Measuring Binding Kinetics of Ligands with Tethered Receptors by Fluorescence Polarization and Total Internal Reflection Fluorescence

Kwok, K. L.; Cheung, Nai Ho

doi:10.1021/ac1002245

Cited by 20 publications

(38 citation statements)

References 30 publications

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“…Calculated RBAs, compared to E2 set at 100%, indicated weak binding of most ligands tested except TAM. These values correspond to the range of RBAs (or IC 50 values) that have been previously reported based on radioligand, polarization anisotropy and fluorescence-based assays (Table 1) (Bolger et al, 1998; Kuiper et al, 1998a; Kuiper et al, 1998b; Matthews et al, 2000; Nikov et al, 2000; Parker et al, 2000; Nikov et al, 2001; Kuramitz et al, 2002; Ohno et al, 2002; Ohno et al, 2003; Mueller et al, 2004; Olsen et al, 2005; Matsui, 2007; Freyberger et al, 2010; Kwok and Cheung, 2010; McLachlan et al, 2011). In general, our assay produces slightly increased affinities for tested xenoestrogen ligands compared to radioligand assays, a result that is likely due to the fact that FP is a homogeneous assay.…”

Section: Discussionsupporting

confidence: 74%

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Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens

Smith

Clark

Bisesi

et al. 2016

Comparative Biochemistry and Physiology Part D: Genomics and Pr

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The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p < 0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in both human and aquatic species.

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Section: Discussionsupporting

confidence: 74%

“… a Ohno et al (2003). b Nikov et al (2001). c Kwok et al (2010). d Matsui et al (2006). e Matthews et al (2000). f Kuiper et al (1998a, 1998b). …”

Section: Figmentioning

confidence: 99%

Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens

Smith

Clark

Bisesi

et al. 2016

Comparative Biochemistry and Physiology Part D: Genomics and Pr

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“…These results are in good agreement with the experimental binding affinities and bioactivity trends. Both the natural E 2 and the synthetic estrogen DES have potent transcriptional activities with equivalent binding affinity to ERα, which in turn is ∼70-fold higher than GEN. 45,46 W is observed experimentally to have a 433-fold lower binding affinity than E 2 to ERα, and shows no ER-dependent transcriptional activity. 21 3.3.…”

Section: Methodsmentioning

confidence: 95%

Characterization of Agonist Binding to His524 in the Estrogen Receptor α Ligand Binding Domain

Gao

Ågren

et al. 2012

J. Phys. Chem. B

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The bioactivities of the natural steroidal estrogen 17β-estradiol (E2), the synthetic estrogen diethylstilbestrol (DES), and the phytoestrogen genistein (GEN) are intimately associated with their binding to the estrogen receptor α ligand binding domain (ERα LBD) and accordingly allostery. Molecular modeling techniques have been performed on agonists in complex with the LBD, focusing on the pivotal role of His524 modeled as the ε-tautomer and the protonated form (depending on pH). It is found that E2 binds to the active LBD with the aid of Leu525, showing existing stable patterns of an H-binding network with Glu419 via His524 in all models. The main difference seen in the effect is that the full agonists E2 and DES have higher binding energies to the protonated His524 than the partial agonists GEN and Way-169916 (W), which is in line with noted experimental transcriptional activities. In conclusion, the study demonstrates that the phytoestrogen GEN interacts differently with the LBD than what E2 and DES do, which explains the observed signaling differences.

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“…The importance of drug stereoselectivity is gaining greater attention in recent years,32 and differential pharmacological effects have been reported between ginsenoside Rg3 stereoisomers 9, 11, 12. It is because although the enantiomers of Rg3 possess the same functional group at C20, the difference in three‐dimensional structure may affect the binding affinity to the receptor binding site.…”

Section: Discussionmentioning

confidence: 99%

Ginsenoside Rg3 stereoisomers differentially inhibit vascular smooth muscle cell proliferation and migration in diabetic atherosclerosis

Guo

Xiao

et al. 2018

J Cellular Molecular Medi

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Ginsenoside 20(R/S)‐Rg3, as a natural peroxisome proliferator‐activated receptor gamma (PPARγ) ligand, has been reported to exhibit differential biological effects. It is of great interest to understand the stereochemical selectivity of 20(R/S)‐Rg3 and explore whether differential PPARγ activation by Rg3 stereoisomers, if it exists, could lead to differential physiological outcome and therapeutic effects in diabetic atherosclerosis. Here, we investigated the binding modes of 20(R/S)‐Rg3 stereoisomers in the PPARγ ligand‐binding domain (PPARγ‐LBD) using molecular modelling and their effects on smooth muscle cell proliferation and migration induced by advanced glycation end products (AGEs). The results revealed that 20(S)‐Rg3 exhibited stronger antiproliferative and antimigratory effects due to stronger PPARγ activation. To validate the in vitro results, we used a mice model with diabetic atherosclerosis and obtained that 20(S)‐Rg3 markedly reduced the plaque size secondary to reducing the proliferation and migration of VSMCs, while the plaques were more stable due to improvements in other plaque compositions. The results shed light on the structural difference between Rg3 stereoisomers that can lead to significant differential physiological outcome, and the (S)‐isomer seems to be the more potent isomer to be developed as a promising drug for diabetic atherosclerosis.

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Measuring Binding Kinetics of Ligands with Tethered Receptors by Fluorescence Polarization and Total Internal Reflection Fluorescence

Cited by 20 publications

References 30 publications

Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens

Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens

Characterization of Agonist Binding to His524 in the Estrogen Receptor α Ligand Binding Domain

Ginsenoside Rg3 stereoisomers differentially inhibit vascular smooth muscle cell proliferation and migration in diabetic atherosclerosis

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