DOI: 10.1002/cmdc.201300250 Blocking the Peroxisome Proliferator-Activated Receptor (PPAR): An Overview Alessandra Ammazzalorso, Barbara De Filippis, Letizia Giampietro, and Rosa Amoroso* [a] Introduction Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that play a key role in the transcriptional regulation of genes involved in lipid and glucose metabolism, inflamma- tion responses, and modulation of the cardiovascular system. [1] The PPAR family consists of three subtypes encoded by sepa- rate genes, PPARa, PPARg, and PPARb/d, with each having dif- ferent tissue distribution, selectivity, and responsiveness to ligands. [2] The three PPAR isoforms share a common general structure, which allows these receptors to recognize a variety of similar ligands and to have the same basic mechanism of action. The highly conserved DNA binding domain (DBD) recognizes spe- cific response elements in the target gene, named peroxisome proliferator response elements (PPREs); the ligand binding domain (LBD), composed of 13 a-helices and a four-stranded b-sheet, incorporates a specific ligand binding function, a dime- rization function, and a ligand-dependent activation function (AF-2). [3] The general mode of action of PPARs and other nucle- ar receptors is well established: when the heterodimer of the PPAR and the retinoid X receptor (RXR) is bound to a co- repressor, the transcription machinery is repressed. The dissoci- ation of the co-repressor and the recruitment of a co-activator result in conformational changes that lead to gene transcrip- tion, which is involved in the control of lipid and glucose ho- meostasis (Figure 1). [4] Different co-repressors and co-activators have been identified, some of which show cell-type and tissue specificity. [5] The crystal structures of three human PPAR LBDs have been solved in complex with different ligands and in the absence of them, to show a common three-dimensional folding structure; the three isoforms share a large Y-shaped binding site that is able to accommodate a wide variety of structures. [6] Crystallo- graphic studies revealed critical insight into the mechanisms involved in the nuclear signaling, including the ligand binding affinity and specificity and the differential recruitment of co- regulators. Helix 12 (H12), a substructure of the LBD, has been proven to be a very important structural feature that is impli- cated in the activation or inactivation of PPARs. In the H12- folding inhibition hypothesis, proposed by Hashimoto et al., the correct folding of H12 allows the recruitment of co-activa- tors, which starts the transcriptional process; if a compound in- terferes with the folding of H12, the conformational change does not occur and the transcription is repressed. [7] In more detail, H12 takes an open conformation in the absence of ligand, whereas it covers the ligand binding pocket in a closed conformation when a ligand is present. The repositioning of H12, promoted by an agonist, creates a hydrophobic groove that favors co-activator binding. Compounds that bind with the LBD but interfere with H12 folding should act as full antag- onists. Instead, compounds inducing the H12 misfolding make the receptor inactive, but they allow the possible binding of Figure 1. The activated heterodimer PPAR–RXR induces target-gene tran- scription. Peroxisome proliferator-activated receptors (PPARs) have been studied extensively over the last few decades and have been assessed as molecular targets for the development of drugs against metabolic disorders. A rapid increase in understanding of the physiology and pharmacology of these receptors has occurred, together with the identification of novel chemical structures that are able to activate the various PPAR subtypes. More recent evidence suggests that moderate activation of these receptors could be favorable in pathological situations due to a decrease in the side effects brought about by PPAR agonists. PPAR partial agonists and antagonists are interesting tools that are currently used to better elucidate the biological processes modulated by this family of nuclear receptors. Herein we present an overview of the various molecular struc- tures that are able to block each of the PPAR subtypes, with a focus on promising therapeutic applications. [a] Dr. A. Ammazzalorso, Dr. B. De Filippis, Dr. L. Giampietro, Prof. R. Amoroso Dipartimento di Farmacia, UniversitȤ “G. d’Annunzio” via dei Vestini 31, 66100 Chieti (Italy) E-mail : [email protected]# 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00,1–9 &1& These are not the final page numbers! ÞÞ CHEMMEDCHEM MINIREVIEWS
Transcript
DOI: 10.1002/cmdc.201300250
Blocking the Peroxisome Proliferator-Activated Receptor(PPAR): An OverviewAlessandra Ammazzalorso, Barbara De Filippis, Letizia Giampietro, and Rosa Amoroso*[a]
Introduction
Peroxisome proliferator-activated receptors (PPARs) are nuclearreceptors that play a key role in the transcriptional regulationof genes involved in lipid and glucose metabolism, inflamma-tion responses, and modulation of the cardiovascular system.[1]
The PPAR family consists of three subtypes encoded by sepa-rate genes, PPARa, PPARg, and PPARb/d, with each having dif-ferent tissue distribution, selectivity, and responsiveness toligands.[2]
The three PPAR isoforms share a common general structure,which allows these receptors to recognize a variety of similarligands and to have the same basic mechanism of action. Thehighly conserved DNA binding domain (DBD) recognizes spe-cific response elements in the target gene, named peroxisomeproliferator response elements (PPREs); the ligand bindingdomain (LBD), composed of 13 a-helices and a four-strandedb-sheet, incorporates a specific ligand binding function, a dime-rization function, and a ligand-dependent activation function(AF-2).[3] The general mode of action of PPARs and other nucle-ar receptors is well established: when the heterodimer of thePPAR and the retinoid X receptor (RXR) is bound to a co-repressor, the transcription machinery is repressed. The dissoci-ation of the co-repressor and the recruitment of a co-activatorresult in conformational changes that lead to gene transcrip-tion, which is involved in the control of lipid and glucose ho-meostasis (Figure 1).[4] Different co-repressors and co-activatorshave been identified, some of which show cell-type and tissuespecificity.[5]
The crystal structures of three human PPAR LBDs have beensolved in complex with different ligands and in the absence ofthem, to show a common three-dimensional folding structure;
the three isoforms share a large Y-shaped binding site that isable to accommodate a wide variety of structures.[6] Crystallo-graphic studies revealed critical insight into the mechanismsinvolved in the nuclear signaling, including the ligand bindingaffinity and specificity and the differential recruitment of co-regulators. Helix 12 (H12), a substructure of the LBD, has beenproven to be a very important structural feature that is impli-cated in the activation or inactivation of PPARs. In the H12-folding inhibition hypothesis, proposed by Hashimoto et al. ,the correct folding of H12 allows the recruitment of co-activa-tors, which starts the transcriptional process; if a compound in-terferes with the folding of H12, the conformational changedoes not occur and the transcription is repressed.[7] In moredetail, H12 takes an open conformation in the absence ofligand, whereas it covers the ligand binding pocket in a closedconformation when a ligand is present. The repositioning ofH12, promoted by an agonist, creates a hydrophobic groovethat favors co-activator binding. Compounds that bind withthe LBD but interfere with H12 folding should act as full antag-onists. Instead, compounds inducing the H12 misfolding makethe receptor inactive, but they allow the possible binding of
Figure 1. The activated heterodimer PPAR–RXR induces target-gene tran-scription.
Peroxisome proliferator-activated receptors (PPARs) have beenstudied extensively over the last few decades and have beenassessed as molecular targets for the development of drugsagainst metabolic disorders. A rapid increase in understandingof the physiology and pharmacology of these receptors hasoccurred, together with the identification of novel chemicalstructures that are able to activate the various PPAR subtypes.More recent evidence suggests that moderate activation of
these receptors could be favorable in pathological situationsdue to a decrease in the side effects brought about by PPARagonists. PPAR partial agonists and antagonists are interestingtools that are currently used to better elucidate the biologicalprocesses modulated by this family of nuclear receptors.Herein we present an overview of the various molecular struc-tures that are able to block each of the PPAR subtypes, witha focus on promising therapeutic applications.
[a] Dr. A. Ammazzalorso, Dr. B. De Filippis, Dr. L. Giampietro, Prof. R. AmorosoDipartimento di Farmacia, Universit� “G. d’Annunzio”via dei Vestini 31, 66100 Chieti (Italy)E-mail : [email protected]
co-activators, which start the transcriptional process. Thesecompounds function as partial antagonists or agonists, de-pending on the presence of suitable co-activators (Figure 2).
The wide variety of metabolic pathways controlled by PPARactivation makes these receptors attractive targets for thedevelopment of drugs againstmetabolic disorders. Selectiveagonists for each isoform havebeen disclosed, and each ofthem represents an attractive re-search area focused on the man-agement of cardiovascular andmetabolic diseases.[8] The mostrecent combination strategy isaddressed toward simultaneous-ly activating different receptorsubtypes, to provide maximal ef-ficacy and to minimize undesiredside effects. Explored ap-proaches include the develop-ment of a/g dual agonists, butalso g/d and a/d dual agonists and a/g/d agonists (pan-ago-nists).[9] The major challenge in this strategy is to identify mole-cules with a balanced potency at each receptor subtype, anoverall efficacy in the target tissues, and an improved safetyprofile.
The need for a deeper understanding of the biological ac-tions controlled by PPARs gave rise, more recently, to specialattention toward compounds that are able to differently mod-ulate the receptor activity. Evidence of beneficial effects on in-sulin resistance and obesity, obtained by decreasing PPARg ac-tivation, started a new research field focused on full characteri-zation of antagonists and inverse agonists. This review coversthe current knowledge on selective PPAR antagonists, withspecial attention given to the possible therapeutic applicationsof this new class of PPAR modulators.
PPARa Antagonists
PPARa is expressed mostly in metabolically active tissues, suchas the liver, kidneys, and skeletal and cardiac muscles, in whichit regulates lipid transport, gluconeogenesis, and fatty acid oxi-dation.[10] This receptor represents a target of intense interestfor the treatment of metabolic diseases because of its functionin lipid metabolism. A decreased synthesis of very-low-densitylipoproteins (VLDLs) and apolipoprotein CIII and a rise in thelevel of high-density lipoproteins (HDLs) contribute to a hypoli-pidemic effect.[11] The down regulation of vascular cell adhe-sion molecule 1 (VCAM-1) and the inhibition of nuclear fac-tor kB (NFkB) and other inflammation mediators make PPARa
an attractive target to contrast the initiation and progressionof atherosclerosis.[12] Classical fibrates, such as fenofibrate, be-zafibrate, and gemfibrozil, have been widely used in the treat-ment of dyslipidemia associated with atherosclerosis.[13] In thelast few years, a very large number of new fibrate derivativeshave been synthesized and tested, with different degrees ofpotency and subtype selectivity.[14]
To date, only a few selective PPARa antagonists have beendescribed (Figure 3), and their therapeutic role remains un-known. No in vivo data have been reported for these com-pounds, but the strategy of blocking PPARa activation hasfound applications in the elucidation of some biological path-ways.
The first PPARa antagonist, MK886 (1), was originally identi-fied as a potent inhibitor of leukotriene biosynthesis, and itwas used for its ability to induce apoptosis in some biologicalsystems.[15] During a study to evaluate the mechanism of anti-apoptotic activity, a marked inhibitory activity against PPARa
was observed.[16] The dose-response data from a reporter assay,as well as the lack of any agonistic activity, suggested a non-competitive inhibition toward PPARa, without effects on the b/d and g isoforms. MK886 has been used for the last ten yearsin functional studies aimed at verifying the involvement ofPPARa in some signaling pathways; examples are the sPLA2-IIAexpression induced by tumor necrosis factor a (TNFa),[17] theincrease of apoA-I gene transcription,[18] the up-regulation ofuncoupling protein-2 (UCP-2) expression,[19] and the inhibitionof cyclooxygenase-2 (COX-2) expression by 20-hydroxyeicosa-
Figure 2. Schematic effect of agonist and antagonist binding on H12 fold-ing. a) H12 assumes a closed conformation upon agonist (in dark gray) bind-ing to the LBD. The hashed circle represents the hydrophobic groove, whichis responsible for co-activator recruitment. b) When an antagonist (in black)binds to the LBD, H12 changes to an open conformation, which does notallow binding of co-activators or subsequent transcriptional activation. c) Ifthe antagonist induces a misfolded conformation of H12, there are variouspossibilities based on the ability to recruit co-activators. Compounds actingin this manner could be full antagonists or partial agonists/antagonists.
tetraenoic acid (20-HETE).[20] The therapeutic potential ofMK886 in chronic lymphocytic leukemia (CLL) has also been in-vestigated: at concentrations in the 5–10 mm range, MK886kills resting CLL cells and causes proliferating cells to undergoimmunogenic death.[21]
In 2002, researchers at Glaxo reported the crystal structureof a ternary complex containing the PPARa LBD bound to thel-tyrosine-based molecule GW6471 (2) and the nuclear co-repressor protein SMRT.[22] The chemical structure of GW6471 issimilar to that of the potent PPARa agonist GW409544, exceptthat the carboxylic function is substituted by an ethylamidegroup. In the complex with PPARa, GW6471 adopts a U-shaped conformation and protrudes into the volume normallyoccupied by H12, which promotes the binding of co-repress-ors; the final effect is to block receptor activation. The antago-nistic properties of GW6471 significantly prevent cardiomyo-cyte differentiation in murine embryonic stem cells[23] and abol-ish the effects of palmitoylethanolamide (PEA) in neurosteroi-dogenesis,[24] pain processing,[25] and the vascular response ofendocannabinoids.[26] GW6471 antagonizes the anti-inflamma-tory effect of simvastatin on spinal cord trauma[27] and the anti-platelet actions of PPARa-associated statins and fibrates withprotein kinase Ca (PKCa).[28] New studies also suggest an inter-esting role for GW6471 in glioblastoma models in reducinglipid droplets, which are related to the tumor-malignancygrade.[29]
Two other compounds with antagonistic activity on PPARa
were identified by Eli Lilly and Company in a survey of carbox-ylic acids containing a triazolone function. The isosteric substi-tution of the carboxylic moiety with an N-acylsulfonamidegroup produced compounds 3 and 4, with high binding affini-ties on PPARa and without receptor activation in a cell-basedtransactivation assay.[30]
In the search for novel compounds acting on PPARa, the au-thors of this review recently reported some N-phenyl- and N-methylsulfonylamides containing a benzothiazole scaffold thatwere found to be PPARa inhibitors. Compound 5 was found tobe the best N-phenylsulfonamide and showed a dose-depen-dent inhibition profile on PPARa activation promoted byGW7647, with a micromolar potency (IC50 = 6.5 mm) ; it was ableto decrease CPT1A gene expression in a dose-dependentmanner.[31] Among the N-methylsulfonylamides, good results interms of antagonistic activity were achieved with compound 6,with data from a transactivation assay (IC50 = 0.8 mm) and fluo-rescence resonance energy transfer (FRET) analysis (IC50 =
0.6 mm) ; this compound also showed a good selectivity profiletoward the PPARa isoform, although these were just prelimina-ry data.[32]
PPARg Antagonists
PPARg was discovered in mammals and Xenopus species onthe basis of its similarity to PPARa.[33] It plays a crucial role inadipocyte differentiation and glucose homeostasis, as well asin cell-cycle regulation and cell differentiation.[34] The thiazolidi-nediones (TZDs) are PPARg agonists widely used in the treat-ment of type 2 diabetes for their beneficial effects on glucose
metabolism and insulin sensitivity.[35] Other PPARg agonistshave been studied for their beneficial effects on glycemic con-trol[36] and for their role in many diseases, such as Alzheimer’sdisease,[37] cardiovascular disease,[38] lung disease,[39] and auto-immune encephalomyelitis.[40] However, full agonists are re-sponsible for undesired effects, including weight gain, periph-eral edema, and in general, an increased risk of cardiovascularheart failure. In addition, a carcinogenic potential emerged forsome PPARg agonists, which has made deeper studies essentialprior to advancing new compounds in clinical development.[41]
These observations emphasize the need to develop novelPPARg-modulating drugs. In the last few years, dual a/g activa-tors, pan-agonists, partial agonists, and antagonists have beenstudied, in attempts to improve the biological effects and ther-apeutic value.[9]
Molecular and pharmacological studies have indicated thata moderate decrease in PPARg activity can prevent insulin re-sistance and obesity induced by a high-fat diet.[42] Recently,new antidiabetic PPARg ligands have shown a different bio-chemical function; these new drugs block the obesity-linkedphosphorylation of PPARg by cyclin-dependent kinase 5(Cdk5).[43]
Therefore, the discovery of PPARg antagonists (Figure 4) isnow of great interest in the treatment of diabetes and obesi-ty.[44] PPARg antagonists represent a new drug class that holdspromise as a broadly applicable therapeutic approach forcancer treatment.[45]
The first synthetic PPARg antagonist was the bisphenol A di-glycidyl ether 7 (BADGE), a synthetic compound used in theproduction of polycarbonate and plastics with industrial appli-cations; competition radioligand binding and functional stud-ies demonstrated that BADGE is a PPARg ligand with micromo-lar affinity.[46] It antagonizes TZDs, such as rosiglitazone, tostimulate the transcriptional activity of PPARg, although it haslow solubility and affinity. BADGE is also a PPARg agonist ina human urinary bladder carcinoma cell line and in a macro-phage-like cell line, and it shows suppression of TNFa produc-tion.[47] These studies indicate that PPARg activation or inhibi-tion may have greater cell-type specificity.
The phosphonophosphate 8 (SR-202) was identified as a se-lective PPARg antagonist that is able to inhibit both TZD-induced transcriptional activity and TZD-stimulated recruit-ment of the steroid receptor co-activator-1.[48] SR-202 enhancesinsulin sensitivity in diabetic ob/ob mice but also preventshigh-fat-diet-induced insulin resistance and improves the lipidprofile.
The nitrobenzanilide 9 (GW9662) and its analogue 10(T0070907) are irreversible and selective full PPARg antagonistsin cell-based assays and in cell-free assays.[49] GW9662 inhibitsgene expression in adipocyte differentiation and the growth ofhuman mammary tumor cell lines.[50] It prevents rosiglitazone-mediated PPARg activation, but it enhances rather than revers-es rosiglitazone-induced growth inhibition, probably througha PPARg-independent pathway related to the anticancer effectsof PPARg ligands.[51] T0070907 blocks adipocyte differentiationand displays anticancer effects in hepatocellular, esophageal,and squamous cell carcinoma.[52] Moreover, T0070907 decreas-
es cellular proliferation, migration, and invasion in two breastcancer cell lines (MB-231 and MCF-7) through a PPARg-depen-dent mechanism. Changes in migration and proliferation inbreast cancer cells might also be caused by an “off-target”action of T0070907 through suppression of the focal adhesionkinase/mitogen-activated protein kinase (FAK–MAPK) pathway.These data suggest that inhibition of endogenous PPARg sig-naling by T0070907 could be used to decrease the growth andmetastasis of breast cancer.[53]
PD068235 (11) inhibits the PPARg transcriptional activity in-duced by rosiglitazone and prevents association with the ago-nist-induced co-activator SRC-1. It is able to arrest adipocytedifferentiation at an early stage, but it does not revert the phe-notype of terminally differentiated adipocytes.[54]
LG100641 (12) selectively and competitively represses thia-zolidinedione-induced PPARg activation and adipocyte conver-sion.[55] It may induce a conformational change in PPARg thatresults in a failure to recruit co-activators; therefore, LG100641bound to PPARg is transcriptionally silent. It increases glucoseuptake in 3T3-L1 adipocytes and antagonizes target-gene acti-vation, as well as repression, in agonist-treated 3T3-L1 adipo-cytes. LG100641 is structurally similar to RXR-selective ligandsbut binds very weakly to the RXR.
In 2006, Ye et al. developed a computational virtual screen-ing approach to search for new PPARg antagonists ; they select-ed G3335 (13), a tryptophan-containing dipeptide derivative,as the starting point for further structural optimization.[56] The3D structure of the PPARg LBD indicates that the antagonisticbinding site consists of three subsites, which form a T-shapedgroove: a small hydrophobic binding pocket (site A), a largershallow hydrophilic pocket (site B), and a deep hydrophobiccavity (site C) that connects sites A and B. The binding modelsindicate that the indole group, the side chain of the C termi-nus, and the free amino acid group at the C terminus of G3335occupy subpockets B, C, and A, respectively. Thirty-five dipep-tide analogues of G3335 were synthesized with the following
modifications: shortening and lengthening of the side chain ofthe C terminus, replacement of the indole group with phenylor naphthyl rings, substitution of the carboxylic acid in theside chain of the C terminus with amide, guanidyl, or isobutylgroups, and esterification of the carboxylic groups to improvelipophilicity and stability.[57] These compounds were tested byusing the yeast two-hybrid system, based on the fact thatPPARg interacts with the co-activator CREB binding protein(CBP) in a ligand-dependent manner, and six of them showedstrong PPARg antagonistic activity.[58]
CDDO-Me (14) is a synthetic terpenoid that antagonizesPPARg.[59] It was reported to improve the diabetic conditionand to produce beneficial effects in high-fat-diet-fed type 2 di-abetic mice. It is considered as a potential antidiabetic agentwhen used at a low concentration. Indeed, oral administrationdecreases total body fat, free fatty acid levels, and plasma tri-glycerides. Furthermore, it improves glucose and insulin toler-ance in rats. When administered at high concentrations,CDDO-Me can inhibit cancer cell growth and proliferation ina wide variety of cell lines, such as ovarian, cervical, liver,breast, leukemia, and lung cancers. For this reason, this com-pound is in phase 1/2 clinical trials for cancer treatment.[60]
Indomethacin (15) is a COX inhibitor belonging to the non-steroidal anti-inflammatory drugs (NSAIDs) and is also de-scribed as a PPARg antagonist with a COX-independent mecha-nism.[61] For these reasons, indomethacin could be involved inchronic proliferative and inflammatory conditions for whichPPARg ligands hold promise as novel anti-inflammatory agents.
Recently, the novel 3-thiazolidine-modified benzoic acid de-rivative 16 (HL005) was synthesized as a potent PPARg-specificantagonist. Much evidence exists for the overexpression ofPPARg in many tumor cells, even if the biological significanceof its role in cancer remains controversial.[62] HL005 inhibits theproliferation of the MCF-7 cell line in a concentration-depen-dent manner, induces cell cycle arrest at the G2/M phase, andinterferes with cell adhesion.[63]
Cyclic phosphatidic acid (CPA,17) is a naturally occurringPPARg antagonist. It is a lyso-phosphatidic acid (LPA) ana-logue generated from lysophos-pholipids by signal-dependentactivation of phospholipase D2(PLD2).[64] CPA is a high-affinityligand with an apparent dissoci-ation constant (Kd) value in thehundred-nanomolar range,which is similar to the Kd valuesof TZDs. CPA binds the PPARg
LBD and stabilizes the bindingof the co-repressor SMRT; itthereby prevents activation byTZDs and the endogenousphospholipid agonist AGP. Theproliferative effect of rosiglitazone could be blocked by co-ad-ministration of CPA, which indicates that this compound canexert antagonistic effects in vivo.[65] PPARg inhibition could beuseful in cancer treatment,[66] but the modulation of PPARg incolon cancer is controversial. Recent studies demonstrate theeffects of CPA on cell-growth inhibition in the human coloncancer line HT-29, which makes this compound a new drugcandidate for the treatment of colon cancer.[67]
PPARb/d Antagonists
PPARb/d has the broadest expression pattern of all three PPARsubtypes. It is ubiquitously expressed and, for this reason, itwas initially considered to be a “housekeeping gene”. PPARb/dis involved in a large number of biological processes, includinglipid and glucose metabolism, cell differentiation, proliferation,apoptosis, and immune regulation.[68] It has also been associat-ed with related pathophysiological processes, such as inflam-mation,[69] obesity, dyslipidemia, diabetes,[70] cancer,[71] and car-diovascular diseases.[72] The crystal structure of the PPARb/dLBD revealed an exceptionally large pocket ~1300 � across.This pocket is similar to that of PPARg but is much larger thanthe pockets of other nuclear receptors. This may partially ex-plain the great variety of natural and synthetic ligands thatbind with and activate this receptor.[73]
To fully understand the biology and pharmacology ofPPARb/d, synthetic ligands are needed as investigative tools.Several high-affinity and subtype-specific PPARb/d agonistshave been developed, and they are currently in clinical trialsfor the treatment of metabolic diseases.[ 70a, 74] The results ofrecent studies show that PPARb/d inhibition, rather than acti-vation, may be beneficial under certain pathophysiologicalconditions. Thus, some high-affinity and subtype-specificPPARb/d inhibitors have been developed, but none of themhave been launched on the market (Figure 5). A selective an-tagonist of PPARb/d would also provide a valuable pharmaco-logical tool for elucidating the role of PPARb/d in cellulargrowth and homeostasis.
Compound 18 (GSK0660), a 2-methoxycarbonyl thiophenederivative identified by high-throughput screening, is a sub-type-specific inhibitor; the mechanism of action is not fully un-derstood, and it shows low bioavailability and potency.[75]
GSK0660 binds to PPARb/d with high potency in an in vitroligand displacement assay, whereas it is nearly inactive onPPARa and PPARg. Despite the potent binding, GSK0660 is in-active on all three PPARs in a standard cell-based transactiva-tion assay, which suggests that it is an antagonist of PPARb/d.
A series of high-affinity ligands derived from 18 have beensynthesized and structure–activity relationship (SAR) studieswere carried out by systematically varying the structural fea-tures to assess the contribution of the various residues to thebinding affinity. The results of a competitive time-resolved (TR)FRET assay showed that the replacement of the phenyl moietyby a medium-length alkyl chain residue has a tremendouseffect on the binding affinity relative to that of 18. Variation ofthe methoxy-substituted 1,4-diaminobenzene moiety, as wellas substitution of the methylthiophene carboxylate by a meth-ylbenzoate group, led to a significant decrease in affinity forthe PPARb/d LBD. Replacement of the 4’-aminophenyl groupby alkyl or benzyl chains resulted in a clear improvement inbinding affinity. Compounds 19 (ST247), with an n-hexylgroup, and 20 (PT-S58), with a tert-butyl group, have IC50
values that are three time lower than that of 18. Compound20 could be classified as the first full and pure PPARb/d inhibi-tor; this clearly distinguishes it from 19, which was classified asan inverse agonist.[76]
Compound 21 (SR13904),[77] identified from a series of struc-tural analogues of the potent PPARb/d agonist GW501516, andthe pyridylsulfone 22 (GSK3787),[78] derived from a Glaxo-SmithKline collection, are potent and selective PPARb/d ligandsin an in vitro ligand displacement assay. Compound 21 repre-sents a novel small molecule that inhibits PPARb/d agonist-induced transactivation; it shows a much weaker potency onPPARg, inhibitory effects on cellular proliferation, and survivalin several human carcinoma lines, including lung, prostate,breast, and liver cell lines. GSK3787 is also an irreversible an-tagonist of human and murine PPARb/d, with good oral phar-
macokinetic properties. SAR studies showed that the substitu-tion of the arylamide ring does not have a significant effect onthe binding affinity, whereas the length of the aliphatic linkerand the substitution of the pyridine ring are critical for activity.Compound 22 was found to be able to repress the expressionof CPT1A and PDK4, two key PPAR-regulated genes, whichplay an important role in energy homeostasis.[79]
Chemical modifications of TIPP-204, an a-ethylphenylpropa-noic acid with PPARb/d agonistic properties, led to the discov-ery of a new class of antagonists, such as compound 23.[80] Theintroduction of a conformationally restricted biphenylcarboxyl-ic acid framework interferes with the appropriate positioningof the H12 section of PPARb/d and, thereby, results in partialagonist and/or antagonistic activity.[81] These modifications re-sulted in retention of the PPARb/d selectivity if the COOHgroup was present at the 3- or 4-position. The efficacy forPPARb/d decreased in this order: 3-biphenylcarboxylic acid >
4-biphenylcarboxylic acid > 2-biphenylcarboxylic acid.Recently, the phenylacrylonitrile 24 (NSC636948) was identi-
fied as a PPARb/d inhibitor through screening of a chemical-compound library.[82] A comprehensive SAR study revealed thatortho halogenation and the introduction of an N-4-methylpi-perazine moiety greatly improve the binding affinity forPPARb/d and the efficiency of co-repressors. The combinationof these critical modifications led to the discovery of the piper-azinephenylacrylonitrile 25 (DG172), which is able to repressboth agonist-induced and basal-level PPRE-dependent tran-scription in cells. Most importantly, DG172 has good oral phar-macokinetic properties and represents a novel tool to investi-gate the biological and pathophysiological functions of PPAR-b/d.
Compound 26 has been identified as a PPARb/d antagonistin a virtual screening workflow, based on a combination ofpharmacophore modeling with 3D-shape and electrostatic-sim-ilarity screening techniques. It shows an interesting dose-dependent antagonism on PPARb/d and decreases the tran-scriptional activity induced by the positive control L165041.[83]
To explain the antagonistic PPARb/d activity of compound 26,an induced-fit docking study was performed. The resultsshowed that the antagonistic activity could be related to thedisplacement of residues in the PPARb/d ligand binding pocketby compound 26, which could inhibit the stabilization of theactive conformation of the H12 section, as known for otherPPAR antagonists.
Conclusions
In the last few years, the discovery of PPAR-selective antago-nists has proven to be a useful strategy for a better under-standing of metabolic pathways regulated by these receptors.However, an increasing body of data suggests that PPAR an-tagonists could also be used as therapeutic tools, because ofinteresting beneficial effects in many diseases, such as diabe-tes, cardiovascular and inflammatory pathologies, and cancers.Although some pharmacological results have been fully clari-fied, this new class of PPAR modulators represents, to date,a challenging therapeutic option for the future.
Acknowledgements
This study was supported by local grants from the University“G. d’Annunzio” of Chieti (Italy).
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&& –&&
Blocking the Peroxisome Proliferator-Activated Receptor (PPAR): AnOverview
Right on target: Peroxisome prolifera-tor-activated receptors (PPARs) havebeen studied extensively in recent de-cades and assessed as molecular targetsfor the development of drugs againstmetabolic disorders. Herein we presentan overview of the various chemicalstructures that are able to block thePPAR subtypes, through different mech-anisms, with a focus on promising ther-apeutic applications.
