Conformational Diversity of the Helix 12 of the Ligand Binding Domain of PPARγ and Functional Implications

Fluorescent labeling coupled with bipartite tetracysteine display enables the surveillance of ligand-mediated helix 12 transitions within the estrogen receptor α ligand-binding domain.

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X-ray crystal structure analysis of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), bound to estrogen receptor alpha ligand binding domain.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15647 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15647-e15647

Author(s):

Sean W. Fanning ◽

Geoffrey Greene ◽

Maureen G. Conlan

Keyword(s):

Breast Cancer ◽

Crystal Structure ◽

Estrogen Receptor ◽

Ligand Binding ◽

Binding Pocket ◽

Binding Domain ◽

Ligand Binding Domain ◽

Ligand Binding Pocket ◽

X Ray ◽

Helix 12

e15647 Background: Antiestrogens are a mainstay of treatment for estrogen receptor positive (ER+) breast cancer in both the adjuvant and the advanced/metastatic settings. Elacestrant is a mixed activity selective estrogen receptor (SER) alpha (ERα) antagonist, acting as a SER modulator (SERM) at low doses and a SER degrader (SERD) at high doses. It has shown activity in hormone sensitive wild type (WT) ERα and insensitive estrogen receptor gene 1 (ESR1) mutation-harboring (Y537S and D538G) ERα breast cancer, both in preclinical models and in clinical studies. It also possesses a unique pharmacology compared to other competitive ER antagonists in its ability to cross the blood brain barrier. Competitive ERα antagonists are typically comprised of a core that sits in the ligand binding pocket and an arm that manipulates the structure to achieve SERM or SERD activities. In these molecules, the arm is attached in the same position as the triphenylethylene core of tamoxifen. However, elacestrant possesses a novel site of attachment. As such, we hypothesized that elacestrant adopts an alternative binding orientation in the ERα ligand binding pocket to achieve its unique pharmaceutical profiles. Methods: X-ray crystallography was used to solve a co-crystal structure of elacestrant in complex with WT ERα ligand binding domain to 2Å. Results: Overall, elacestrant promotes the formation of a canonical ERα ligand binding domain antagonist conformation, whereby helix 12 (H12) is docked into the activating function-2 cleft. However, elacestrant adopts a novel vector in the ERα ligand binding pocket that places it in close proximity to helix 12. As a result, it forms a bifurcated hydrogen bond that is not observed in other competitive antiestrogens and samples a chemical space known to increase H12 mobility and induce SERD activity. This novel vector also places it near positions 537 and 538, the two most common sites of somatic mutation. Conclusions: The high-resolution x-ray crystal structure of elacestrant suggests that the unique binding mode it adopts enables novel pharmacology and positions it to achieve potency in the WT and activating somatic ERα mutated breast cancer setting.

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Molecular Dynamics Simulation of the Ligand Binding Domain of Farnesoid X Receptor. Insights into Helix-12 Stability and Coactivator Peptide Stabilization in Response to Agonist Binding

Journal of Medicinal Chemistry ◽

10.1021/jm049182o ◽

2005 ◽

Vol 48 (9) ◽

pp. 3251-3259 ◽

Cited By ~ 25

Author(s):

Gabriele Costantino ◽

Antonio Entrena-Guadix ◽

Antonio Macchiarulo ◽

Antimo Gioiello ◽

Roberto Pellicciari

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Farnesoid X Receptor ◽

Dynamics Simulation ◽

Agonist Binding ◽

Helix 12

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Targeted mutation of key residues at the start of helix 12 in the hERα ligand-binding domain identifies the role of hydrogen-bonding and hydrophobic interactions in the activity of the protein

The Journal of Steroid Biochemistry and Molecular Biology ◽

10.1016/j.jsbmb.2005.06.036 ◽

2006 ◽

Vol 98 (1) ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Changqing Zhao ◽

Judith Abrams ◽

Debra F. Skafar

Keyword(s):

Hydrogen Bonding ◽

Ligand Binding ◽

Hydrophobic Interactions ◽

Binding Domain ◽

Ligand Binding Domain ◽

Targeted Mutation ◽

Helix 12 ◽

Key Residues

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The SERM/SERD Bazedoxifene Disrupts ESR1 Helix 12 to Overcome Acquired Hormone Resistance in Breast Cancer Cells

10.1101/306472 ◽

2018 ◽

Author(s):

Sean W. Fanning ◽

Rinath Jeselsohn ◽

Venkatasubramanian Dharmarajan ◽

Christopher G. Mayne ◽

Mostafa Karimi ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Ligand Binding ◽

Hormone Replacement ◽

Acquired Resistance ◽

Metastatic Breast ◽

Binding Domain ◽

Ligand Binding Domain ◽

Breast Cancers ◽

Helix 12

AbstractAcquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in theESR1(estrogen receptor alpha (ERα) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER+ breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We find BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show that BZA’s selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.SignificanceBazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ERα that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ERα ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.

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Structural Characterization of the Human Androgen Receptor Ligand-binding Domain Complexed with EM5744, a Rationally Designed Steroidal Ligand Bearing a Bulky Chain Directed toward Helix 12

Journal of Biological Chemistry ◽

10.1074/jbc.m705524200 ◽

2007 ◽

Vol 282 (42) ◽

pp. 30910-30919 ◽

Cited By ~ 42

Author(s):

Line Cantin ◽

Frédérick Faucher ◽

Jean-François Couture ◽

Karine Pereira de Jésus-Tran ◽

Pierre Legrand ◽

...

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Structural Characterization ◽

Binding Domain ◽

Ligand Binding Domain ◽

Receptor Ligand ◽

Helix 12 ◽

Human Androgen Receptor

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Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

Biological Chemistry ◽

10.1515/bc.2004.004 ◽

2004 ◽

Vol 385 (1) ◽

pp. 21-30 ◽

Cited By ~ 20

Author(s):

S. Przibilla ◽

W. W. Hitchcock ◽

M. Szécsi ◽

M. Grebe ◽

J. Beatty ◽

...

Keyword(s):

Dna Binding ◽

Fusion Protein ◽

Ligand Binding ◽

Binding Pocket ◽

Negative Influence ◽

Binding Domain ◽

Ligand Binding Domain ◽

Ecdysteroid Receptor ◽

Ligand Binding Pocket ◽

Helix 12

AbstractThe functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acidbinding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.

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