A yeast two-hybrid technology-based system for the discovery of PPARγ agonist and antagonist

Chen, Qing; Chen, Jing; Sun, Tao; Shen, Jianhua; Shen, Xu; Jiang, Hualiang

doi:10.1016/j.ab.2004.09.004

Cited by 30 publications

(21 citation statements)

References 35 publications

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“…CBP overexpression was able to abrogate PPARg-mediated AT1R gene transcription suppression, [37,38] and PPARg agonists were able to promote PPARg-CBP binding affinity. [39,40] In our previous work [41] we had exploited an efficient approach in the discovery of PPARg agonists and antagonists by the yeast two-hybrid system, based on the fact that PPARg interacts with the coactivator CBP in ligand-dependent fashion. We successfully employed the MEL1 reporter gene to evaluate the protein-protein interactions by conducting a con- venient a-galactosidase assay in the AH109 yeast strain with genes for PPARg-LBD and CBP introduced at the N terminus.…”

Section: Resultsmentioning

confidence: 99%

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The Dipeptide H‐Trp‐Glu‐OH Shows Highly Antagonistic Activity against PPARγ: Bioassay with Molecular Modeling Simulation

Zhang

Luo

et al. 2006

ChemBioChem

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The peroxisome proliferator-activated receptor gamma (PPARgamma) is an important therapeutic drug target for several conditions, including diabetes, inflammation, dyslipidemia, hypertension, and cancer. It is shown that an antagonist or partial agonist of PPARgamma has attractive potential applications in the discovery of novel antidiabetic agents that may retain efficacious insulin-sensitizing properties and minimize potential side effects. In this work, the dipeptide H-Trp-Glu-OH (G3335) was discovered to be a novel PPARgamma antagonist. Biacore 3000 results based on the surface plasmon resonance (SPR) technique showed that G3335 exhibits a highly specific binding affinity against PPARgamma (K(D) = 8.34 microM) and is able to block rosiglitazone, a potent PPARgamma agonist, in the stimulation of the interaction between the PPARgamma ligand-binding domain (LBD) and RXRalpha-LBD. Yeast two-hybrid assays demonstrated that G3335 exhibits strong antagonistic activity (IC50 = 8.67 microM) in perturbing rosiglitazone in the promotion of the PPARgamma-LBD-CBP interaction. Moreover, in transactivation assays, G3335 was further confirmed as an antagonist of PPARgamma in that G3335 could competitively bind to PPARgamma against 0.1 microM rosiglitazone to repress reporter-gene expression with an IC50 value of 31.9 muM. In addition, homology modeling and molecular-docking analyses were performed to investigate the binding mode of PPARgamma-LBD with G3335 at the atomic level. The results suggested that residues Cys285, Arg288, Ser289, and His449 in PPARgamma play vital roles in PPARgamma-LBD-G3335 binding. The significance of Cys285 for PPARgamma-LBD-G3335 interaction was further demonstrated by PPARgamma point mutation (PPARgamma-LBD-Cys285Ala). It is hoped our current work will provide a powerful approach for the discovery of PPARgamma antagonists, and that G3335 might be developed as a possible lead compound in diabetes research.

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Section: Resultsmentioning

confidence: 99%

“…Yeast two-hybrid based assay: The yeast two-hybrid system used for PPARg antagonist screening was constructed by the method published by Chen et al [41] with certain modifications. By the PCR technique, mCBP (aa.…”

mentioning

confidence: 99%

The Dipeptide H‐Trp‐Glu‐OH Shows Highly Antagonistic Activity against PPARγ: Bioassay with Molecular Modeling Simulation

Zhang

Luo

et al. 2006

ChemBioChem

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“…That a similar mechanism subsequent to COX-2 induction may also account for the investigated chemotherapeutics is supported by our finding that apoptosis elicited by paclitaxel, cisplatin or 5-FU was likewise impaired when PPARg was knocked down or blocked. Finally, troglitazone, a PPARg agonist with an about 10-fold lower affinity than 15d-PGJ 2 (Chen et al, 2004), mimicked the apoptotic response of PGD 2 , 15d-PGJ 2 and chemotherapeutics in HeLa cells. These results suggest that COX-2-dependently synthesized PGD 2 activates PPARg and thereby induces apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Decisive role of cyclooxygenase-2 and lipocalin-type prostaglandin D synthase in chemotherapeutics-induced apoptosis of human cervical carcinoma cells

2007

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“…It has to be noted that none of the methods used to study protein-protein interactions are better than any other and typically lead to the identifi cation of different subsets of interactions [35][36][37][38]. Analysis of protein complexes using affi nity purifi cation followed by MS typically identifi es directly and indirectly associated proteins, whereas Y2H analyses and identifi es direct, binary protein-protein interactions [35].…”

Section: Proteomics a Still Developing Complex Fi Eldmentioning

confidence: 98%

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

et al. 2011

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A yeast two-hybrid technology-based system for the discovery of PPARγ agonist and antagonist

Cited by 30 publications

References 35 publications

The Dipeptide H‐Trp‐Glu‐OH Shows Highly Antagonistic Activity against PPARγ: Bioassay with Molecular Modeling Simulation

The Dipeptide H‐Trp‐Glu‐OH Shows Highly Antagonistic Activity against PPARγ: Bioassay with Molecular Modeling Simulation

Decisive role of cyclooxygenase-2 and lipocalin-type prostaglandin D synthase in chemotherapeutics-induced apoptosis of human cervical carcinoma cells

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

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