Protocol

A High-Throughput Screening Method to Identify Small Molecule Inhibitors of Thyroid Hormone Receptor Coactivator Binding

See allHide authors and affiliations

Science's STKE  27 Jun 2006:
Vol. 2006, Issue 341, pp. pl3
DOI: 10.1126/stke.3412006pl3

Abstract

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Introduction

The thyroid hormone receptor (TR) is part of the superfamily of nuclear hormone receptors (NRs) whose major transcriptional activity is controlled by the thyroid hormone 3,5,3′-triiodo-L-thyronine (T3) (1, 2). As a heterodimer with the retinoid X receptor (RXR), TR binds to specific thyroid response elements (TREs) on DNA (3). Gene regulation mediated by TR is especially important during growth and development, as well as general metabolism (46). Abnormal levels of T3 are responsible for medical conditions such as obesity, high plasma cholesterol levels, type II diabetes, high blood pressure, and increased risk of heart failure (711). The binding of T3 occurs at the ligand-binding pocket present in the ligand-binding domain (LBD) of the receptor (12). This binding event induces a conformational change of the TR to enable the formation of the solvent-exposed hydrophobic pocket that contains the transactivation function 2 (AF2) domain. The formation of a competent AF2 allows recruitment of coregulatory proteins that strongly influence transcriptional regulation, because they link the receptor to the transcriptional machinery. Several coactivators of the TR have been identified (13).

Modulation of the TR-stimulated transcription by small molecules has focused on the development of T3 analogs (1419). Although some antagonists have now been reported, it remains unclear if such analogs can overcome the undesirable side effects on cardiac stimulation (20, 21). The interface formed by the TR AF2 domain and its coactivators offers the possibility to modulate TR-dependent gene transcription in the presence of its natural hormone. α-helical proteomimetics have been reported to inhibit this interaction in competition binding assays but, to date, none of these inhibitors regulated NR signaling in cellular systems (2225).

High-throughput screening (HTS) is frequently used to discover small molecules that inhibit protein-protein interactions, (26) although only a limited number of compounds have been reported that are active in cellular models. (27, 28). We applied the technique of fluorescence polarization (29) and developed a competitive NR coactivator binding assay to discover small molecules with the ability to bind to TR in the presence of T3 and block binding of coregulators (30). This method is very time efficient, and 140K small molecules were screened in 2 to 3 weeks using a semiautomated screening protocol. The hit verification by secondary assays is an important part of the protocol and identifies if this inhibition has biological relevance in cellular signal transduction by inhibiting the transcriptional activity of the TR in the presence of T3.

The process for developing and executing an HTS campaign required multiple sequential steps (Fig. 1). The procedure starts with the expression and purification of TR protein and the synthesis of fluorescently labeled coactivator peptide. The assay development phase included the measurement of the binding constant of TR for quality control (QC) and determination of the screening concentration (about 2× Kd). The HTS involved liquid handling (transfer of liquids by a laboratory automation workstation) and the measurement of fluorescence intensity (FI) and fluorescence polarization (FP). The FP values of the positive and negative controls determined 0% and 100% inhibition of coactivator recruitment. Compounds with inhibitory ability of >50% and FI variations of less than 10% in comparison to the controls were identified as hit compounds. These hit compounds were validated in secondary assays. The dose response analysis using a competition coactivator binding assay determined the IC50 value of the compounds. The ability to inhibit the interaction between full length TR and coactivator was investigated by a glutathione-S-transferase (GST) pull-down assay. The hormone displacement assay ensures that the small molecule is competing with the coactivator and not with T3. Finally, the transcription assay determines if the small molecules are capable to penetrate the cell membrane and inhibit signal transduction in a cellular environment.

Fig. 1.

The workflow of the high-throughput method includes assay development (synthesis of labeled coactivator peptide, protein expression and purification, and protein-binding assay), high-throughput screening, and the secondary assays (dose-response analysis, pull-down assay, hormone displacement assay, and transcription assay).

The following methods detail the steps for identifying and validating compounds that inhibit TR activity in the presence of the TR ligand T3. These methods can be adapted to screening for compounds that influence the activity of other NRs by using a labeled peptide that binds with high affinity to the NR under investigation or by screening for compounds that compete for co-repressor binding instead of coactivator binding to the NR.

Materials

Many of the procedures rely on the same reagents and chemicals. A list of reagents and chemicals common to two or more procedures is listed first. Each individual subsection lists additional reagents specific to that procedure.

Common Reagents and Chemicals

96-well opaque plate (Costar #3365)

Ampicillin (anhydrous basic, 96.0 to 100.5%; Sigma)

BCA assay (Pierce)

Dimethyl sulfoxide (DMSO; ACS grade, 99.9%; Aldrich)

Dithiothreitol (DTT; Aldrich)

E. coli BL21 strain (Stratagene)

EDTA ethylenediaminetetraacetic acid disodium salt dihydrate (ACS reagent; Aldrich)

Glycerol (ACS reagent; Aldrich)

Isopropyl-beta-D-thiogalactopyranoside (IPTG; Sigma)

KCl, potassium chloride (Aldrich)

LB agar EZMix powder (Sigma)

LB broth EZMix powder (Sigma)

Liquid nitrogen

Lysozyme (from chicken egg white, lyophilized powder, 50,000 U/mg; Sigma)

Methanol (MeOH; for HPLC, 99.9%; Aldrich)

MgCl2 (hexahydrate; Aldrich)

NaCl (Sigma)

NP-40 (Aldrich)

NuPAGE 10% bis-tris gel with MOPS (Invitrogen)

NuPAGE LDS sample loading buffer (Invitrogen)

Phosphate-buffered saline (PBS; Sigma)

Phenylmethylsulfonyl fluoride (PMSF; Sigma)

Poly-Prep Column (BioRad, #731-1550)

Thyroid hormone, 3,5,3′-triiodo-L-thyronine (T3; Sigma)

TNT T7 quick coupled transcription/translation system (Promega)

Tris(hydroxymethyl)aminomethane, hydrochloride (Tris-HCl; Sigma)

Tween 20 (Sigma)

SRC2-2 Labeling

5-iodoacetamidofluorescein (Molecular Probes)

Acetonitrile (for HPLC, 99.9%; Aldrich)

Coactivator SRC2-2 peptide (CLKEKHKILHRLLQDSSSPV) (crude; BIO SYNTHESIS, Lewisville, TX)

Dimethyl formamide (DMF; ACS grade, 98.8%; Aldrich)

Ethanethiol (Aldrich)

Trifluoroacetic acid (TFA; spectrophotometric grade; Aldrich)

Protein Expression and Purification of the Thyroid Receptor

Antipain (Sigma)

E. coli strain BL21(DE3)pLsyE (Invitrogen)

hTRα LBD (His6 E148-V410) cloned into the expression vector pET DUET-1 (Novagen)

hTRβ LBD (His6 T209-D461) cloned into the expression vector pET DUET-1 (Novagen)

Note: Both constructs were cloned into the BamHI and HindIII restriction sites downstream of the hexahistidine tag of the expression vector pET DUET-1. The replacement of C309 for alanine (A) in the hTRβ LBD construct was performed with the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene). The sequences of all constructs were verified by DNA sequencing and the plasmids are available upon request.

Imidazole (Sigma)

Leupeptin (Sigma)

Pepstatin (Sigma)

Talon protein purification column (Metal Affinity Chromatography; BD Biosciences)

Protein-Binding Assay, High-Throughput Screen, and Dose-Response Analysis

384-well black plate (Costar, #3710)

384-well opaque plate (Costar, #3702)

Centrifugal filter units (Ultra-15, 10K NMWL; Amicon)

Compounds 3 and 11 (10 mM in DMSO) were a gift from Tim Geistlinger (23)

SRC2-2 peptide, labeled with fluorescein (see Instructions, below)

TRβ LBD (see Instructions, below)

Glutathione-S-Transferase Pull-Down Assay

35S-methionine (1000 MCi; Perkin Elmer)

Acetic acid (ACS grade; Aldrich)

Bovine serum albumin (BSA; Sigma, #A-2153)

Glutathione Sepharose 4B (GE, #17-0756-01)

GST-TRβ full length (available on request)

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes; Sigma)

Plasmid SRC2 (available on request)

Protease inhibitor cocktail (set II; Calbiochem)

Triton X (Aldrich)

Hormone Displacement Assay

125I-T3, 3,5,3′-triiodo-L-thyronine (2200 Ci/mmol; Perkin Elmer)

Column rack and trough (Fisher)

G25 Sephadex (Aldrich)

Histone from calf thymus (Sigma)

Microscintillation fluid (Beckman)

Monothioglycerol (MTG; Aldrich)

Phosphate, dibasic, hexahydrate (Aldrich)

Plasmid, full-length TRβ containing a CMV promoter (available on request)

Scintillation vials (7 ml; Fisher)

Transcription Assay

96-well plate (white, flat bottom with lid, tissue culture-treated, Costar #3917)

Bovine calf serum (defined; HyClone)

Cell culture-treated PS dish, 150 mm (Corning)

Dextran-coated charcoal (Sigma-Aldrich)

Dual luciferase reporter assay kit (Promega)

Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham with L-glutamine and 15 mM Hepes, without sodium bicarbonate and phenol red (DMEM/Ham’s F12; powder; Sigma)

Dulbecco's Modified Eagle’s H-21 containing 4.5 g/liter glucose, 584 mg/liter glutamine, 3.7 g/liter sodium bicarbonate and phenol red (DMEM/H12; HyClone)

Electroporation cuvette (4 mm gap; Eppendorf)

Glucose 10% (HyClone)

PBS buffer, without Mg2+ and Ca2+ ions (MediaTech)

Penicillin-streptomycin 100× (penicillin 6.37 g/liter, streptomycin 10 g/liter; HyClone)

Plasmid hTRβ (available on request)

pRL-CMV Vector (Promega)

Reporter plasmid [synthetic TR response element (DR-4) containing two copies of a direct repeat spaced by four nucleotides (AGGTCA-cagg-AGGTCA) cloned immediately upstream of a minimal (−32 to +45) thymidine kinase promoter linked to luciferase coding sequence] (available on request)

Sodium bicarbonate (NaHCO3; Aldrich)

Trypsin 0.05%, versene 0.02%, in saline A (HyClone)

U2OS cells (human bone osteosarcoma epithelial cells; ATCC, #HTB-96)

Equipment

Many of the procedures rely on the same equipment. A list of equipment common to two or more procedures is listed first. Each individual subsection lists additional equipment specific to that procedure.

Common Laboratory Equipment

2.8-liter Fernback flasks

Biomate 3 spectrometer (Thermo)

Centrifuge (Eppendorf 5810R)

Centrifuge, JLA-14 rotor (Avanti J-25; Beckman)

Glass beads (0.5 mm diameter)

Maxi Rotator (Barnstead)

Petri dishes

Plate reader (Analyst AD; LJL Biosystems)

Shaker incubator (Innova 44; New Brunswick Scientific)

Shaker, rotisserie (Thermolyne)

SigmaPlot 8.0 (SPSS; Chicago, IL)

Sonicator (Branson 250)

Ultra-Centrifuge, VTi50 rotor (Optima L-90K; Beckman)

Water bath

SRC2-2 Labeling

Analytical HPLC-MS system (Alliance HT, Micromass ZQ 4000; Waters)

HT-4X evaporator (Genevac)

Preparative HPLC system (Delta 600; Waters)

RP-C18 Xterra column 5 μm, 19 mm × 50 mm (Waters)

RP-C18 Xterra column 5 μm, 6 mm × 50 mm (Waters)

Protein Expression and Purification of the Thyroid Receptor

Incubator (Thermo)

Protein-Binding Assay, High-Throughput Screen, and Dose-Response Analysis

Assay Explorer: In-house screening software written in Pipeline Pilot 4.5.1 (Scitegic)

Note: This Web-based program automates the process of joining experimental data to compound information, flagging suspicious plates based on low Z-factors, extracting compounds with statistically significant activity, and annotating hits with additional information (for example, chemical similarity to known bioactive compounds, known genotoxic or cytotoxic molecules, or available compounds, and profiles from ADME models). Data are available online at http://www.stjuderesearch.org/guy/AssayReporter/TR_SCREEN/

Multi-channel pipette (12 channels)

Multimek (Beckman)

Note: This instrument is a laboratory automation workstation that performs liquid handling, including pipetting, diluting, and dispensing.

Wellmate (Matrix)

Note: This instrument can dispense liquids into 96-well and 384-well microplates with a tubing cartridge.

Glutathione-S-Transferase Pull-Down Assay

Centrifugal concentrator (CentriVap; Labconco)

PhosphorImager (Storm; GE)

Hormone Displacement Assay

Equipment to work with radiolabeled compounds emitting gamma rays (lead protection)

Scintillation counter (Beckman)

Transcription Assay

Cell culture hood

Genepulser (BioRad)

Hemocytometer

Incubator, 37°C, 5% CO2 (MCO 36M; Sanyo)

Microscope

Recipes

SRC2-2 Labeling

Recipe 1: TFA in Water

Dissolve 2 ml of TFA in 4000 ml of deionized water.

Note: TFA is a corrosive and volatile liquid and should be stored and handled in a well-ventilated place (fume hood).

Recipe 2: TFA in Acetonitrile

Dissolve 2 ml of TFA in 4000 ml of acetonitrile.

Recipe 3: TFA in MeOH

Dissolve 2 ml of TFA in 4000 ml of methanol.

Recipe 4: 20 mM Tris Buffer, pH 9.0

Dissolve 3.15 g of Tris-HCl in 80 ml of deionized water, adjust pH to 9.0 with NaOH (aq), and adjust volume to 100 ml with deionized water.

Protein Expression and Purification of the Thyroid Receptor

Recipe 5: 1 M Tris Buffer, pH 7.0

Dissolve 157.6 g of Tris-HCl in 800 ml of deionized water, adjust to pH 7.0 with NaOH (aq), and adjust volume to 1000 ml with deionized water.

Recipe 6: 3 M NaCl

Dissolve 174 g of NaCl in 1000 ml of deionized water.

Recipe 7: 100 mM DTT

Dissolve 154.2 mg of DTT in 10 ml of deionized water. Store in 1 ml aliquots at −20°C.

Recipe 8: 100 mM PMSF

Dissolve 174.2 mg of PMSF in 10 ml of ethanol. Store in 1 ml aliquots at −20°C.

Recipe 9: 10 mM T3

Dissolve 9.76 mg of T3 in 2 ml of DMSO; store at −20°C.

Recipe 10: 10 mM Leupeptin

Dissolve 49.3 mg of leupeptin in 10 ml of deionized water. Store in 0.5 ml aliquots at −20°C.

Recipe 11: 10 mM Pepstatin

Dissolve 8.5 mg of pepstatin in 10 ml of methanol. Store in 0.5 ml aliquots at −20°C.

Recipe 12: 10 mM Antipain

Dissolve 67.7 mg of antipain in 10 ml of DMSO. Store in 0.5 ml aliquots at −20°C.

Recipe 13: Sonication Buffer

Stock solution Volume Final concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)5 ml50 mM
3 M NaCl (Recipe 6)5 ml150 mM
100 mM PMSF (Recipe 8)0.1 ml0.1 mM
10 mM T3 (Recipe 9)0.1 ml0.01 mM
10 mM leupeptin (Recipe 10)0.5 ml0.05 mM
10 mM pepstatin (Recipe 11)0.5 ml 0.05 mM
10 mM antipain (Recipe 12)0.5 ml 0.05 mM
Glycerol10 ml10%

Add deionized water to 80 ml, adjust to pH 7.5 if necessary with NaOH (aq), and adjust volume to 100 ml with deionized water.

Note: Protease inhibitor cocktail tablets (complete, EDTA-free) from Roche may be used instead of leupeptin, pepstatin, and antipain.

Recipe 14: 1 M Imidazole

Dissolve 6.8 g imidazole in 80 ml of deionized water, adjust to pH 7.5 with HCl (aq), and adjust volume to 100 ml with deionized water.

Recipe 15: Talon Buffer 1

Stock solution Volume Final concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)5 ml50 mM
3 M NaCl (Recipe 6)10 ml300 mM
Glycerol10 ml10%

Add deionized water to 80 ml, adjust to pH 7.5 if necessary with NaOH (aq), and adjust volume to 100 ml with deionized water.

Recipe 16: Talon Buffer 2

Stock solution Volume Final concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)5 ml50 mM
3 M NaCl (Recipe 6)5 ml150 mM
Glycerol10 ml10%
100 mM DTT (Recipe 7)0.2 ml0.2 mM
100 mM PMSF (Recipe 8)0.1 ml0.1 mM
10 mM T3 (Recipe 9)0.1 ml 0.01 mM
1 M Imidazole (Recipe 14)3 ml30 mM

Add deionized water to 80 ml, adjust to pH 7.5 if necessary with NaOH (aq), and adjust volume to 100 ml with deionized water.

Recipe 17: Talon Buffer 3

Stock solution Volume Final concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)5 ml50 mM
3 M NaCl (Recipe 6)5 ml150 mM
Glycerol10 ml10%
1 M Imidazole (Recipe 14)7.5 ml75 mM
Add deionized water to 80 ml, adjust to pH 8.0 if necessary with NaOH (aq), and adjust volume to 100 ml with deionized water.

Recipe 18: Talon Buffer 4

Stock solution Volume Final concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)1 ml50 mM
3 M NaCl (Recipe 6)1 ml150 mM
Glycerol2 ml10%
1 M Imidazole (Recipe 14)10 ml500 mM
Adjust to pH 8.0 if necessary with NaOH (aq), and adjust volume to 20 ml with deionized water.

Recipe 19: 1000× Ampicillin

Dissolve 1 g of ampicillin in 10 ml of deionized water. Store in 1 ml aliquots at −20°C.

Recipe 20: 1× LB Broth

Dissolve 20 g of LB powder in 1000 ml of deionized water. Autoclave and store at room temperature.

Recipe 21: 2× LB Broth

Dissolve 40 g of LB powder in 1000 ml of deionized water. Autoclave and store at room temperature.

Recipe 22: 0.5 M IPTG

Dissolve 2.38 g of IPTG in 20 ml of deionized water. Sterilize through a 0.22-μm filter and store at 4°C in 1 ml aliquots.

Recipe 23: Assay Buffer

Compound Amount Final concentration
NaCl23.2 g100 mM
Tris12.5 g20 mM
EDTA1.48 g1 mM
DTT306 mg1 mM
Dissolve in 3.5 l deionized water, adjust to pH 7.0 with NaOH (aq), adjust volume to 3.6 liters with deionized water, and add:
NP-40 0.4 ml0.01%
Glycerol400 ml10%

Protein-Binding Assay, High-Throughput Screen, and Dose-Response Analysis

Note: In addition to the following recipes, this procedure requires Assay Buffer (Recipe 23).

Recipe 24: Protein Cocktail

1 μM hTRβ LBD

1 μM T3

0.025 μM SRC2-2 labeled with fluorescein

Prepare in Assay Buffer (Recipe 23).

Note: The concentration of TR (1 μM) in the Protein Cocktail represents 2 × Kd. The solution of labeled SRC2-2 (25 nM in buffer) should give a 100-fold increased fluorescence intensity signal compared to straight buffer. Because fluorescent molecules can degrade after several freeze-thaws and exposure to light, we advise checking the fluorescence intensity of the solution before starting the experiment.

Recipe 25: Positive Control

Mix 1 μl of 10 mM compound 3 (10 μM) into 999 μl Protein Cocktail (Recipe 24). Prepare just before use.

Recipe 26: Negative Control

Mix 1 μl of 10 mM compound 11 (10 μM) into 999 μl Protein Cocktail (Recipe 24). Prepare just before use.

Glutathione-S-Transferase Pull-Down Assay

Note: In addition to the following recipes, this procedure requires 1 M Tris Buffer, pH 7.0 (Recipe 5), 3 M NaCl (Recipe 6), 100 mM DTT (Recipe 7), and 100 mM PMSF (Recipe 8).

Recipe 27: TST Buffer

Stock solutionVolumeFinal concentration
1 M Tris Buffer, pH 7.0 (Recipe 5)25 ml50 mM
3 M NaCl (Recipe 6)25 ml150 mM
Add deionized water to 400 ml, adjust to pH 7.5 if necessary with NaOH (aq), adjust volume to 500 ml with deionized water, and add 0.25 ml of Tween 20 for a final concentration of 0.05%.

Recipe 28: Protease Inhibitor Cocktail (1000×)

Dissolve protease inhibitor cocktail tablet in 1 ml of DMSO. Add 4 ml of deionized water to a final volume of 5 ml and store at −20°C in 1 ml fractions.

Recipe 29: 0.5 M Hepes Buffer

Dissolve 11.9 g of Hepes in 80 ml of deionized water, adjust to pH 7.9 with NaOH (aq), and adjust to final volume of 100 ml with deionized water.

Recipe 30: 3 M KCl

Dissolve 222 g of KCl in 1000 ml of deionized water.

Recipe 31: 1 M MgCl2

Dissolve 20.3 g of MgCl2 in 100 ml of deionized water.

Recipe 32: A-150

Stock solution Volume Final concentration
0.5 M Hepes Buffer (Recipe 29)20 ml20 mM
3 M KCl (Recipe 30)25 ml150 mM
1 M MgCl2 (Recipe 31)5 ml10 mM
Glycerol5 ml1%
Add deionized water to 400 ml, adjust to pH 8.0 with NaOH (aq), and adjust volume to 500 ml with deionized water.

Recipe 33: IPAB Buffer

Stock solution Volume Final concentration
100 mM DTT (Recipe 7)0.2 ml0.2 mM
100 mM PMSF (Recipe 8)0.1 ml0.1 mM
Protease Inhibitor Cocktail (Recipe 28)0.1 ml
Adjust to 100 ml with A-150 (Recipe 32).

Recipe 34: PBB

Stock solution Volume Final concentration
1% Triton-X in PBS2 ml
1% NP-40 in PBS2 ml
100 mM DTT (Recipe 7)0.25 ml1 mM
100 mM PMSF (Recipe 8)0.12 ml0.5 mM
Protease Inhibitor Cocktail (Recipe 28)25 μl
2 g/liter BSA0.24 ml0.016 g/l
Adjust to 25 ml with A-150 (Recipe 32).
Note: 25 ml is enough for two or three gels. Prepare immediately before use and store on ice.

Recipe 35: 1.5 mM T3

Dissolve 7.32 mg of T3 in 10 ml of DMSO and store at −20°C in 1 ml aliquots.

Recipe 36: Gel Fixer

60 ml acetic acid

100 ml MeOH

Adjust to 500 ml with deionized water.

Hormone Displacement Assay

Note: In addition to the following recipes, this procedure requires 100 mM PMSF (Recipe 8).

Recipe 37: 1 mM T3

Dissolve 9.76 mg of T3 in 20 ml of DMSO; store at −20°C.

Recipe 38: Hormone Assay Buffer

Compound Amount Final concentration
KCl29.8 g400 mM
Sodium phosphate (dibasic)5.36 g20 mM
EDTA186 mg0.5 mM
MgCl2203 mg1 mM
Dissolve in 700 ml of deionized water, adjust to pH 8.0 with NaOH (aq), adjust volume to 900 ml with deionized water, and add 100 ml of glycerol for a final concentration of 10%.

Recipe 39: 5 mg/ml Histones

Dissolve 5 mg of histones in 1 ml of deionized water. Prepare just before use.

Recipe 40: 50% MTG

Dissolve 1 g of MTG in 1 ml of deionized water. Prepare just before use.

Recipe 41: Hormone Assay Cocktail

Stock solution Volume Final concentration
100 mM PMSF (Recipe 8)0.75 μl1 mM
5 mg/ml Histones (Recipe 39)7.5 μl0.05 mg/ml
50% MTG (Recipe 40)1.5 μl0.1%
TNT reaction (about 25 fM protein)120 μl
125I-T334 μl
Adjust to 750 μl with Hormone Assay Buffer (Recipe 38).
Note: The volume of 125I-T3 required will change with time. Upon delivery, the concentration is ~145 nM, but it will decay over time. Therefore, calculate the required amount based on the formula

$$mathtex$$\[^{125}I\ to\ add\ =\ starting\ concentration\ {\times}\ 1/2^{(days\ since\ initial\ concentration\ measured/60\ days)}\]$$mathtex$$

Transcription Assay

Recipe 42: Growth Medium

500 ml DMEM/H21

50 ml newborn calf serum, heat-inactivated (see note) (10%)

5 ml penicillin-streptomycin 100×

Note: To heat-inactivate newborn calf serum, thaw the serum slowly to 37°C and mix the contents of the bottle thoroughly. Place the bottle for 30 min in a water bath at 56°C and swirl every 10 min. Cool immediately in an ice bath and store at 4°C.

Recipe 43: Assay Medium

1 package (for 1 liter) DMEM/Ham’s F12 powder

1.338 g NaHCO3 (16 mM)

Adjust to 1000 ml with deionized water. Filter-sterilize, then add:

100 ml newborn calf serum, heat-inactivated, hormone-depleted (see note) (10%)

10 ml penicillin-streptomycin 100×

Note: To deplete heat-inactivated newborn calf serum of hormones, treat 500 ml of heat-inactivated newborn calf serum for 2 hours with 5 g of dextran-coated charcoal at 25°C in the dark. Centrifuge for 10 min at 8000g. Filter-sterilize and store at 4°C.

Recipe 44: Electroporation Buffer

Combine 500 ml of PBS (without Mg2+ and Ca2+) with 5 ml of glucose 10% for a final concentration of 0.1%.

Recipe 45: 10 μM T3

Dissolve 10 μl of 1 mM T3 (Recipe 37) in 990 μl of DMSO.

Instructions

SRC2-2 Labeling

SRC2-2 is a peptide derived from the p160 family of nuclear receptor coactivators. The SRC2-2 peptide was utilized for screening for inhibitors of TR, because it had the tightest binding to TRβ (0.44 μM) of all the NR-box peptides investigated (31). Because the high-throughput screen is based on displacement of this coactivator from the purified NR (in this case TR LBD) using fluorescence polarization, the first step is to create a fluorescently tagged form of the coactivator (in this case SRC2-2) peptide.

1. Dissolve 5 mg of crude SRC2-2 peptide in 3 ml of DMF:PBS, pH 7.0 (1:1).

2. Dissolve 30 mg of 5-iodoacetamidofluorescein in 300 μl of DMF.

3. Add fluorescein solution to peptide solution and stir at room temperature for 2 hours in the dark.

4. Add 100 μl of ethanethiol to inactivate excess 5-iodoacetamidofluorescein, and continue stirring for 5 min.

5. Purify fluorescently labeled peptide by preparative RP-HPLC [gradient: 100% TFA in Water (Recipe 1) to 20% TFA in Water (Recipe 1) and 80% TFA in Acetonitrile (Recipe 2), linear gradient over 30 min; flow rate: 20 ml/min; total run time: 30 min; UV detection: 215 and 280 nm].

6. Check the purity of fractions by HPLC–ESMS [gradient: 100% TFA in Water (Recipe 1) to 20% TFA in Water (Recipe 1) and 80% TFA in MeOH (Recipe 3), linear gradient over 8 min; flow rate: 1 ml/min; run time: 10 min]. Analyze labeled peptide by photodiode array, total ion count, and expected mass (m/z).

7. Evaporate the pure fraction using the Genevac evaporator

Note: Lyophilization may be used as an alternative method to dehydrate the samples.

8. Dissolve each fraction in 1 ml of DMSO.

9. Dissolve 1 μl of the DMSO solution in 1 ml of 20 mM Tris Buffer, pH 9.0 (Recipe 4).

10. Measure absorption at 492 nm with spectrometer.

11. Calculate concentration using the extinction coefficient 78000 cm−1 M−1 and the formula below. Aλ = ε × c × d (ε = 78,000 cm−1 M−1, Aλ = measured absorbance, d = diameter of cell, M = mol/l)

Protein Expression and Purification of the Thyroid Receptor

To perform the high-throughput screen and subsequent validation assays, purified NR or NR LBD are required. In this case, the following procedure, which is based on purification of histidine-tagged TR peptides using nickel columns or beads, can be used to purify the hTRα LBD, hTRβ LBD, or unliganded TR LBD. To produce unliganded protein, sonicate in Sonication Buffer (Recipe 13) lacking T3, wash in Talon Buffers (Recipes 15, 16, and 17) lacking T3, and reduce imidazole concentration of Talon Buffer 4 (Recipe 18) from 500 mM to 100 mM. We have also used the same procedure for purifying an hTRβ LBD C309A mutant (His6; residues T209 to D461) expressed in E. coli BL21 cells. However, to express and purify this protein, the bacteria must be grown at 18° to 20°C, instead of 22°C during the induction period.

Note: Purification of the TR protein in the presence of T3 increased the yield dramatically, and in protein binding assays, we obtained more consistent Kd values. In the presence of T3, TR remained in solution at concentrations up to 145 μM. Several freeze-thaw cycles (up to five) did not alter the activity of the protein.

Transformation from purified super-coiled plasmid DNA

1. Dissolve 35 g of LB agar in 1000 ml of deionized water.

2. Autoclave and allow to cool to 37°C, then add 1 ml of 1000 × Ampicillin (Recipe 19) and pour into sterile petri dishes until dish surface is completely covered. Leave plates at room temperature for 24 to 48 hours to dry.

3. Thaw E. coli BL21(DE3) strain and DNA stock, hTRβ LBD (His6 T209-D461) and hTRα LBD (His6 E148-V410), on ice.

4. Once thawed, add 1 μl of DNA (20 to 40 ng/ml) to each tube of competent cells and spin briefly to mix.

5. Incubate 30 min on ice.

6. Heat shock at 42°C for 45 s and then incubate 2 min on ice.

7. Add 950 μl of 1 × LB Broth (Recipe 20) to each tube and shake tubes at 37°C for 1 hour.

8. Label plates with cell type, protein, volume plated, and date and add 100 to 200 μl on each plate and spread with glass beads (add 20 to 50 beads and spread by moving the plate).

9. Discard beads and incubate plates at 37°C overnight.

Preculture and culture preparation

1. Pick a single colony from each of the transformation plates and add to 50 ml of 1× LB Broth (Recipe 20) with 1 × Ampicillin diluted from 1000 × Ampicillin (Recipe 19).

2. Grow culture at 37°C for 4 to 6 hours while shaking.

3. Measure the optical density (OD) at absorbance 600 (A600) of 100 μl of precultured bacteria diluted with 900 μl of 1 × LB Broth (Recipe 20).

4. Calculate the volume of the preculture to add to the LB in Fernback flasks using the following formula:

$$mathtex$$\[1.5\ /\ (OD\ {\times}\ 10)\ =\ Volume\ (liters)\ of\ preculture\ to\ inoculate\ in\ 1\ liter\]$$mathtex$$

5. Add the appropriate amount of preculture cells to 1 liter of 2 × LB Broth (Recipe 21) in each Fernback flask and grow at 22°C for 14 to 16 hours (overnight) on an orbital shaker.

Induction

1. Starting at 13 hours, measure the OD (A600) of 100 μl samples diluted with 900 μl of 1 × LB (Recipe 20).

2. For bacteria expressing hTRβ LBD, when OD (A600) reaches 0.6, induce protein expression using 1 ml of 0.5 M IPTG (Recipe 22) (final concentration 500 μM) for each liter and incubate at 22°C for about 4 hours on an orbital shaker. For bacteria expressing hTRα LBD, induce expression with IPTG when the OD (A600) reaches 1.2.

3. Transfer culture to 1-liter centrifugation bottles and spin at 8000g for 20 min.

4. Decant supernatant and transfer into 50-ml conical tubes.

Note: Supernatant should be clear. If it is cloudy, the cells may have lysed or died. In that case, discard culture and start over again.

5. Flash-freeze bacteria in liquid nitrogen and store at −80°C.

Protein purification

1. Combine the stored cell pellets and resuspend in 20 ml of Sonication Buffer (Recipe 13) with 20 mg lysozyme per liter of original cell culture.

2. Sonicate on ice using until the suspension is no longer "gooey," using the following parameters:

Duty cycle: 70; timer 12 min (3 min intervals); output control: 6

3. Centrifuge 1 hour at 4°C at 100,000g.

4. To a conical tube (50 ml) add Talon resin (0.5 to 0.75 ml per liter cell culture) and wash the resin two times with 15 ml of Talon Buffer 1 (Recipe 15).

Note: All buffers for the purification are stored on ice. The wash and elution steps were carried out by resuspending the Talon resin in the conical tube, centrifuging it for 5 min at 4°C at 50g, and decanting the supernatant. It is very important that the resin remain in the bottom of the tube.

5. Decant Talon Buffer 1 (Recipe 15) and add protein supernatant to Talon resin (40 ml of supernatant for each conical tube) and rotate gently for at least 1 hour at 4°C using the rotisserie shaker.

6. Pellet the resin by centrifuging for 5 min.

7. Wash the resin three times with 15 ml of Talon Buffer 2 (Recipe 16).

8. Wash the resin with 15 ml of Talon Buffer 3 (Recipe 17).

9. Elute with 5 × 3 ml of Talon Buffer 4 (Recipe 18).

10. Analyze samples of the eluted protein by SDS-PAGE using standard methods.

11. Pool fractions containing protein and dialyze overnight against 4 liters of Assay Buffer (Recipe 23).

12. Measured the protein concentration using a BCA assay (usually around 50 to 100 μM).

13. Measure protein functionality by a direct binding assay or store at −80°C in 1-ml aliquots.

Protein-Binding Assay, High-Throughput Screen, and Dose-Response Analysis

The fitted binding curve for the binding assay not only provides the estimated Kd, but also the expected saturation at high and low concentrations of protein. If no saturation is detectable at higher concentrations, this indicates that protein is nonfunctional. We obtained the following binding constants (Kd): hTRβ, Kd = 0.44 μM; hTRα LBD, Kd = 0.17 μM; hTRβ LBD (C309A), Kd = 0.17 μM.

Protein-binding assay

Note: This assay is conducted in quadruplicate.

1. Concentrate protein to 50 μM, using an Amicon centrifugal filter unit if necessary.

2. To 100 μl of concentrated protein, add 1 μl of 10 mM T3 (Recipe 9).

3. Transfer 50 μl of this mixture into each of well A1 and A2 of a 96-well opaque plate.

4. Add 50 μl of Assay Buffer (Recipe 23) to wells A2 through A12.

5. Create serial dilutions in wells A2 through A12 by sequentially transferring 50 μl (50 μl from A2 into A3, 50 μl from A3 into A4, … 50 μl from A11 into A12).

6. Prepare 800 μl of a 20 nM solution of the labeled SRC2-2 in Assay Buffer (Recipe 23).

7. Add 50 μl of SRC2-2 (20 nM) into wells B1 through B12.

8. Transfer 10 μl of each well of row A (96-well plate) into each well of rows A and B of a black 384-well plate with a 12-channel pipette.

9. Transfer 10 μl of each well of row B (96-well plate) into each well of rows A and B of a black 384 well plate with a 12-channel pipette.

Note: Total volume of 384 wells is 20 μl. Visually inspect each well for air bubbles. Careful pipetting and spinning the assay plate for 5 min at 400g can improve the standard deviation. Sometimes, poking the well with a 10-μl pipette tip removes air bubbles.

10. Equilibrate for 30 min.

11. Measure binding using fluorescence polarization (excitation λ 485 nm, emission λ 530 nm) with plate reader.

Note: Fluorescence polarization may be read up to 8 hours after assay without significant lost of reproducibility.

12. Analyze data using SigmaPlot 8.0 and obtain the Kd value by fitting the data to the following equation:

$$mathtex$$\[\mathit{y}\ =\ min\ +\ (max\ {-}\ min)/1\ +\ (\mathit{x}/\mathit{K}d)\ {\times}\ Hill\ slope\]$$mathtex$$

High-throughput screen

A library comprising 138,000 compounds (ChemRX, 28K; ChemDiv, 53K; ChemBridge, 24K; SPECS, 31K; Microsource, 2K) was screened in 384-well format in a single point format. The complete composition of this library is available from the BASC Web site (www.ucsf.edu/basc). Compounds are screened for their ability to compete for coactivator binding to the TR in the presence of the T3 ligand.

Note: The protocol for the liquid handling using the Multimek should be carefully developed before running the screen. Mixing by trituration (repeated aspirating and dispensing) can create air bubbles, especially when using air gaps in the automation protocol. Sufficient mixing should be investigated using a colored compound, such as fluorescein.

1. Add 34 μl of Assay Buffer (Recipe 23) containing 5.9% DMSO to each well, except columns 1, 2, 11 and 12, of an opaque 384-well plate using a WellMate.

2. Add 6 μl of compound solutions (dissolved in 1 mM DMSO) using a Multimek equipped with a 96-channel head, and mix by trituration. (The concentration of compounds in these dilution plates is 150 μM.)

3. Transfer 6 μl from the dilution plates into 384-well black plates using a Multimek.

4. Add 24 μl of Protein Cocktail (Recipe 24) using a WellMate. The final concentration of compound is 30 μM with DMSO content of about 4%.

Note: Visually inspect each well for air bubbles. Careful pipetting and spinning the assay plate for 5 min at 400g can improve the standard deviation. Sometimes, poking the well with a 10-μl pipette tip removes air bubbles.

5. Add 30 μl of Positive Control (Recipe 25) to wells A1, A2, B1, and B2 of each plate using a micropipettor (by hand).

6. Add 30 μl of Negative Control (Recipe 26) to wells C1, C2, D1, and D2 of each plate using a micropipettor (by hand).

7. Incubate for 2 hours at room temperature.

8. Measure fluorescence polarization (excitation λ 485 nm, emission λ 530 nm) and fluorescence intensity with a plate reader.

9. Analyze data using "Assay Explorer."

Note: The data may also be analyzed using ActivityBase (idbs, Guildford, Surrey, UK).

Dose-response analysis

Note: This assay is conducted in quadruplicate.

1. Using a 96-well plate, prepare a serial dilution of compound (1000 to 0.48 μM in DMSO) in wells A1 through A12.

2. Add 90 μl of Assay Buffer (Recipe 23) to wells B1 through B12.

3. Transfer 10 μl of row A to row B and mix by trituration.

4. Add 50 μl of Protein Cocktail (Recipe 24) to wells C1 through C12.

5. Transfer 10 μl of every well of row B (96 well plate) into each well of rows A and B (black 384 well plate) with a 12-channel pipette.

6. Transfer 10 μl of each well of row C (96 well plate) into each well of rows A and B (black 384 well plate) with a 12-channel pipette.

Note: Total volume of 384 wells is 20 μl. Visually inspect each well for air bubbles. Careful pipetting and spinning the assay plate for 5 min at 400g can improve the standard deviation. Sometimes, poking the well with a 10-μl pipette tip removes air bubbles.

7. Add 20 μl of Positive Control (Recipe 25) to wells C1 through C4.

8. Add 20 μl of Negative Control (Recipe 26) to wells C5 through C8.

9. Incubate for 3 hours at room temperature.

10. Measure inhibition using fluorescence polarization (excitation λ 485 nm, emission λ 530 nm) with an Analyst AD plate reader.

11. Analyze data using SigmaPlot 8.0 and obtain the Kd value by fitting the data to the following equation:

$$mathtex$$\[\mathit{y}\ =\ min\ +\ (max\ {-}\ min)/1\ +\ (\mathit{x}/\mathit{K}d)\ {\times}\ Hill\ slope\]$$mathtex$$

The final compound concentrations are 50 to 0.024 μM.

Hit Validation by Glutathion-S-Transferase Pull-Down Assay

The GST pull-down assay fulfills two functions: (i) It represents a secondary assay to validate those hit compounds that were positive in the high-throughput screen for their ability to bind to the full-length TR; and (ii) it determines whether the small molecules can inhibit the interaction between full-length TR and full-length coactivator protein. Therefore, GST-TR protein was expressed in E. coli using the steps up to the preparation of the bacterial pellet described in "Protein expression and purification of the thyroid receptor," above. Radiolabeled SRC2 full-length protein was obtained by using a TNT T7 quick coupled transcription/translation system.

Transformation and preparation of bacterial pellet

1. Transform bacteria with the plasmid DNA that includes the full GST-TRβ construct and prepare a bacterial culture.

2. Resuspend bacterial pellet on ice in TST Buffer (Recipe 27) (20 ml per liter of bacterial culture) and add 0.2 ml of 100 mM DTT (Recipe 7), 0.2 ml of 100 mM PMSF (Recipe 8), and 0.02 ml of Protease Inhibitor Cocktail (Recipe 28).

3. Store pellet at −80°C.

Protein purification

1. Thaw pellet and add 10 mg of lysozyme per liter of bacteria culture.

2. Incubate on ice for 15 min.

3. Sonicate using the following parameters:

Duty cycle: 70, timer 12 min (3 min intervals), output control: 6

4. Reserve a 10-μl aliquot to verify protein weight by SDS-PAGE.

5. Centrifuge twice for 30 min each at 100,000g at 4°C, decanting supernatant between spins.

6. Prepare Glutathione Sepharose 4B beads (0.5 ml/liter of culture) by washing it three times with 10 ml of TST Buffer (Recipe 27).

Note: All buffers are stored on ice.

7. Incubate the supernatant with beads and shake for 1 hour at 4°C using the rotisserie shaker.

8. Dispense beads and supernatant into a Poly-Prep column and wash twice with 5 ml of PBS containing 0.1% NP-40.

Note: Liquid will flow slowly through the column.

9. Suspend the beads with 0.6 ml of IPAB Buffer (Recipe 33) in the Poly-Prep column and transfer into microcentrifuge tubes.

10. Add glycerol to the bead slurry to a final concentration of 20% and shake to mix. Total volume should be 1.6 ml for each liter of culture.

11. Take an aliquot of the suspension and measure protein concentration using BCA method.

12. Snap-freeze with liquid nitrogen and store at −20°C.

GST pull-down assay

Note: This part of the procedure should be performed in a cold room.

1. Add 127.5 μl of PBB (Recipe 34) to each of eight microcentrifuge tubes.

2. Add 2 μl of DMSO to tube 1 (Control 1).

3. Add 1 μl of DMSO and 1 μl of 1.5 mM T3 (Recipe 35) (10 μM final concentration) to tube 2 (Control 2).

4. Prepare a serial dilution of hit compound (50 to 0.39 μM) in DMSO (final concentrations are 7.5 to 0.075 μM).

5. Add 1 μl of 1.5 mM T3 (Recipe 35) and 1 μl of hit compound of each concentration to tubes 3 through 8.

6. Add 10 μl of sepharose bead slurry corresponding to 3 μg of GST-TRβ fusion protein to tubes 2 through 8.

7. Perform in vitro translation reaction using 0.5μg SRC2 plasmid in the presence of 35S-methionine according to manufacturer's instructions.

Note: From this point on, precautions for handling radioactivity must be taken.

8. Dilute 50 μl of in vitro translation reaction with 450 μl of PBB (Recipe 34).

9. Add 10 μl of diluted translation reaction to each tube.

10. Rock for 90 min at 4°C.

11. Spin in a microcentrifuge at 13,000 rpm for 10 to 15 s.

12. Remove 120 μl of supernatant with a pipette and discard supernatant properly as radioactive waste.

Note: Do not try to remove all of the supernatant; you may dislodge and remove the beads.

13. Add 200 μl of IPAB Buffer (Recipe 33) to the beads.

14. Mix by inversion and microcentrifuge for 10 to 15 s.

15. Remove 200 μl of supernatant with a pipette and discard as radioactive waste.

16. Wash three more times with 200 μl of IPAB Buffer (Recipe 33), pelleting by pulsing in a microcentrifuge and removing supernatant carefully with a pipette.

17. After the last wash, remove as much supernatant as possible without disturbing the beads.

Note: You may wish to use a narrow-tip pipette tip to avoid removing the beads.

18. Dry the beads in a vacuum centrifuge (such as a CentriVap) for 5 to 10 min at "high."

19. Add 20 μl of NuPAGE loading buffer and denature at 70°C for 10 min.

Note: Take care to seal tubes or use cap locks to prevent caps from popping off during denaturation.

20. Microcentrifuge at maximum speed for 3 min and remove 15 μl to analyze by SDS-PAGE (NuPAGE).

21. For input lane use 1 μl of diluted translation reaction diluted with 22 μl of NuPAGE loading buffer.

22. Run gel at 200 V.

23. Incubate the gel for 15 min in Gel Fixer (Recipe 36).

24. Scan gel with PhosphorImager.

Note: Typhoon (GE) would be an alternative instrument.

Hormone Displacement Assay

This assay is used to validate that the hit compounds do not compete with T3 for binding to the TR. This assay is conducted in triplicate for T3 and hit compounds.

1. Produce full-length hTRβ using a TNT T7 quick-coupled transcription translation system according to the manufacturer's instructions.

2. In wells A1 through A5 of a 96-well plate, prepare a 1:10 dilution (1000 to 0.1 μΜ) of T3 starting with 1 mM T3 (Recipe 37) in DMSO.

3. In wells B1 through B5, prepare a 1:10 dilution (1000 to 0.1 μΜ) of hit compound in DMSO.

4. Add 50 μl Hormone Assay Buffer (Recipe 38) to the wells of rows C through H, columns 1 through 5.

5. Transfer 1 μl from each well of row A to the corresponding wells in rows C, D, and E and mix by trituration (A1 to C1, D1, E1; A2 to C2, D2, E2, …and A5 to C5, D5, E5).

6. Transfer 1 μl from each well of row B to the corresponding wells in rows F, G, and H and mix by trituration (B1 to F1, G1, H1; B2 to F2, G2, H2, …and B5 to F5, G5, H5).

7. Add 50 μl of Hormone Assay Cocktail (Recipe 41) to rows C through H and incubate 3 hours at room temperature with gentle shaking.

Note: The Hormone Assay Cocktail contains 125I-T3, so take precautions appropriate to the use of gamma-emitting radioisotopes. Use lead shielding and proper disposal methods.

8. Prepare 30 Poly-Prep columns by placing them in a column rack above the trough.

9. Gently stir 2 g of G25 Sephadex in 40 ml of Hormone Assay Buffer (Recipe 38) for 10 min.

10. Add 5 ml of the G25 Sephadex suspension to each column to obtain a 2-ml bed volume.

11. Store columns with a slight excess of Hormone Assay Buffer (Recipe 38).

12. Apply one reaction mixture to the top of each column and, using 3 × 500 μl of Hormone Assay Buffer (Recipe 38), elute directly into 7 ml scintillation vials.

13. Add 4 ml of microscintillation fluid to each vial.

14. Read vials in scintillation counter.

15. Analyze data using SigmaPlot 8.0 and obtain the Kd value by fitting the data to the following equation:

$$mathtex$$\[\mathit{y}\ =\ min\ +\ (max\ {-}\ min)/1\ +\ (\mathit{x}/\mathit{K}\mathrm{d})\ {\times}\ Hill\ slope\]$$mathtex$$

Note: Normalize the calculated Kd for the compound by assuming a Kd of 0.081 nM for T3, which takes into account the variation of the actual concentration of radiolabeled thyroid hormone.

Transcription Assay

This assay tests whether the hit compounds can penetrate the cell membrane and inhibit TR activity in a cellular context. This procedure uses the human bone osteosarcoma epithelial cell line U2OS, which are transfected with a plasmid encoding TRβ and a reporter plasmid containing a synthetic TR response element (DR-4). DR-4 contains two copies of a direct repeat spaced by four nucleotides (AGGTCA-cagg-AGGTCA), and it is cloned immediately upstream of a minimal thymidine kinase promoter that is linked to the luciferase coding sequence. Cells should be grown in a 37°C, 5% CO2 humidified incubator and handled using sterile technique in a laminar flow hood. The assay is conducted in triplicate.

1. Thaw 1 ml of U2OS starter culture at room temperature.

2. Add thawed cells to a 150-mm cell culture dish containing 25 ml of Growth Medium (Recipe 42).

3. Grow cells to a confluence of no more that 80% (usually within 2 days).

4. Remove growth medium and wash cells with 10 ml of PBS without Ca2+ and Mg2+, prewarmed to 37°C.

5. Add 5 ml trypsin solution and incubate for 3 to 5 min at 37°C, 5% CO2.

6. Add detached cells to 5 ml of Assay Medium (Recipe 43) and count the cells using a hemocytometer.

7. Pellet the cells by centrifugation (800g) and remove medium.

8. Resuspend the cells in appropriate amount of Electroporation Buffer (Recipe 44) to achieve 2 × 106 cells per 0.5 ml.

9. Add 1.5 μl of the TRβ-CMV plasmid (2.5 μg), 0.5 μl of the pRL-CMV plasmid (2.5 μg), and 5 μl of the DR-4 reporter plasmid (5 μg) into all microcentrifuge tubes, and add 0.5 ml of the buffer containing U2OS cells (step 8, above).

10. Mix gently and add to electroporation cuvettes.

11. Electroporate at a potential of 0.25 kV and capacity of 960 mF.

12. Thoroughly mix cells within the cuvette for 2 min.

Note: Thorough mixing is essential to ensure adequate and even resuspension of the cell pellet. Uneven resuspension can produce a low signal-to-noise ratio during luminance detection.

13. Transfer cells into conical tube and dilute with Assay Medium (Recipe 43) to a concentration of 200,000 cells/ml.

14. Add 100 μl of cell suspension to each well of a 96-well cell culture plate and incubate for 4 to 6 hours at 37°C, 5% CO2.

15. In wells A1 through A12 of a 96 opaque well plate, prepare a serial dilution (10 μl) of hit compound (500 to 0.24 μM) in 10 μM T3 (Recipe 45) solution.

16. Use wells B1 through B6 for controls: to wells B1 and B2, add 10 μl of 10 μM T3; to wells B3 and B4, add 10 μl of DMSO; and to wells B5 and B6, add 10 μl of hit compound (500 μM).

17. Using a 12-channel pipette, transfer 1 μl from each well of row A (96 opaque well plate) to each well of rows A, B, and C of the cell culture plate.

18. Transfer 1 μl from each well of B1 through B6 (96 opaque well plate) to the wells of row D of the cell culture plate.

19. Incubate for 16 hours at 37°C, 5% CO2.

20. Remove the medium from the cell culture plates and wash the cells with 100 μl of PBS without Ca2+ and Mg2+.

21. Add 20 μl of lysis buffer provided with Dual Luciferase Assay kit to each well and rock culture plate for 15 min at room temperature.

22. Add 100 μl of the kit’s Luciferase reagent and read luminance with plate reader

23. Add 100 μl of the kit’s Stop & Glo reagent and read luminance again.

24. Analyze data using SigmaPlot 8.0 and normalize to basal expression (treatment with equal amounts of DMSO, but no T3) and fully induced expression (treatment with equal amounts of DMSO and T3).

Related Techniques

There are two related high-throughput screening techniques. (i) Alfascreen is a heterogeneous assay that relies on hydrogel-coated donor and acceptor beads that are conjugated to interacting proteins. The beads come in close proximity when binding occurs and are excited by a laser, producing singlet oxygen that migrates from the donor bead to react with chemiluminescers on the acceptor bead. The chemiluminescers then activate fluorophores, emitting light at 520 to 620 nm. (ii) Förster resonance energy transfer (FRET) measures the energy transfer between two interacting fluorescently labeled proteins.

The fluorescence polarization assay has the advantage that it is a cost-efficient, reliable, and robust process that is broadly applicable to many protein-protein interactions. The secondary assays described herein are only a few examples of possible alternative assays that can be used to validate hit compounds.

Notes and Remarks

The described high-throughput screening method can be used to discover small molecules capable of inhibiting the interactions of nuclear receptors and their coactivators. The goal is to develop drugs for hormone-dependent diseases and research tools to investigate the functional changes in signaling by the targeted receptor and overall changes in transcriptional regulation in the cellular environment.

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
View Abstract

Stay Connected to Science Signaling

Navigate This Article