Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPARγ Agonism

SummaryLigand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators

Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARλ 3 full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARλ 3, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARλ 3 provides a rationale for the different activation of the ligand towards PPARα and PPARλ 3, suggesting a novel basis for ligand design

Progress and challenges of selective Farnesoid X Receptor modulation.

Bile acids are amphipathic molecules that were previously known to serve as fat solubilizers in the intestine in postprandial conditions. In the last two decades, bile acids have been recognized as signaling molecules regulating energy metabolism pathways via, amongst others, the farnesoid X receptor (FXR). Upon bile acid activation, FXR controls expression of genes involved in bile acid, lipid, glucose and amino acid metabolism. In addition, FXR activation has been shown to limit the inflammatory response. The central role of FXR in various aspects of metabolism and inflammation makes FXR an attractive drug target for several diseases, such as obesity, metabolic syndrome, non-alcoholic steatohepatitis, cholestasis and chronic inflammatory diseases of the liver and intestine. However, most of the currently available compounds impact on all discovered FXR-mediated functions and may have, on top of beneficial effects, undesired biological actions depending on the disease. Therefore, research efforts are increasingly focused on the development of selective FXR modulators, i.e. selective bile acid receptor modulators (SBARMs), aimed at limiting the potential side-effects of conventional full FXR agonists upon chronic treatment. Here, we review the rationale for the design of SBARMs comprising dissociation between metabolic and inflammatory signaling, gene-selective and tissue-specific targeting. We discuss the potential structural mechanisms underlying the binding properties of dissociating ligands of FXR in light of ongoing efforts on the generation of dissociated ligands for otxher nuclear receptors, as well as their pharmacological and therapeutic potential

Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis

Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (\u3e90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis

Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis

Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (\u3e90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis

Computational Studies and Design of PPARγ and GLUT1 Inhibitors

The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor of the nuclear receptor superfamily that controls the expression of a variety of genes involved in fatty acid metabolism, adipogenesis, and insulin sensitivity. PPARγ is a target for insulin-sensitizing drugs, and it plays a significant function in prostate cancer. PPARγ antagonists have anti-proliferative effects in a broad range of hematopoietic and epithelial cell lines. The ligand binding domain (LBD) of PPARγ is large and has orthosteric and allosteric binding sites. Several PPARγ-ligand co-crystal structures show two bound molecules, one to the orthosteric pocket and a second to the allosteric site. We ran docking studies against the orthosteric and allosteric binding sites to determine the most favorable binding site for PPARγ antagonists. We found that Glide docking performed well in predicting PPARγ antagonist binding affinities, and that the allosteric site of PPARγ was the most favorable binding site for antagonists. We also investigated PPARγ ligand-protein interactions to better define a structural basis for the binding selectivity of PPARγ antagonists. We found that Phe282, Arg288, and Lys367 interact with antagonists more than with agonists and partial agonists. We then identified several potential PPARγ antagonists by virtual screening of the PPARγ allosteric pocket. The glucose transporter 1 (GLUT1) is a uniporter protein that facilitates the transport of glucose across the plasma membranes of mammalian cells. As GLUT1 is overexpressed in numerous tumors, this transporter is a potential target for cancer treatment. GLUT1 works through conformational switching from an outward-open (OOP) to an inward-open (IOP) conformation passing through an occluded conformation. We sought to determine which conformation is favored for ligand binding by molecular docking studies of known GLUT1 inhibitors with the different GLUT1 conformers. Our data revealed that the IOP is the preferred conformation and that residues Phe291, Phe379, Glu380, Trp388, and Trp412 may play important roles in ligand binding to GLUT1. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against an NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and anti-proliferative activities. From these, we identified four compounds that inhibited cell viability in an HCT116 colon cancer cell line. We also determined that one of these, NSC295720, inhibited GLUT1 in a biochemical assay

Biostructural studies on PPAR nuclear receptors

The peroxisome proliferator-activated receptors (PPARs) represent a family of nuclear receptors that function as ligand-activated transcription factors, regulating genes involved in cell differentiation and various metabolic processes, especially lipid and glucose homeostasis. The PPAR family comprises three isoforms: PPARα, PPARβ/δ and PPARγ, with different tissue distribution, ligand specificitiy and physiological role. Because of their wide range of actions on glucose homeostasis, lipid metabolism and vascular inflammation, PPARs represent promising targets for the development of new drugs for the treatment of metabolic disorders such as type 2 diabetes mellitus (T2DM), dyslipidemia and atherosclerosis. Among all the subtypes, despite the undesiderable side effects associated to the drug treatment, PPARγ is still the most widely studied for its crucial role in the complex cross-talk between metabolically active tissues essential for energy balance. Then, new combination strategies using dual or pan agonists, as well as selective modulators, are currently in development. This study is aimed to understand in deep the dynamic personality of the nuclear receptors PPAR in complex with both natural and synthetic ligands that, interacting with different regions of the LBD, confer a differentiated biological response in cellular and animal models. PPARs could be then described as a ‘functionally pluripotent’ proteins being their activity mediated by ligands that, causing the functional site to adopt an active/inactive conformations, activate different structural and biological pathways depending on the co-activator/co-repressor recruited. Through a structural approach we propose to get more insights on how the biological response is variably affected by ligands depending on their binding mode and even the mutation of a single residue responsible for a structural destabilization of the LBD could be associated to rare genetic disorder. The understanding of such a mechanism required the use of more than one biophysical technology. X-ray diffraction was used as the main approach to investigate the binding mode of the selected ligands. In addition, the binding has been also characterized using other biophysical techniques such as Isotermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) to obtain thermodynamic and kinetic parameters of the binding

The application of molecular modelling in the safety assessment of chemicals: A case study on ligand-dependent PPARγ dysregulation.

The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q(2)cv=0.610, Nopt=7, SEPcv=0.505, r(2)pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development

Effect of Synthetic Magnolol Derivatives on Activity of Nuclear Receptors PPARγ and RXRα

Univerzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Katedra biochemických věd University of Vienna Faculty of Life Sciences Department of Pharmacognosy Kandidát: Andrea Dymáková Školitelé: Mag. Simone Latkolik, doc. Ing. Barbora Szotáková, Ph. D. Název diplomové práce: Vliv syntetických derivátů magnololu na aktivitu nukleárních receptorů PPARγ and RXRα Mezi terapeutické cíle v léčbě civilizačních onemocnění, jako jsou například metabolický syndrom nebo diabetes mellitus typu II, patří nukleární receptory, receptor aktivovaný peroxisomovými proliferátory (PPAR) a jeho heterodimerizační partner retinoidní X receptor (RXR). V současnosti užívané léky však mají řadu nežádoucích účinků, proto se hledají noví PPAR agonisté, kteří by disponovali lepšími vlastnostmi než tyto stávající léky (Berger et al. 2002, Berger et al. 2005). Právě magnolol byl již dříve popsán jako duální agonista PPAR a RXR (Fakhrudin et al. 2010, Zhang et al. 2011). Na základě bi-arylové struktury jeho molekuly byly navrženy a syntetizovány tzv. spojené magnolol dimery. Cílem této diplomové práce bylo studium agonistického potenciálu těchto derivátů na výše zmíněné nukleární receptory PPAR a RXR, výsledky byly porovnávány s magnololem. Aktivita těchto sloučenin byla studována v trans-aktivačním modelu v...Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Vienna Faculty of Life Sciences Department of Pharmacognosy Candidate: Andrea Dymáková Supervisors: Mag. Simone Latkolik, doc. Ing. Barbora Szotáková, Ph. D. Title of diploma thesis: Effect of Synthetic Magnolol Derivatives on Activity of Nuclear Receptors PPARγ and RXRα The nuclear receptors, peroxisome proliferator-activated receptor γ (PPARγ) and its heterodimerization partner retinoid X receptor α (RXRα) are drug targets in the treatment of diseases like the metabolic syndrome and diabetes mellitus type 2. The effort has been made to develop new agonists for PPARγ to obtain ligands with more favourable properties than currently used drugs (Berger et al. 2002, Berger et al. 2005). Magnolol was previously described as a dual agonist of PPARγ and RXRα (Fakhrudin et al. 2010, Zhang et al. 2011). Based on the bi- aryl structure of magnolol, the effort has been made to design and synthesize linked magnolol dimers. The aim of this thesis was to investigate the agonistic potential of these compounds with respect of the nuclear receptors PPARγ and RXRα in comparison to magnolol. We evaluated the ligand binding properties of the compounds and their functionality as PPARγ agonists in vitro...Department of Biochemical SciencesKatedra biochemických vědFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Molecular and Structural Insights into Nuclear Hormone Receptor Repression Mediated by the Corepressor NCOR

Nuclear hormone receptors comprise a large family of ligand-sensitive transcription factors that can directly bind and regulate target genes to affect various physiological processes including development, differentiation, circadian rhythm and metabolism. Classically, activation of transcription by nuclear receptors (NRs) is due to a ligandinduced switch from corepressor- to coactivator-bound states. Highly analogous corepressors including NCoR and SMRT facilitate repression by NR via recruitment and activation of the histone deacetylase HDAC3. Liver X Receptor (LXR) is an NR that functions to regulate diverse physiological processes including cholesterol metabolism, lipid homeostasis, immunity and inflammation. Selective modulators of LXR to specifically target pathways for peripheral cholesterol efflux were developed and observed to function as partial agonists of LXR. We determined that the selective partial agonism of LXR by these ligands was indeed related to differential recruitment of the corepressor NCoR. Secondly, to understand the structural basis for physiological v repression of NR by NCoR we co-crystallized a small peptide comprising a region of the interaction domain of NCoR with the ligand-binding domain of Rev-erba. This revealed that the relative structural requirements for the previously reported antagonized PPARa- bound SMRT were distinct from that of the Rev-erba:NCoR complex. Altogether, these studies provide novel molecular insight into the function of NCoR in regulating transcription by nuclear hormone receptors