Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPAR<i>γ</i>)

Ma, Jingjing; Zhang, Tao; Fang, Ning; Zou, Yan; Gong, Qihai; Yu, Limei; Chen, Daixiong

doi:10.1111/j.2042-7158.2012.01462.x

Cited by 10 publications

(4 citation statements)

References 29 publications

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“…Human embryonic kidney (HEK) 293 cells have been widely used for the screening of PPARγ agonists 58 , suggesting that the endogenous factor of HEK293 cells do not affect the transcriptional activity of PPARγ. The lipid mediators tested were not metabolized by HEK 293 cells (Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes

Song

Kim

et al. 2018

Nat Commun

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Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

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Section: Methodsmentioning

confidence: 99%

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes

Song

Kim

et al. 2018

Nat Commun

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“…Importantly, a large body of evidence demonstrates that these pathways can regulate the activity of pro-inflammatory TFs as well as drive differentiation of immune cell subsets [33][34][35]. In addition, studies suggest that HEK293 cell lines express and are influenced by AhR and PPARγ [36,37]. Consequently, activation of these pathways may contribute to the ability of LMWF5A to suppress the release of pro-inflammatory cytokines from activated immune cells.…”

Section: Pparγ and Ahr Antagonists Reduce Lmwf5a Drug Potencymentioning

confidence: 99%

LMWF5A suppresses cytokine release by modulating select inflammatory transcription factor activity in stimulated PBMC

et al. 2020

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Background Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-β, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. Methods ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1β relative potency were then employed to confirm the involvement of enriched pathways and TFs. Results LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1β, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1β release. Conclusion In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

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“…Hitherto, several screening pipelines have been designed to search for novel PPARγ ligands ( 23 – 25 ). Transactivation assays and combined approaches using transactivation and binding assays are the best-established and used methods to screen for compounds that activate PPARγ ( 26 , 27 ). However, only a restricted number of screening techniques have been designed and applied to find PPARγ non-agonists ( 28 , 29 ).…”

Section: Introductionmentioning

confidence: 99%

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties

et al. 2018

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Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879’s mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.

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Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPARγ)

Abstract: These data indicated that the HEK293T cells could be stably transfected with hPPARγ. This cell-based drug screening platform could be used targeting specific nuclear receptor of hPPARγ with effectiveness and specificity for hPPARγ agonists discovery.

Cited by 10 publications

References 29 publications

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes

LMWF5A suppresses cytokine release by modulating select inflammatory transcription factor activity in stimulated PBMC

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties

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