Molecular and cellular pharmacology
Identification and characterisation of a prototype for a new class of competitive PPARγ antagonists

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Abstract

Understanding of the physiological role of peroxisome proliferator-activated receptor gamma (PPARγ) offers new opportunities for the treatment of cancers, immune disorders and inflammatory diseases. In contrast to PPARγ agonists, few PPARγ antagonists have been studied, though they do exert immunomodulatory effects. Currently, no therapeutically useful PPARγ antagonist is commercially available. The aim of this study was to identify and kinetically characterise a new competitive PPARγ antagonist for therapeutic use.

A PPARγ-dependent transactivation assay was used to kinetically characterise (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB) in kidney, T and monocytic cell lines. Cytotoxic effects were analysed and intracellular accumulation of MTTB was assessed by tandem mass spectrometry (LC-MS/MS). Potential interactions of MTTB with the PPARγ protein were suggested by molecular docking analysis.

In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), MTTB exhibited competitive antagonism against rosiglitazone in HEK293T and Jurkat T cells, with IC50 values in HEK293T cells of 4.3 µM and 1.6 µM, using the PPARγ ligand binding domain (PPARγ-LBD) and the full PPARγ protein, respectively. In all cell lines used, however, MTTB showed much higher intracellular accumulation than GW9662. MTTB alone exhibited weak partial agonistic effects and low cytotoxicity. Molecular docking of MTTB with the PPARγ-LBD supported direct interaction with the nuclear receptor.

MTTB is a promising prototype for a new class of competitive PPARγ antagonists. It has weak partial agonistic and clear competitive antagonistic characteristics associated with rapid cellular uptake. Compared to commercially available PPARγ modulators, this offers the possibility of dose regulation of PPARγ and immune responses.

Introduction

The ligand-dependent, activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily (Issemann and Green, 1990). It is predominantly expressed in adipose tissue and cells of the immune system (Chawla et al., 1994, Tontonoz et al., 1994, Tontonoz et al., 1995). As with other members of the nuclear hormone receptor superfamily, the tissue- and ligand-specific activation and transcriptional target gene regulation of PPARγ is a multistep process (Kliewer et al., 1994, Seargent et al., 2004). It involves specific binding of several natural and synthetic ligands (Forman et al., 1995, Kliewer et al., 1995), heterodimerisation with the retinoid X receptor alpha (RXRα), the recognition and interaction of sequence-specific PPARγ response elements (PPREs) in the promoter region of their target genes and finally the recruitment of cofactors and other nuclear coregulatory proteins (Chawla et al., 2001, Kliewer et al., 2001, Nolte et al., 1998, Spiegelman and Heinrich, 2004).

Because of its role during adipogenesis and in glucose metabolism, in which PPARγ predominantly induces gene expression of adipocyte protein 2 (aP2, also called fatty acid binding protein 4, FABP4), cluster of differentiation 36 (CD36), lipoprotein lipase (LPL) or glucose transporter 4 (GLUT4), for example, it has been studied intensively as a therapeutic target (Lehmann et al., 1995, Nakano et al., 2006, Tontonoz et al., 1995). The most prominent synthetic PPARγ agonists with high receptor affinity are the thiazolidinediones (TZDs). These are used in type-2 diabetes mellitus, in which they improve insulin sensitivity and cause a concomitant reduction of free glucose levels (Rangwala and Lazar, 2004, Staels and Fruchart, 2005). PPARγ can also bind directly to other proteins and inhibits signal transduction. This capability, called transrepression, is mainly mediated by direct protein–protein interactions between PPARγ and other transcription factors, such as the nuclear factor “kappa-light-chain-enhancer“ of activated B cells (NFkB), nuclear factor of activated T cells (NFAT) or the activator protein 1 (AP-1) (Chen et al., 2003, Wang et al., 2001, Yang et al., 2000). In this way, PPARγ inhibits pro-inflammatory signalling and induces an anti-inflammatory response (Pascual and Glass, 2006, Ricote and Glass, 2007). In this context, activation of PPARγ by TZDs is effective in reducing inflammation. In contrast to the proven beneficial effects and widespread use of the TZDs in vivo, they are also associated with a number of deleterious side-effects, serious tolerability and safety issues, including significant weight gain, peripheral oedema, congestive heart failure and bone fracture (Nesto et al., 2003, Nissen and Wolski, 2007).

On the other hand, PPARγ also induces apoptosis and thereby exerts immunosuppressive activity. Recently, PPARγ antagonism, especially by the PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662) (Leesnitzer et al., 2002), has guided the development of new drugs and therapeutic strategies for a wide range of cancer types, such as breast cancer (Burton et al., 2008, Seargent et al., 2004), for regulation of adiposity (Nakano et al., 2006) and modulation of immunity and inflammatory diseases (Schmidt et al., 2011). Because of the irreversible binding of GW9962 to the PPARγ protein, it is not suitable for therapeutic use. The increased understanding of the physiological role and clinical relevance of PPARγ has emphasised the critical need for the discovery, identification and characterisation of new PPARγ agonists, antagonists or selective PPARγ modulators (SPPARγMs), while avoiding the known side-effects.

In this report, the identification and characterisation of (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl) benzyl)oxy)-benzylidene) hexanoic acid (MTTB) as a prototype for a new class of competitive PPARγ antagonists is described. Compared to the reference compound, GW9662, MTTB showed properties of a competitive PPARγ antagonist, with weak partial agonism, high intracellular uptake and low cytotoxicity in vitro. Because of its apparently reversible binding to the PPARγ protein, compounds such as MTTB may be well suited for controlled therapeutic use in immune disorders.

Section snippets

Chemicals and reagents

All chemicals and reagents were of the highest grade of purity and if not indicated otherwise, commercially available from AppliChem GmbH (Darmstadt, Germany), Carl Roth GmbH (Karlsruhe, Germany), Merck KGaA (Darmstadt, Germany) and Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). The PPARγ antagonist, GW9662, the SPPARγMs, N-(9-fluorenylmethoxycarbonyl)-l-leucine (FMOC-l-leucine) and netoglitazone (MCC-555) were acquired from Cayman Chemical Company (Ann Arbor, USA) and the PPARγ agonist,

MTTB docks to the PPARγ-LBD

In a previous study, we showed that similarity of the co-crystallised ligand is crucial for predictive modelling of receptor-bound ligand conformation due to the pronounced induced fit known for PPARγ (Weber et al., 2012). Therefore, we chose for comparison, in the present study, the X-ray structure of a potent sulfoncarboxamide antagonist in complex with the PPARγ-LBD (PDB code: 2HFP) (Hopkins et al., 2006). The corresponding ligand incorporates all the structural features of MTTB (Fig. 2A):

Discussion

The extensive clarification of PPARγ molecular interactions and signal transduction pathways, as well as its activation in molecular, cellular and clinical settings, has provided invaluable insights into the design of therapeutically useful PPARγ agonists and SPPARγMs (Balint and Nagy, 2006, Sporn et al., 2001, Willson et al., 2000). While PPARγ activation by natural and synthetic ligands is well established (Forman et al., 1995, Kliewer et al., 1995), relatively little is known about PPARγ

Conclusions

Because of the strong antagonistic activity of GW9662, it is widely used as a research tool in cell culture systems and animal models to study the role of PPARγ in biological processes. In contrast to the irreversible nature of GW9662 binding, MTTB appears to bind reversibly to the PPARγ protein, still inhibiting the expression of target genes. Taken together, the competitive antagonism, negligible partial agonism, low cytotoxicity and high intracellular uptake are properties which would allow

Author contribution

T.K., B.B., M.J.P. and A.v.K. conceived the principal question and designed the experiments.

T.K., E.P., B.B., M.J.P. and A.v.K. wrote the manuscript.

T.K., D.F., S.L., N.F. M.W. and E.P. conducted the experiments.

T.K., L.K.S., L.K., A.K.G., S.L., N.F., T.S. and E.P. analysed the data.

D.F., S.L., N.F., M.W., E.P. and M.S.-Z. contributed reagents/materials/analysis tools.

Statement of conflict of interest

The authors declare no competing interests. Michael J. Parnham is a consultant for Xellia Pharmaceuticals ApS and LEO Pharma A/S.

Acknowledgements

This research was supported by the research funding programe Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) of the State of Hessen, Germany and the Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation – Pharma (TRIP). We thank members of the Fraunhofer IME Project Group Translational Medicine and Pharmacology TMP and of the Institute of Biochemistry I – Pathobiochemistry for their technical help and support.

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