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Structural and functional profiling of environmental ligands for estrogen receptors.

Delfosse V, Grimaldi M, Cavaillès V, Balaguer P, Bourguet W - Environ. Health Perspect. (2014)

Bottom Line: However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable.We observed xenoestrogens binding to both ERs-with affinities ranging from subnanomolar to micromolar values-and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ.The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment.

View Article: PubMed Central - PubMed

Affiliation: Inserm (Institut national de la santé et de la recherche médicale) U1054, Montpellier, France.

ABSTRACT

Background: Individuals are exposed daily to environmental pollutants that may act as endocrine-disrupting chemicals (EDCs), causing a range of developmental, reproductive, metabolic, or neoplastic diseases. With their mostly hydrophobic pocket that serves as a docking site for endogenous and exogenous ligands, nuclear receptors (NRs) can be primary targets of small molecule environmental contaminants. However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable.

Objectives: We conducted a correlative analysis of structural and functional data to gain insight into the mechanisms by which 12 members of representative families of pollutants bind to and activate the estrogen receptors ERα and ERβ.

Methods: We used a battery of biochemical, structural, biophysical, and cell-based approaches to characterize the interaction between ERs and their environmental ligands.

Results: Our study revealed that the chemically diverse compounds bound to ERs via varied sets of protein-ligand interactions, reflecting their differential activities, binding affinities, and specificities. We observed xenoestrogens binding to both ERs-with affinities ranging from subnanomolar to micromolar values-and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ.

Conclusions: The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment.

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Related in: MedlinePlus

The relative activity of xenoestrogens relies on their different binding modes. (Left) Dose–response curves corresponding to the HGELN-ERα, -∆AB-ERα, -ERβ, and -∆AB-ERβ, luciferase assays of E2 and xenoestrogens. The maximal activity (100%) was obtained with 10 nM E2; values are mean ± SD from three separate experiments. (Right) The interaction networks of E2 and xenoestrogens with LBD residues of ERα. Key for structures: red, oxygen; blue, nitrogen; cyan, carbon; yellow, sulfur; green, chlorine; black dashed lines, hydrogen bonds; red spheres, water molecules. The electron density represents a Fo–Fc simulated annealing omit map contoured at 3σ.
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f2: The relative activity of xenoestrogens relies on their different binding modes. (Left) Dose–response curves corresponding to the HGELN-ERα, -∆AB-ERα, -ERβ, and -∆AB-ERβ, luciferase assays of E2 and xenoestrogens. The maximal activity (100%) was obtained with 10 nM E2; values are mean ± SD from three separate experiments. (Right) The interaction networks of E2 and xenoestrogens with LBD residues of ERα. Key for structures: red, oxygen; blue, nitrogen; cyan, carbon; yellow, sulfur; green, chlorine; black dashed lines, hydrogen bonds; red spheres, water molecules. The electron density represents a Fo–Fc simulated annealing omit map contoured at 3σ.

Mentions: Compound activities on ERα and ERβ. We monitored the agonistic potential of the compounds using stably transfected HGELN-ERα and -ERβ cell lines, allowing for a comparison of the effect of compounds on both human ER subtypes in a similar cellular context. All compounds were first tested on the HGELN parental cell line containing only the reporter gene. We observed some cytotoxicity at ligand concentrations of ≥ 10 μM but no unspecific modulation of luciferase expression (data not shown). We then characterized the activity of the compounds on HGELN-ER cell lines containing full-length (FL) ERα or ERβ. As shown in Figures 2 and 3A, the agonistic potentials depend on the receptor subtype and vary drastically among molecules that range from full agonists to weak agonists or antagonists. Whereas BP-2 acted as a full agonist of both ER subtypes, ferutinine was a selective activator of ERα, and α-ZA efficiently activated both receptors with a slight preference for ERα. The remainder of the compounds can be considered partial agonists with graded effects, with 4-OP being the most active (~ 80% of the transactivation seen with E2) and TCBPA inducing only 17% activity in the HGELN-ERβ cell line. Interestingly, some of these compounds displayed different activation capabilities of the two receptor subtypes, as illustrated by TCBPA and chlordecone, which were significantly more efficacious for ERα (50% and 39%, respectively) than for ERβ (17% and 19%, respectively). In total, three compounds activated the two ER subtypes equally (BP-2, 4-OP, and BBP), two compounds activated ERβ more efficiently than ERα (BPA and butylparaben), and seven compounds activated ERα more efficiently than ERβ (ferutinine, α-ZA, BPC, TCBPA, DDE, chlordecone, and HPTE). The EC50 (median effective concentration) values derived from the transactivation curves suggest that the compounds bound to both ER subtypes with similar affinities (see Supplemental Material, Table S1). To validate this observation, we performed competitive binding assays with (3H)-E2 (see Supplemental Material, Figure S1 and Table S2), which showed a wide array of affinities ranging from subnanomolar to micromolar values. Together, these experiments show that, in the context of HeLa cells, all the molecules bound to FL-ERα and FL-ERβ without subtype selectivity, whereas the functional outcomes of these interactions were, in most cases, subtype specific.


Structural and functional profiling of environmental ligands for estrogen receptors.

Delfosse V, Grimaldi M, Cavaillès V, Balaguer P, Bourguet W - Environ. Health Perspect. (2014)

The relative activity of xenoestrogens relies on their different binding modes. (Left) Dose–response curves corresponding to the HGELN-ERα, -∆AB-ERα, -ERβ, and -∆AB-ERβ, luciferase assays of E2 and xenoestrogens. The maximal activity (100%) was obtained with 10 nM E2; values are mean ± SD from three separate experiments. (Right) The interaction networks of E2 and xenoestrogens with LBD residues of ERα. Key for structures: red, oxygen; blue, nitrogen; cyan, carbon; yellow, sulfur; green, chlorine; black dashed lines, hydrogen bonds; red spheres, water molecules. The electron density represents a Fo–Fc simulated annealing omit map contoured at 3σ.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256047&req=5

f2: The relative activity of xenoestrogens relies on their different binding modes. (Left) Dose–response curves corresponding to the HGELN-ERα, -∆AB-ERα, -ERβ, and -∆AB-ERβ, luciferase assays of E2 and xenoestrogens. The maximal activity (100%) was obtained with 10 nM E2; values are mean ± SD from three separate experiments. (Right) The interaction networks of E2 and xenoestrogens with LBD residues of ERα. Key for structures: red, oxygen; blue, nitrogen; cyan, carbon; yellow, sulfur; green, chlorine; black dashed lines, hydrogen bonds; red spheres, water molecules. The electron density represents a Fo–Fc simulated annealing omit map contoured at 3σ.
Mentions: Compound activities on ERα and ERβ. We monitored the agonistic potential of the compounds using stably transfected HGELN-ERα and -ERβ cell lines, allowing for a comparison of the effect of compounds on both human ER subtypes in a similar cellular context. All compounds were first tested on the HGELN parental cell line containing only the reporter gene. We observed some cytotoxicity at ligand concentrations of ≥ 10 μM but no unspecific modulation of luciferase expression (data not shown). We then characterized the activity of the compounds on HGELN-ER cell lines containing full-length (FL) ERα or ERβ. As shown in Figures 2 and 3A, the agonistic potentials depend on the receptor subtype and vary drastically among molecules that range from full agonists to weak agonists or antagonists. Whereas BP-2 acted as a full agonist of both ER subtypes, ferutinine was a selective activator of ERα, and α-ZA efficiently activated both receptors with a slight preference for ERα. The remainder of the compounds can be considered partial agonists with graded effects, with 4-OP being the most active (~ 80% of the transactivation seen with E2) and TCBPA inducing only 17% activity in the HGELN-ERβ cell line. Interestingly, some of these compounds displayed different activation capabilities of the two receptor subtypes, as illustrated by TCBPA and chlordecone, which were significantly more efficacious for ERα (50% and 39%, respectively) than for ERβ (17% and 19%, respectively). In total, three compounds activated the two ER subtypes equally (BP-2, 4-OP, and BBP), two compounds activated ERβ more efficiently than ERα (BPA and butylparaben), and seven compounds activated ERα more efficiently than ERβ (ferutinine, α-ZA, BPC, TCBPA, DDE, chlordecone, and HPTE). The EC50 (median effective concentration) values derived from the transactivation curves suggest that the compounds bound to both ER subtypes with similar affinities (see Supplemental Material, Table S1). To validate this observation, we performed competitive binding assays with (3H)-E2 (see Supplemental Material, Figure S1 and Table S2), which showed a wide array of affinities ranging from subnanomolar to micromolar values. Together, these experiments show that, in the context of HeLa cells, all the molecules bound to FL-ERα and FL-ERβ without subtype selectivity, whereas the functional outcomes of these interactions were, in most cases, subtype specific.

Bottom Line: However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable.We observed xenoestrogens binding to both ERs-with affinities ranging from subnanomolar to micromolar values-and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ.The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment.

View Article: PubMed Central - PubMed

Affiliation: Inserm (Institut national de la santé et de la recherche médicale) U1054, Montpellier, France.

ABSTRACT

Background: Individuals are exposed daily to environmental pollutants that may act as endocrine-disrupting chemicals (EDCs), causing a range of developmental, reproductive, metabolic, or neoplastic diseases. With their mostly hydrophobic pocket that serves as a docking site for endogenous and exogenous ligands, nuclear receptors (NRs) can be primary targets of small molecule environmental contaminants. However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable.

Objectives: We conducted a correlative analysis of structural and functional data to gain insight into the mechanisms by which 12 members of representative families of pollutants bind to and activate the estrogen receptors ERα and ERβ.

Methods: We used a battery of biochemical, structural, biophysical, and cell-based approaches to characterize the interaction between ERs and their environmental ligands.

Results: Our study revealed that the chemically diverse compounds bound to ERs via varied sets of protein-ligand interactions, reflecting their differential activities, binding affinities, and specificities. We observed xenoestrogens binding to both ERs-with affinities ranging from subnanomolar to micromolar values-and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ.

Conclusions: The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment.

Show MeSH
Related in: MedlinePlus