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Functional method for generating or screening for ligands which modulate steroid hormone receptors

A functional, receptor technology that can be used in compound screening, chemical instruments and methods, biological testing, etc., and can solve problems such as low overall homology

Inactive Publication Date: 2007-04-11
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the overall homology between the ligand-binding domains (LBDs) of ERα and ERβ is less than 55%

Method used

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  • Functional method for generating or screening for ligands which modulate steroid hormone receptors
  • Functional method for generating or screening for ligands which modulate steroid hormone receptors
  • Functional method for generating or screening for ligands which modulate steroid hormone receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] Example 1: Combination of MNAR and VDR

[0197] method

[0198] Reagent

[0199] 1,25(OH) 2 Vit D3 was purchased from Sigma. Biotinylated peptides corresponding to different MNAR LXXLL motifs were synthesized and purified by peptide chemistry at Wyeth Research. Glutathione Sepharose beads were obtained from Sigma. Anti-Phosphotyrosine Antibody, SuperSignal Elisa Pico Peroxidase Substrate and Reacti-Bind TM Neutr Avidin TM Coated microtiter plates were from Pierce.

[0200] GST pull dow interaction analysis

[0201] A GST fusion of the VDR ligand-binding domain (i.e., amino acids 110-427) (designated GST-VDR-LBD) was expressed in BL-21 cells and bound to Glutathione Sepharose-4B (Amersham Biosciences, Piscataway, NJ). .Wild-type MNAR was transcribed / translated with the Promega TNT Quick CoupledTranscription / Translation System (Madison, WI) and performed 35 S radiolabeled, then in 100 nM 1,25(OH) in binding buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM P...

Embodiment 2

[0209] Example 2: VDR activity is activated by agonists of MNAR

[0210] method

[0211] Western blot analysis

[0212] UMR-106 cells were transfected with control vector or MNAR expression vector using Lipofectamine 2000 reagent according to the manufacturer's recommended method. After transfection, cells were cultured for an additional 48 h in medium supplemented with 2% charcoal-adsorbed FBS. After 48 hours, cells were treated with 10 nM 1,25(OH)2D3 for the indicated times. Cells were rinsed and harvested in cold PBS, then centrifuged and the supernatant discarded. Use 2 volumes of lysis buffer (20mM Tris-HCl pH7.5, 150mM NaCl, 1mMNa2EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na 2 VO 4 , 1 μg / μl leupeptin) to lyse cells. Cell debris was removed by centrifugation and equal amounts of protein were subjected to SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and the levels of MNAR, ERK and p-ERK were dete...

Embodiment 3

[0221] Example 3: Regulation of PI3 Kinase in ER by MNAR

[0222] method

[0223] MNAR was recently shown to affect PI3 / Akt kinase-mediated signaling in the ER by forming an ER / MNAR / PI3 kinase ternary complex. MNARs are expected to have similar effects on PI3 / Akt kinases that signal through the VDR.

[0224] Transfection and cell lysate preparation

[0225] MCF-7 cells were transfected with control, MNAR expression vector, non-specific siRNA or MNAR-specific siRNA using Lipofectamine 2000 reagent following the manufacturer's recommended method. After transfection, cells were cultured for an additional 48 hours in medium supplemented with 2% charcoal-adsorbed FBS. After 48 hours, cells were treated with 10 nM 17[beta]-estradiol for the indicated times. Cells were rinsed and harvested in cold PBS, then centrifuged and the supernatant discarded. Use 2 volumes of lysis buffer (20mM Tris-HCl pH7.5, 150mM NaCl, 1mM Na 2 EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1m...

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Abstract

This application presents the discovery that the Vitamin D3 receptor (VDR), a member of the nuclear hormone ligand-activated receptor superfamily, interacts with a MNAR, a scaffolding protein. This interaction results in the formation of a ternary complex between VDR, MNAR, and the Src or PI3 kinase families of tyrosine kinases to mediate cell signaling, especially in osteoblast cells.

Description

field of invention [0001] The present invention provides methods for the functional design or identification by screening of vitamin D receptor (VDR) selective ligands useful in the treatment of osteoporosis or other disorders involving signaling through the VDR. The present invention relates to the discovery that VDR interacts with the scaffold protein MNAR, previously demonstrated to interact with other nuclear hormone receptors, and that the ligand-dependent interaction between VDR and MNAR leads to activation of the Src / MAP kinase pathway. Background of the invention [0002] nuclear hormone receptor [0003] Nuclear hormone receptors are a superfamily of ligand-inducible transcription factors that, as a class, are involved in the ligand-dependent transcriptional control of gene expression. Binding of specific ligands induces conformational changes in the receptor molecule, thereby affecting the interaction of the receptor with other transcription factors and ultimately...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/10C07K14/00G01N33/53C07K7/06G01N33/567G01N33/82
CPCG01N2500/04C07K7/06G01N33/82G01N2800/108A61P19/10A61P3/02
Inventor F·巴莱塔G·奥唐奈B·J·切斯基斯
Owner WYETH LLC
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