RESUMO
Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.
Assuntos
Ácidos e Sais Biliares/metabolismo , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linfócitos T/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Receptor Constitutivo de Androstano , Doença de Crohn/metabolismo , Feminino , Ileíte/metabolismo , Inflamação/metabolismo , Interleucina-10/biossíntese , Interleucina-10/genética , Intestino Delgado/citologia , CamundongosRESUMO
Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded efficacy receptor conformations predicted by receptor theory has been limited but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). For all ligands, agonist functional efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state toward an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency and affinity are also correlated to efficacy and conformation, indicating ligand residence times among related analogs may influence receptor conformation and function. Our results derived from quantitative graded activity-conformation correlations provide experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.
Assuntos
PPAR gama/química , Sítios de Ligação , Células HEK293 , Humanos , PPAR gama/agonistas , PPAR gama/metabolismo , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Tiazolidinedionas/química , Tiazolidinedionas/farmacologiaRESUMO
The FP (Fbxo7/PI31) domains found in the F-box protein Fbxo7 and the proteasome inhibitor PI31 mediate the homodimerization and heterodimerization of Fbxo7 and PI31. Fbxo7 is the substrate-recognition subunit of the SCF(Fbxo7) (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also interacts with proteins that are not substrates of the ubiquitin proteasome system, such as Cdk6 and PI31. Here, the crystal structure of the Fbxo7 FP domain is reported at 2.0â Å resolution. The Fbxo7 FP domain adopts an α/ß-fold similar to that of the PI31 FP domain. However, an α-helix and three ß-strands in the Fbxo7 FP domain are longer than their counterparts in the PI31 FP domain. The differences in these secondary-structural elements are spatially clustered to define a more structured and extended C-terminal end of the Fbxo7 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein-protein interaction. The inter-domain interface of the Fbxo7 FP domain is defined by the α-helical surface in one protomer and the ß-sheet surface in the other protomer, whereas for the PI31 domain it is defined by either the α-helical surfaces or the ß-sheet surfaces in both protomers. The inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The Fbxo7 FP domain also has the potential to bind two protein partners simultaneously using the α-helical and ß-sheet surfaces. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with other regulatory proteins via the FP domain.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Estrutura Terciária de ProteínaRESUMO
Lung cancer is one of the most lethal malignancies worldwide. Peroxisome proliferator-activated receptor gamma (PPARγ, NR1C3) is a ligand-activated transcriptional factor that governs the expression of genes involved in glucolipid metabolism, energy homeostasis, cell differentiation, and inflammation. Multiple studies have demonstrated that PPARγ activation exerts anti-tumor effects in lung cancer through regulation of lipid metabolism, induction of apoptosis, and cell cycle arrest, as well as inhibition of invasion and migration. Interestingly, PPARγ activation may have pro-tumor effects on cells of the tumor microenvironment, especially myeloid cells. Recent clinical data has substantiated the potential of PPARγ agonists as therapeutic agents for lung cancer. Additionally, PPARγ agonists also show synergistic effects with traditional chemotherapy and radiotherapy. However, the clinical application of PPARγ agonists remains limited due to the presence of adverse side effects. Thus, further research and clinical trials are necessary to comprehensively explore the actions of PPARγ in both tumor and stromal cells and to evaluate the in vivo toxicity. This review aims to consolidate the molecular mechanism of PPARγ modulators and to discuss their clinical prospects and challenges in tackling lung cancer.
Assuntos
Neoplasias Pulmonares , PPAR gama , Humanos , Apoptose , Diferenciação Celular , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , PPAR gama/agonistas , Fatores de Transcrição/agonistas , Microambiente TumoralRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous lipids and synthetic ligands, including covalent antagonist inhibitors such as GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent antagonist inhibitors weaken-but do not prevent-the binding of other synthetic ligands via an allosteric mechanism rather than direct ligand clashing. The covalent ligands shift the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with and reduces the orthosteric binding affinity of non-covalent synthetic ligands. Crystal structures reveal different non-covalent synthetic ligand-specific cobinding mechanisms ranging from alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent ligand binding pose. Our findings not only highlight the significant flexibility of the PPARγ orthosteric pocket and its ability to accommodate multiple ligands simultaneously, but also demonstrate that GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPARγ.
RESUMO
Nuclear receptors (NRs) are thought to dynamically alternate between transcriptionally active and repressive conformations, which are stabilized upon ligand binding. Most NR ligand series exhibit limited bias, primarily consisting of transcriptionally active agonists or neutral antagonists, but not repressive inverse agonists-a limitation that restricts understanding of the functional NR conformational ensemble. Here, we report a NR ligand series for peroxisome proliferator-activated receptor gamma (PPARγ) that spans a pharmacological spectrum from repression (inverse agonism) to activation (agonism) where subtle structural modifications switch compound activity. While crystal structures provide snapshots of the fully repressive state, NMR spectroscopy and conformation-activity relationship analysis reveals that compounds within the series shift the PPARγ conformational ensemble between transcriptionally active and repressive conformations that are populated in the apo/ligand-free ensemble. Our findings reveal a molecular framework for minimal chemical modifications that enhance PPARγ inverse agonism and elucidate their influence on the dynamic PPARγ conformational ensemble.
RESUMO
A library of 31 natural neo-clerodanes isolated from Ajuga decumbens was assayed for antiosteoporosis. This results in 18 neo-clerodane osteoclastogenesis inhibitors, and compound 3 prevents bone loss in vivo. Further mechanistic studies demonstrated that these compounds inhibit osteoporosis by antagonizing peroxisome proliferator-activated receptor-γ (PPARγ). We designed and synthesized 17 compounds by chemically modifying the natural neo-clerodane 19 (highly potent and the major composition of A. decumbens extract) by means of structure-based drug design techniques. Among these neo-clerodane derivatives, compound 34 is the most potent osteoporosis inhibitor with a 46-fold improvement in inhibiting osteoclastogenesis (IC50 = 0.042 vs 1.92 µM), 11-fold increased activity in PPARγ antagonism (EC50 = 0.75 vs 8.35 µM), 66-fold enhancement in receptor affinity (KD = 0.27 vs 17.7 µM), and enhanced osteogenic promotion compared to 19. This underscores the potential of neo-clerodane diterpenoids as promising leads for osteoporosis treatment by targeting PPARγ.
RESUMO
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.
Assuntos
Piridinas , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Proteínas Virais de Fusão , Humanos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/genética , Piridinas/farmacologia , Camundongos , Animais , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/antagonistas & inibidores , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/genética , Antivirais/farmacologia , Antivirais/química , Piperidinas/farmacologia , Piperidinas/química , Camundongos Endogâmicos BALB C , Conformação Proteica , DibenzocicloeptenosRESUMO
Fbxo7 is a conserved protein in higher eukaryotes that belongs to the F-box protein family. Fbxo7 is the substrate-recognition component of the SCFFbxo7 (Skp1-Cul1-Fbox protein) E3 ubiquitin ligase. Besides the F-box motif, Fbxo7 also contains a C-terminal proline-rich region, an N-terminal ubiquitin-like domain and a novel FP (Fbxo7/PI31) domain preceding the F-box motif. The FP domains of Fbxo7 and the PI31 proteasome inhibitor mediate interaction between the two proteins. For structure determination of the FP domain of Fxbo7, a protein construct (amino acids 181-335) corresponding to the FP domain was expressed, purified and crystallized. The native and selenomethionine-labeled proteins crystallized in different crystal forms. Native and single-wavelength anomalous dispersion data sets with diffraction to 2.1 and 2.0â Å resolution, respectively, have been collected and structure determination is in progress.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/isolamento & purificação , Cristalização , Cristalografia por Raios X , Humanos , Estrutura Terciária de ProteínaRESUMO
Small molecule compounds that activate transcription of Nurr1-retinoid X receptor alpha (RXRα) (NR4A2-NR2B1) nuclear receptor heterodimers are implicated in the treatment of neurodegenerative disorders, but function through poorly understood mechanisms. Here, we show that RXRα ligands activate Nurr1-RXRα through a mechanism that involves ligand-binding domain (LBD) heterodimer protein-protein interaction (PPI) inhibition, a paradigm distinct from classical pharmacological mechanisms of ligand-dependent nuclear receptor modulation. NMR spectroscopy, PPI, and cellular transcription assays show that Nurr1-RXRα transcriptional activation by RXRα ligands is not correlated with classical RXRα agonism but instead correlated with weakening Nurr1-RXRα LBD heterodimer affinity and heterodimer dissociation. Our data inform a model by which pharmacologically distinct RXRα ligands (RXRα homodimer agonists and Nurr1-RXRα heterodimer selective agonists that function as RXRα homodimer antagonists) operate as allosteric PPI inhibitors that release a transcriptionally active Nurr1 monomer from a repressive Nurr1-RXRα heterodimeric complex. These findings provide a molecular blueprint for ligand activation of Nurr1 transcription via small molecule targeting of Nurr1-RXRα.
Assuntos
Proteínas de Transporte , Receptor X Retinoide alfa , Ligantes , Ligação Proteica , Receptor X Retinoide alfa/metabolismo , Domínios Proteicos , Ativação TranscricionalRESUMO
Autoimmune diseases affect 50 million Americans, predominantly women, and are thought to be one of the top 10 leading causes of death among women in age groups up to 65 years. A central role for TH17 cells has been highlighted by genome-wide association studies (GWAS) linking genes preferentially expressed in TH17 cells to several human autoimmune diseases. We and others have reported that the nuclear receptors REV-ERBα and ß are cell-intrinsic repressors of TH17 cell development and pathogenicity and might therefore be therapeutic targets for intervention. Herein, we describe detailed SAR studies of a novel REV-ERBα-selective scaffold. Metabolic stability of the ligands was optimized allowing for in vivo interrogation of the receptor in a mouse model of multiple sclerosis (EAE) with a ligand (34). Reduction in frequency and number of T-cells in the CNS as well as key REV-ERB target genes is a measure of target engagement in vivo.
Assuntos
Estudo de Associação Genômica Ampla , Esclerose Múltipla , Camundongos , Animais , Humanos , Feminino , Masculino , Fatores de Transcrição/genética , Diferenciação Celular , Esclerose Múltipla/tratamento farmacológico , Relação Estrutura-Atividade , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/agonistas , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
Ligands bind to an occluded orthosteric ligand-binding pocket within the nuclear receptor ligand-binding domain. Molecular simulations have revealed theoretical ligand entry/exit pathways to the orthosteric pocket; however, it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Here, using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and surface plasmon resonance analysis, we provide evidence that structurally distinct agonists bind peroxisome proliferator-activated receptor γ (PPARγ) via a two-step induced fit mechanism involving an initial fast kinetic step followed by a slow conformational change. The agonist encounter complex binding pose is suggested in crystal structures where ligands bind to a surface pore suggested as a ligand entry site in molecular simulations. Our findings suggest an activation mechanism for PPARγ whereby agonist binding occurs through an initial encounter complex followed by a transition of the ligand into the final binding pose within the orthosteric pocket, inducing a transcriptionally active conformation.
Assuntos
PPAR gama/química , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Simulação de Dinâmica Molecular , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Ligação ProteicaRESUMO
Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBß (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBß ligand-binding domain (LBD). We solved two crystal structures of REV-ERBß LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
RESUMO
Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations-supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations.
Assuntos
Benzamidas/metabolismo , Proteínas Correpressoras/metabolismo , PPAR gama/química , PPAR gama/metabolismo , Piridinas/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Proteínas Correpressoras/química , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Mutação , Coativadores de Receptor Nuclear/química , Coativadores de Receptor Nuclear/metabolismo , PPAR gama/agonistas , PPAR gama/genética , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Pioglitazone (Pio) is a Food and Drug Administration-approved drug for type-2 diabetes that binds and activates the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), yet it remains unclear how in vivo Pio metabolites affect PPARγ structure and function. Here, we present a structure-function comparison of Pio and its most abundant in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in co-regulator recruitment assays. X-ray crystallography and molecular docking analysis of PioOH-bound PPARγ ligand-binding domain revealed an altered hydrogen bonding network, including the formation of water-mediated bonds, which could underlie its altered biochemical phenotype. NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry analysis coupled to activity assays revealed that PioOH better stabilizes the PPARγ activation function-2 (AF-2) co-activator binding surface and better enhances co-activator binding, affording slightly better transcriptional efficacy. These results indicating that Pio hydroxylation affects its potency and efficacy as a PPARγ agonist contributes to our understanding of PPARγ-drug metabolite interactions.
Assuntos
Hipoglicemiantes/farmacologia , PPAR gama/metabolismo , Pioglitazona/farmacologia , Sítios de Ligação , Células HEK293 , Humanos , Ligação de Hidrogênio , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Simulação de Acoplamento Molecular , Pioglitazona/química , Pioglitazona/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , EstereoisomerismoRESUMO
Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinson's disease and schizophrenia.
Assuntos
Simulação de Acoplamento Molecular , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/química , Sítios de Ligação , Ácidos Graxos Insaturados/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Ligação ProteicaRESUMO
Crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPARγ ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega (Ω)-loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative biochemical functional and cellular assays reveal that synthetic ligand and fatty acid cobinding can form a 'ligand link' to the Ω-loop and synergistically affect the structure and function of PPARγ. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.
Assuntos
Simulação de Dinâmica Molecular , PPAR gama/química , PPAR gama/metabolismo , Conformação Proteica , Sítios de Ligação , Cristalografia por Raios X , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Ligantes , Estrutura Molecular , Oxazóis/química , Oxazóis/metabolismo , Oxazóis/farmacologia , PPAR gama/agonistas , Ligação Proteica , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas/química , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacologiaRESUMO
Peroxisome proliferator-activated receptors (PPARs) are pharmacological targets for the treatment of metabolic disorders. Previously, we demonstrated the anti-diabetic effects of SR1664, a PPARγ modulator lacking classical transcriptional agonism, despite its poor pharmacokinetic properties. Here, we report identification of the antagonist SR11023 as a potent insulin sensitizer with significant plasma exposure following oral administration. To determine the structural mechanism of ligand-dependent antagonism of PPARγ, we employed an integrated approach combining solution-phase biophysical techniques to monitor activation helix (helix 12) conformational dynamics. While informative on receptor dynamics, hydrogen/deuterium exchange mass spectrometry and nuclear magnetic resonance data provide limited information regarding the specific orientations of structural elements. In contrast, label-free quantitative crosslinking mass spectrometry revealed that binding of SR11023 to PPARγ enhances interaction with co-repressor motifs by pushing H12 away from the agonist active conformation toward the H2-H3 loop region (i.e., the omega loop), revealing the molecular mechanism for active antagonism of PPARγ.
Assuntos
Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/química , Células 3T3-L1 , Animais , Sítios de Ligação , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Cristalografia por Raios X , Medição da Troca de Deutério , Desenho de Fármacos , Células HEK293 , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
RORγt is well recognized as the lineage-defining transcription factor for T helper 17 (TH17) cell development. However, the cell-intrinsic mechanisms that negatively regulate TH17 cell development and autoimmunity remain poorly understood. Here, we demonstrate that the transcriptional repressor REV-ERBα is exclusively expressed in TH17 cells, competes with RORγt for their shared DNA consensus sequence, and negatively regulates TH17 cell development via repression of genes traditionally characterized as RORγt dependent, including Il17a. Deletion of REV-ERBα enhanced TH17-mediated pro-inflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE) and colitis. Treatment with REV-ERB-specific synthetic ligands, which have similar phenotypic properties as RORγ modulators, suppressed TH17 cell development, was effective in colitis intervention studies, and significantly decreased the onset, severity, and relapse rate in several models of EAE without affecting thymic cellularity. Our results establish that REV-ERBα negatively regulates pro-inflammatory TH17 responses in vivo and identifies the REV-ERBs as potential targets for the treatment of TH17-mediated autoimmune diseases.
Assuntos
Autoimunidade , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Colite/imunologia , Colite/patologia , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Células HEK293 , Humanos , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Regulação para CimaRESUMO
Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity. X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism. NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPARγ for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide routes for developing corepressor-selective repressive PPARγ ligands.