; Imoto, H.; Momose, Y.;

Odaka, H. and Sawada, H. (2004) Eur. J. Pharmacol., 495, 17-26.[34] Watkins, R. E.; Wisely, G. B.; Moore, L. B.; Collins, J. L.;

Lambert, M. H.; Williams, S. P.; Willson, T. M.; Kliewer, S. A.and Redinbo, M. R. (2001) Science, 292, 2329-33.

[35] Chai, X.; Chen, W. and Napoli, J. L. (2001) Gene, 274, 27-33.[36] Willson, T. M. (2002) Structure (Camb.), 10, 1605-6.[37] Kallen, J.; Schlaeppi, J. M.; Bitsch, F.; Delhon, I. and Fournier, B.

(2004) J. Biol. Chem., 279, 14033-8.[38] Wang, Z.; Benoit, G.; Liu, J.; Prasad, S.; Aarnisalo, P.; Liu, X.; Xu,

H.; Walker, N. P. and Perlmann, T. (2003) Nature, 423, 555-60.[39] Gampe, R. T., Jr.; Montana, V. G.; Lambert, M. H.; Miller, A. B.;

Bledsoe, R. K.; Milburn, M. V.; Kliewer, S. A.; Willson, T. M. andXu, H. E. (2000) Mol. Cell, 5, 545-55.

[40] Escriva, H.; Delaunay, F. and Laudet, V. (2000) Bioessays, 22,717-27.

[41] Bourguet, W.; Vivat, V.; Wurtz, J. M.; Chambon, P.; Gronemeyer,H. and Moras, D. (2000) Mol. Cell, 5, 289-98.

[42] Xu, H. E.; Lambert, M. H.; Montana, V. G.; Parks, D. J.;Blanchard, S. G.; Brown, P. J.; Sternbach, D. D.; Lehmann, J. M.;Wisely, G. B.; Willson, T. M.; Kliewer, S. A. and Milburn, M. V.(1999) Mol. Cell, 3, 397-403.

[43] Nagy, L. and Schwabe, J. W. (2004) Trends Biochem. Sci., 29, 317-24.

[44] Rocchi, S.; Picard, F.; Vamecq, J.; Gelman, L.; Potier, N.; Zeyer,D.; Dubuquoy, L.; Bac, P.; Champy, M. F.; Plunket, K. D.;Leesnitzer, L. M.; Blanchard, S. G.; Desreumaux, P.; Moras, D.;Renaud, J. P. and Auwerx, J. (2001) Mol. Cell, 8, 737-47.

[45] Agostini, M.; Gurnell, M.; Savage, D. B.; Wood, E. M.; Smith, A.G.; Rajanayagam, O.; Garnes, K. T.; Levinson, S. H.; Xu, H. E.;Schwabe, J. W.; Willson, T. M.; O'Rahilly, S. and Chatterjee, V. K.(2004) Endocrinology, 145, 1527-38.

[46] Forman, B. M.; Chen, J. and Evans, R. M. (1997) Proc. Natl. Acad.

Sci. USA, 94, 4312-7.[47] Krey, G.; Braissant, O.; L'Horset, F.; Kalkhoven, E.; Perroud, M.;

Parker, M. G. and Wahli, W. (1997) Mol. Endocrinol., 11, 779-91.[48] Kliewer, S. A.; Sundseth, S. S.; Jones, S. A.; Brown, P. J.; Wisely,

G. B.; Koble, C. S.; Devchand, P.; Wahli, W.; Willson, T. M.;Lenhard, J. M. and Lehmann, J. M. (1997) Proc. Natl. Acad. Sci.

USA, 94, 4318-23.[49] Liu, K. G.; Smith, J. S.; Ayscue, A. H.; Henke, B. R.; Lambert, M.

H.; Leesnitzer, L. M.; Plunket, K. D.; Willson, T. M.; Sternbach, D.D. (2001) Bioorg. Med. Chem. Lett., 11, 2385-8.

[50] Liu, K. G.; Lambert, M. H.; Ayscue, A. H.; Henke, B. R.;Leesnitzer, L. M.; Oliver, W. R., Jr.; Plunket, K. D.; Xu, H. E.;Sternbach, D. D. and Willson, T. M. (2001) Bioorg. Med. Chem.Lett., 11, 3111-3.

[51] Liu, K. G.; Lambert, M. H.; Leesnitzer, L. M.; Oliver, W., Jr.; Ott,R. J.; Plunket, K. D.; Stuart, L. W.; Brown, P. J.; Willson, T. M.and Sternbach, D. D. (2001) Bioorg. Med. Chem. Lett., 11, 2959-62.

[52] Cronet, P.; Petersen, J. F.; Folmer, R.; Blomberg, N.; Sjoblom, K.;Karlsson, U.; Lindstedt, E. L. and Bamberg, K. (2001) Structure

(Camb.), 9, 699-706.[53] Etgen, G. J.; Oldham, B. A.; Johnson, W. T.; Broderick, C. L.;

Montrose, C. R.; Brozinick, J. T.; Misener, E. A.; Bean, J. S.;Bensch, W. R.; Brooks, D. A.; Shuker, A. J.; Rito, C. J.; McCarthy,


J. R.; Ardecky, R. J.; Tyhonas, J. S.; Dana, S. L.; Bilakovics, J. M.;Paterniti, J. R., Jr.; Ogilvie, K. M.; Liu, S. and Kauffman, R. F.(2002) Diabetes, 51, 1083-7.

[54] Lohray, B. B.; Lohray, V. B.; Bajji, A. C.; Kalchar, S.; Poondra, R.R.; Padakanti, S.; Chakrabarti, R.; Vikramadithyan, R. K.; Misra,P.; Juluri, S.; Mamidi, N. V. and Rajagopalan, R. (2001) J. Med.Chem., 44, 2675-8.

[55] Imoto, H.; Sugiyama, Y.; Kimura, H. and Momose, Y. (2003)Chem. Pharm. Bull. (Tokyo), 51, 138-51.

[56] Devasthale, P. V.; Chen, S.; Jeon, Y.; Qu, F.; Shao, C.; Wang, W.;Zhang, H.; Cap, M.; Farrelly, D.; Golla, R.; Grover, G.; Harrity, T.;Ma, Z.; Moore, L.; Ren, J.; Seethala, R.; Cheng, L.; Sleph, P.; Sun,W.; Tieman, A.; Wetterau, J. R.; Doweyko, A.; Chandrasena, G.;Chang, S. Y.; Humphreys, W. G.; Sasseville, V. G.; Biller, S. A.;Ryono, D. E.; Selan, F.; Hariharan, N. and Cheng, P. T. (2005) J.

Med. Chem., 48, 2248-50.[57] Saad, M. F.; Greco, S.; Osei, K.; Lewin, A. J.; Edwards, C.; Nunez,

M. and Reinhardt, R. R. (2004) Diabetes Care, 27, 1324-9.[58] Skrumsager, B. K.; Nielsen, K. K.; Muller, M.; Pabst, G.; Drake, P.

G. and Edsberg, B. (2003) J. Clin. Pharmacol., 43, 1244-56.[59] Ljung, B.; Bamberg, K.; Dahllof, B.; Kjellstedt, A.; Oakes, N. D.;

Ostling, J.; Svensson, L. and Camejo, G. (2002) J. Lipid Res., 43,1855-63.

[60] Johnson, B. A.; Wilson, E. M.; Li, Y.; Moller, D. E.; Smith, R. G.and Zhou, G. (2000) J. Mol. Biol., 298, 187-94.

[61] Hegarty, B. D.; Furler, S. M.; Oakes, N. D.; Kraegen, E. W. andCooney, G. J. (2004) Endocrinology, 145, 3158-64.

[62] Brand, C. L.; Sturis, J.; Gotfredsen, C. F.; Fleckner, J.; Fledelius,C.; Hansen, B. F.; Andersen, B.; Ye, J. M.; Sauerberg, P. andWassermann, K. (2003) Am. J. Physiol. Endocrinol. Metab., 284,E841-54.

[63] Chakrabarti, R.; Vikramadithyan, R. K.; Misra, P.; Hiriyan, J.;Raichur, S.; Damarla, R. K.; Gershome, C.; Suresh, J. andRajagopalan, R. (2003) Br. J. Pharmacol., 140, 527-37.

[64] Ye, J. M.; Iglesias, M. A.; Watson, D. G.; Ellis, B.; Wood, L.;Jensen, P. B.; Sorensen, R. V.; Larsen, P. J.; Cooney, G. J.;Wassermann, K. and Kraegen, E. W. (2003) Am. J. Physiol.

Endocrinol. Metab., 284, E531-40.[65] Larsen, P. J.; Jensen, P. B.; Sorensen, R. V.; Larsen, L. K.; Vrang,

N.; Wulff, E. M. and Wassermann, K. (2003) Diabetes, 52, 2249-59.

[66] Guo, Q.; Sahoo, S. P.; Wang, P. R.; Milot, D. P.; Ippolito, M. C.;Wu, M. S.; Baffic, J.; Biswas, C.; Hernandez, M.; Lam, M. H.;Sharma, N.; Han, W.; Kelly, L. J.; MacNaul, K. L.; Zhou, G.;Desai, R.; Heck, J. V.; Doebber, T. W.; Berger, J. P.; Moller, D. E.;

Sparrow, C. P.; Chao, Y. S. and Wright, S. D. (2004)Endocrinology, 145, 1640-8.

[67] Seki, N.; Bujo, H.; Jiang, M.; Shibasaki, M.; Takahashi, K.;Hashimoto, N. and Saito, Y. (2005) Atherosclerosis, 178, 1-7.

[68] Adams, A. D.; Hu, Z.; von Langen, D.; Dadiz, A.; Elbrecht, A.;MacNaul, K. L.; Berger, J. P.; Zhou, G.; Doebber, T. W.; Meurer,R.; Forrest, M. J.; Moller, D. E.; Jones, A. B. (2003) Bioorg. Med.

Chem. Lett., 13, 3185-90.[69] Berger, J. P.; Petro, A. E.; Macnaul, K. L.; Kelly, L. J.; Zhang, B.

B.; Richards, K.; Elbrecht, A.; Johnson, B. A.; Zhou, G.; Doebber,T. W.; Biswas, C.; Parikh, M.; Sharma, N.; Tanen, M. R.;Thompson, G. M.; Ventre, J.; Adams, A. D.; Mosley, R.; Surwit, R.S.; Moller, D. E. (2003) Mol. Endocrinol., 17, 662-76.

[70] Picard, F.; Gehin, M.; Annicotte, J.; Rocchi, S.; Champy, M. F.;O'Malley, B. W.; Chambon, P. and Auwerx, J. (2002) Cell, 111,931-41.

[71] Minoura, H.; Takeshita, S.; Ita, M.; Hirosumi, J.; Mabuchi, M.;Kawamura, I.; Nakajima, S.; Nakayama, O.; Kayakiri, H.; Oku, T.;Ohkubo-Suzuki, A.; Fukagawa, M.; Kojo, H.; Hanioka, K.;Yamasaki, N.; Imoto, T.; Kobayashi, Y. and Mutoh, S. (2004) Eur.J. Pharmacol., 494, 273-81.

[72] Lonard, D. M.; Tsai, S. Y. and O'Malley, B. W. (2004) Mol. CellBiol., 24, 14-24.

[73] Misra, P.; Chakrabarti, R.; Vikramadithyan, R. K.; Bolusu, G.;Juluri, S.; Hiriyan, J.; Gershome, C.; Rajjak, A.; Kashireddy, P.;Yu, S.; Surapureddi, S.; Qi, C.; Zhu, Y. J.; Rao, M. S.; Reddy, J. K.and Ramanujam, R. (2003) J. Pharmacol. Exp. Ther., 306, 763-71.

[74] Vikramadithyan, R. K.; Chakrabarti, R.; Misra, P.; Premkumar, M.;Kumar, S. K.; Rao, C. S.; Ghosh, A.; Reddy, K. N.; Uma, C. andRajagopalan, R. (2000) Metabolism, 49, 1417-23.

[75] Olefsky, J. M. (2000) J. Clin. Invest., 106, 467-72.[76] Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.;

Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G. and Moller, D. E.(1999) J. Biol. Chem., 274, 7913-22.

[77] Acton, J. J., 3rd; Black, R. M.; Jones, A. B.; Moller, D. E.; Colwell,L.; Doebber, T. W.; Macnaul, K. L.; Berger, J. and Wood, H. B.(2005) Bioorg. Med. Chem. Lett., 2005, 15, 357-62.

[78] Elangbam, C. S.; Brodie, T. A.; Brown, H. R.; Nold, J. B.;Raczniak, T. J.; Tyler, R. D.; Lightfoot, R. M. and Wall, H. G.(2002) Toxicol. Pathol., 30, 420-6.

[79] Yang, B.; Clifton, L. G.; McNulty, J. A.; Chen, L.; Brown, K. K.and Baer, P. G. (2003) J. Cardiovasc. Pharmacol., 42, 436-41.

[80] Chen, L.; Yang, B.; McNulty, J. A.; Clifton, L. G.; Binz, J. G.;Grimes, A. M.; Strum, J. C.; Harrington, W. W.; Chen, Z.; Balon,T. W.; Stimpson, S. A. and Brown, K. K. (2005) J. Pharmacol.

Exp. Ther., 312, 718-25.

Receive: 18 January, 2005 Revised: 26 April, 2005 Accepted: 09 March, 2005

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