A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Lanchao Sun; Chuanhai Li

doi:10.3791/67327

JoVE Journal > Environment

Environment

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Published: September 20, 2024

doi:

10.3791/67327

Lanchao Sun, Chuanhai Li

¹School of Public Health,Qingdao University

Summary

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Abstract

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPARγ) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

Introduction

A large number of chemicals have been widely detected in the environment and human bodies, raising significant concerns about their impact on the ecological environment and human health¹^,²^,³. Despite their high environmental occurrence, information regarding their toxicological effects is scarce. Therefore, it is urgent to develop high-throughput screening (HTS) methods to facilitate the assessment of chemical toxicity.

Several high-throughput screening (HTS) methods have been reported for chemical toxicity assessment, such as the HTS bioassays used in the Tox21 and ToxCast programs⁴^,⁵. These methods can rapidly identify potential toxicants and provide valuable information on the mechanisms of chemical toxicity. However, these HTS bioassays mainly rely on cell-based systems, which can be complex and expensive. Additionally, high-throughput sequencing methods have also been used for chemical toxicity assessment, but achieving high-throughput evaluation of chemicals remains challenging⁶. Previous studies have developed fluorescence polarization (FP)-based receptor-ligand competitive binding assays to determine the binding potency of several environmental pollutants, including per- and polyfluoroalkyl substances (PFAS)⁷^,⁸^,⁹, bisphenol A (BPA)¹⁰^,¹¹, and particulate matter (PM)¹², with nuclear receptors such as peroxisome proliferator-activated receptor (PPAR)⁷^,⁸^,⁹^,¹⁰^,¹³, farnesoid X receptor (FXR)¹¹^,¹², and thyroid receptor (TR)¹⁴^,¹⁵. This approach is efficient, cost-effective, and provides mechanistic insights.

In this study, the protocol for the receptor-ligand binding assay is described based on detecting the fluorescence polarization (FP) of a small fluorescent probe. The principle of the FP-based receptor-ligand binding assay is illustrated in Figure 1. When a small fluorescent molecule is excited by plane-polarized light, the emitted light becomes highly depolarized due to rapid molecular rotation. However, when the tracer binds to a larger receptor, its rotation is slowed. A high FP value is detected when the tracer is bound to the large receptor, whereas a low FP value is observed when the tracer is free. Peroxisome proliferator-activated receptor gamma (PPARγ) was purified for the binding of the probe to the receptor. Rosiglitazone (Rosi), perfluorooctanesulfonic acid (PFOS), and triphenyl phosphate (TPHP) were used to compete for the binding of the probe with the receptor. Rosi, a specific agonist of PPARγ, was used as a positive control in the receptor competitive binding assays. Additionally, PFOS and TPHP have been previously identified as weak agonists of PPARγ in past studies⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷. Furthermore, they belong to different structural categories of compounds known for environmental exposure and are notable for their relatively high detection rates in human populations. These compounds were used to further validate the broad applicability of the competition binding assay. The procedure consists of four steps: construction and transformation of recombinant vectors, expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor.

Protocol

The details of the reagents and the equipment are listed in the Table of Materials.

1. Construction and transformation of recombinant vectors

NOTE: PPARγ is a ligand-dependent transcription factor with a classical nuclear receptor structure, comprising a DNA-binding domain that regulates target genes and a ligand-binding domain activated by ligands. Upon ligand activation, PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), and binds to response elements of PPARγ, thereby regulating the transcription of downstream target genes⁹^,¹⁶.

Design primers for the PPARγ-LBD (see Table 1) and amplify the PPARγ-LBD DNA segment (see Table 2 and Table 3).
Linearize the His×6-tagged pET28a vector by digesting it with restriction endonucleases XhoI and BamHI¹⁸.
Clone the PPARγ-LBD DNA segment into the His×6-tagged pET28a vector using the commercially available cloning kit, resulting in the recombinant plasmid pET28a-PPARγ-LBD-6×His¹⁸.
Transfect the recombinant expression plasmid pET28a-PPARγ-LBD-6×His into BL21 (DE3) Escherichia coli cells for protein expression¹⁸.
Add 5 µL of the recombinant vector to competent BL21(DE3) cells, incubate on ice for 30 min, perform a heat shock at 42 °C for 45 s, then immediately return to the ice.
Add 900 µL of LB medium, shake at 37 °C for 1 h (160-200 rpm), then centrifuge at ~3000 x g for 5 min at room temperature.
Discard the supernatant, resuspend the bacterial pellet in 100 µL of LB medium, and spread onto a solid medium. Invert the plates and culture at 37 °C for 12-16 h.
Select individual colonies for sequencing identification and subsequent protein expression and purification.

2. Expression and purification of the receptor protein

Incubate the transformed BL21 (DE3) cells in 200 mL LB medium supplemented with 100 µg/mL ampicillin on an orbital shaker (230 rpm) for 1-2 h at 37 °C.
Induce the cells when the OD₆₀₀ reaches 0.4-0.6 absorbance units by adding 10 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubate at 16 °C for 16 h.
Collect the bacterial suspension and centrifuge at 8000 × g, 4 °C, for 10 min.
Lyse the cells in 20 mL soluble lysis buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 10 mM of imidazole, pH 8.0), adding 200 µL of phenylmethylsulfonyl fluoride (PMSF) and 200 µL of lysozyme. Resuspend the bacterial pellet using a 5 mL pipette, then proceed with sonication at 30% power for 20 min.
Add lysis buffer equal to 5 times the column volume to equilibrate the nickel column. Repeat this process twice and set aside.
Centrifuge the sonicated bacterial suspension at 8000 × g for 15 min at 4 °C to obtain the bacterial supernatant lysate (CL).
Load the CL onto the nickel column (for protein adsorption) to obtain the flow-through (FT).
Wash the column with 5 times the column volume of wash buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 20 mM of imidazole, pH 8.0) to obtain the wash eluate (W1-W6).
Wash the column with 1 mL of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0) to elute the target protein and obtain the protein fractions (E1-E5).
Take a 20 µL aliquot of each fraction (CL, FT, W1-W6, and E1-E5) and analyze using SDS-PAGE¹⁸ with Coomassie Brilliant Blue staining. PPARγ-LBD runs as a 34.9 kDa protein on a denaturing gel.

3. Receptor binding assay

NOTE: In this assay, C1-BODIPY-C12 was used as a site-specific fluorescent probe to establish the receptor-ligand binding system. C1-BODIPY-C12, a specific ligand for PPARγ, is a fluorescent analog of fatty acid with the BODIPY fluorescent group incorporated into the fatty acid at the C1 position.

Dilute the purified human PPARγ-LBD in Tris-HCl buffer (20 mM of Tris, 100 mM of NaCl, pH 8.0) to a concentration range of 1 nM to 6400 nM. Also, dilute the C1-BODIPY-C12 probe in Tris-HCl buffer to a concentration of 50 nM.
Mix the diluted PPARγ-LBD solution (55 µL per well) and the C1-BODIPY-C12 probe solution (55 µL per well) in a 96-well black plate. Incubate at room temperature for 5 min.
Measure fluorescence polarization (FP) with the microplate reader.
Plot the FP values against the receptor concentration, fit the curve using the specific binding with the Hill slope equation using statistical and graphing software, and calculate the K_d value.

4. Competitive binding assay

NOTE: In this assay, 800 nM of human PPARγ-LBD and 50 nM C1-BODIPY-C12 probe were used for receptor binding. Rosiglitazone (Rosi), triphenyl phosphate (TPHP), and perfluorooctanesulfonic acid (PFOS) were used to compete with the binding of the probe to PPARγ.

Dilute the three compounds in Tris-HCl buffer within a concentration range from 0-200 µM.
Prepare the receptor-probe binding solution with a final concentration of 800 nM of human PPARγ-LBD and 50 nM of C1-BODIPY-C12 probe.
Mix the receptor-probe binding solution (55 µL per well) and compound solution (55 µL per well) in a 96-well black plate. Incubate at room temperature for 5 min.
Measure fluorescence polarization (FP) with the microplate reader.
Plot the FP values as a function of the ligand concentration. Obtain the half-maximal inhibitory concentration (IC₅₀) of each ligand from the competition curve using the sigmoidal model processed by a graphing and analysis software.

Representative Results

Protein expression and purification of PPARγ-LBD
PPARγ-LBD was heterologously expressed in BL21 (DE3) as a histidine-tagged protein. The protein was detected in the soluble fractions, and the purified PPARγ-LBD showed a single band on SDS-PAGE with an apparent molecular weight of approximately 34.9 kDa (Figure 2), consistent with the predicted molecular weight of the protein.

The binding of C₁-BODIPY-C₁₂ probe to PPARγ-LBD
In the receptor binding assay, C1-BODIPY-C12, a BODIPY-labeled fatty acid that can bind to PPARγ-LBD, was used as a fluorescence probe to study the binding potency of environmental pollutants with hPPARγ-LBD. As shown in Figure 3A, the fluorescence polarization (FP) values increased from 20 to 250 upon the addition of PPARγ-LBD, indicating the binding of C1-BODIPY-C12 to the receptor. The binding curve reached saturation at 800 nM PPARγ-LBD, with a dissociation constant (K_d) of 253.5 nM ± 10.05 nM. Therefore, 800 nM PPARγ-LBD was chosen for the subsequent competitive binding assays.

The competitive binding of environmental pollutants to PPARγ-LBD
Rosiglitazone (Rosi), a specific agonist of PPARγ, was used as a positive control to displace the fluorescent probe in the competitive ligand binding assays. As shown in Figure 3B, Rosi inhibited the binding of the C1-BODIPY-C12 probe to PPARγ-LBD in a dose-dependent manner, with an IC₅₀ of 6.89 µM, demonstrating the feasibility of the assay. Next, the binding affinities of TPHP and PFOS to PPARγ-LBD were determined. As shown in Figure 3C,D, TPHP and PFOS also inhibited the binding of the C1-BODIPY-C12 probe to PPARγ-LBD in a dose-dependent manner, with IC₅₀ values of 60.45 µM and 37.27 µM, respectively.

Figure 1: Schematic illustration of fluorescence polarization (FP)-based receptor competitive binding assays. Please click here to view a larger version of this figure.

Figure 2: SDS-PAGE analysis of the purified PPARγ-LBD. M is the protein molecular weight marker. Cl is the cell lysate, FT is the flow-through fraction, W1-W6 are the wash solutions, E1 is the eluate, and E2-E4 are the purified PPARγ-LBD proteins. Please click here to view a larger version of this figure.

Figure 3: Fluorescence polarization (FP)-based receptor binding curve and competitive binding curves. (A) FP-based binding curve of C₁-BODIPY-C₁₂ to human PPARγ-LBD. (B–D) FP-based competitive binding curves of Rosi, TPHP, and PFOS to Human PPARγ-LBD. Error bars denote the standard deviation (SD) for three independent experiments. Please click here to view a larger version of this figure.

ID	seq
pET28a-Human PPARG-P1	CAAATGGGTCGCGGATCCATGATCGACCAGCTGAATCCAGAGTCC
pET28a-Human PPARG-P2	GTGGTGGTGGTGCTCGAGTCAGTACAAGTCCTTGTAGATCTC
PPARγ-LBD nucleotide sequence	AGACGACATTCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGAT ATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTG GTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAA TGGGTCGCGGATCCATGATCGACCAGCTGAATCCAGAGTCCGCTGAC CTCCGGGCCCTGGCAAAACATTTGTATGACTCATACATAAAGTCCTTCC CGCTGACCAAAGCAAAGGCGAGGGCGATCTTGACAGGAAAGACAACA GACAAATCACCATTCGTTATCTATGACATGAATTCCTTAATGATGGGAGA AGATAAAATCAAGTTCAAACACATCACCCCCCTGCAGGAGCAGAGCAA AGAGGTGGCCATCCGCATCTTTCAGGGCTGCCAGTTTCGCTCCGTGG AGGCTGTGCAGGAGATCACAGAGTATGCCAAAAGCATTCCTGGTTTTG TAAATCTTGACTTGAACGACCAAGTAACTCTCCTCAAATATGGAGTCCA CGAGATCATTTACACAATGCTGGCCTCCTTGATGAATAAAGATGGGGT TCTCATATCCGAGGGCCAAGGCTTCATGACAAGGGAGTTTCTAAAG AGCCTGCGAAAGCCTTTTGGTGACTTTATGGAGCCCAAGTTTGAGT TTGCTGTGAAGTTCAATGCACTGGAATTAGATGACAGCGACTTGGCAA TATTTATTGCTGTCATTATTCTCAGTGGAGACCGCCCAGGTTTGCTGAA TGTGAAGCCCATTGAAGACATTCAAGACAACCTGCTACAAGCCCTGG AGCTCCAGCTGAAGCTGAACCACCCTGAGTCCTCACAGCTGTTTG CCAAGCTGCTCCAGAAAATGACAGACCTCAGACAGATTGTCACGGA ACACGTGCAGCTACTGCAGGTGATCAAGAAGACGGAAACCGACATG AGTCTTCACCCGCTCCTGCAGGAGATCTACAAGGACTTGGACTGG CTCGAGGCCACCCACCC

Table 1: The primer and nucleotide sequence of PPARγ ligand-binding domain.

Experimental Reagents names	Volume/Dose
pET28a-Human PPARG-P1	1 μL
pET28a-Human PPARG-P2	1 μL
2×phanta Max Master Mix	12.5 μL
cDNA	2 μL
ddH₂O	8.5 μL

Table 2: PCR experimental reagent system.

Experiment names	Temperature	Time
Pre-denaturation	95 °C	3 min
Denaturation	95 °C	15 s
Annealing	65 °C	15 s
Extension	72 °C	6 min
Store	12 °C	—

Table 3: PCR experimental reaction program.

Discussion

Fluorescence polarization (FP), surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) are common techniques used for assessing direct binding interactions between proteins and compounds¹⁹^,²⁰. FP has been widely employed in the investigation of molecular interactions for drug discovery and chemical screening²¹^,²²^,²³. In comparison, SPR and NMR assays are expensive and time-consuming, making them less suitable for high-throughput screening (HTS) applications. This protocol describes a receptor-ligand analysis method based on FP with a multi-well plate detection system, enabling the HTS of chemicals.

Choosing the appropriate probe for a receptor binding assay is crucial. The probe should consist of two components: a specific ligand for the nuclear receptor and a fluorescent group. Typically, such probes can be commercially obtained, as exemplified by the C1-BODIPY-C12 probe used in this study. C1-BODIPY-C12 is a fluorescent analog of fatty acid, with the BODIPY fluorescent group incorporated at the C1 position, and is a specific ligand for PPARγ. If a commercially available fluorescent probe is not an option, it should be chemically synthesized by attaching a fluorescent group to a known specific ligand.

The classic structure of a nuclear receptor includes a DNA-binding domain responsible for regulating target gene expression and a ligand-binding domain (LBD), typically located at the receptor's C-terminus²⁴. The coregulator binding site is generally found within the transcriptional activation function domain of the nuclear receptor, where it interacts with nuclear coregulatory factors²⁵. These coregulators can either enhance or suppress the receptor's transcriptional activity, thereby modulating gene expression. The LBD, located in a specific region of the receptor protein, regulates the receptor's conformational changes upon ligand binding, which in turn activates or inhibits its transcriptional activity. Since the LBD is a well-defined domain crucial for recognizing and binding specific small molecule ligands²⁴, only the receptor-LBD was used in the receptor competitive binding assay in this study. This approach, which involved expressing and purifying the ligand-binding domain of PPARγ, reduced assay costs.

The reagents used in this method, including buffers for protein purification, C1-BODIPY-C12 probe, and Tris-HCl, are commercially available and inexpensive, which is a significant advantage for the assay. Fluorescence polarization (FP) detection is performed in a multi-well plate (either 96-well or 384-well), with a rapid read time of 3-5 min per plate, enhancing testing throughput and efficiency. The receptor-ligand binding approach is highly flexible and can be easily adapted to various receptors, provided the receptor proteins are available. This adaptability broadens the scope of research that can be conducted using this method. Overall, the combination of these advantages-ease of use, cost efficiency, high-throughput capacity, rapid processing, and flexibility in receptor adaptation – makes this method a promising option for screening toxic environmental pollutants.

The present results demonstrate that PFOS and TPHP can bind to PPARγ, which is consistent with previous findings from molecular docking and reporter gene assays⁹^,²⁶. Additionally, the method described in this manuscript has been utilized to assess the binding potency of several environmental pollutants with nuclear receptors. For instance, using the FP-based receptor-ligand competitive binding assay, the binding potency of 19 per- and polyfluoroalkyl substances (PFASs) and 7 bisphenol A (BPA) compounds to peroxisome proliferator-activated receptor β/δ (PPARβ/δ)⁷^,¹⁰, 12 PFASs to PPARγ⁹^,¹³, 7 BPA compounds and 3 particulate matter (PM_2.5) components to farnesoid X receptor (FXR)¹¹^,¹², and 8 polybrominated diphenyl ethers (PBDEs) and 6 polychlorinated biphenyls (PCBs) to thyroid receptor (TR)¹⁴^,¹⁵ have been determined. These findings highlight the potential of this method for screening the binding interactions between environmental pollutants and nuclear receptors.

Previous studies have reported fluorescence polarization (FP) assay methods for PPARγ that involve using gel filtration to measure binding, which requires at least 1.5 h to detect the binding of a single compound²³. In contrast, this method allows the detection of binding affinities for at least 12 compounds with PPARγ within 10 min. Additionally, some commercially available assay kits (see Table of Materials) are priced at over $3000 for an 800 × 20 µL format. These methods are notably expensive and time-consuming. This study presents improvements over these existing methods.

One potential limitation of this method is that the fluorescence probe must be a ligand for the receptor, and fluorescence probes do not interact with environmental pollutants directly. Therefore, it is crucial to ensure that the probe and environmental pollutants are kept separate in the solution. Additionally, this assay reflects an in vitro situation and cannot fully capture in vivo interactions, as receptors are heterodimeric in vivo²⁷. Consequently, while this method serves as a rapid screening tool, further investigation of receptor activation and related toxicological mechanisms in vivo is necessary.

Another drawback is the "rightward shift" phenomenon observed in fluorescence polarization (FP) assays when evaluating binding affinity, which may lead to a systematic underestimation of the measured affinities. In previous studies, the binding affinity of rosiglitazone with PPARγ was assessed using the time-resolved fluorescence resonance energy transfer (TR-FRET) assay²⁸, which is a highly sensitive method for measuring ligand-receptor binding affinity. However, the TR-FRET assay is relatively time-consuming and cumbersome, requiring a 4 h incubation of the reaction solution at room temperature before detection. Although the FP assay exhibits lower sensitivity compared to the TR-FRET assay, it is more suitable for high-throughput screening of environmental ligands that bind to nuclear receptors. Nevertheless, the FP assay may miss some weak environmental ligands. Additionally, in previous studies, the binding affinity of PFOS with PPARγ was determined using equilibrium dialysis (EqD)²⁹, another highly sensitive method for measuring ligand-receptor binding affinity. However, EqD relies on expensive analytical equipment (LC-MS/MS), which limits its application and precludes high-throughput screening capabilities.

Although this fluorescence polarization (FP) assay exhibits a "right-shift" phenomenon, which may result in generally higher calculated IC₅₀ values, it does not affect the relative affinity ranking or subsequent risk assessment predictions. Furthermore, the FP assay is cost-effective, time-efficient, and capable of screening a wide range of compounds for their affinity towards multiple nuclear receptors. Overall, FP-based high-throughput screening of environmental ligands facilitates the rapid identification of toxic environmental pollutants.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 82103875).

Materials

C₁-BODIPY-C₁₂ Probe	Thermo Fisher Scientific, China	102209-82-3	Binds to PPARγ-LBD and emits fluorescence.
Coomassie Brilliant Blue R-250	Solarbio, China	6104-59-2	Stain the protein bands.
GraphPad prism	Dotmatics		https://www.graphpad.com/features
imidazole	Solarbio, China	I8090	Prepare buffers for the protein purification process.
Isopropyl β-D-1-thiogalactopyranoside	Solarbio, China	367-93-1	Induce the expression of PPARγ-LBD
Microplate reader	Biotek , USA	Synergy H1	Detecting FP value
NaCl	Shanghai Reagent	7647-15-5	Prepare buffers for the protein purification process.
NaH₂PO₄ · 2H₂O	Shanghai Reagent	13472-35-0	Prepare buffers for the protein purification process.
Ni NTA Beads 6FF	Smart-Lifesciences, China	SA005005	Protein purification.
Origin 8.5	OriginLab, Northampton, MA, U.S.A.
Perfluorooctanesulfonic acid (PFOS)	J&K Scientific Ltd, China	1763-23-1	The detected environmental pollutants
Phenylmethylsulfonyl fluoride (PMSF)	Solarbio, China	P0100	Inhibit protein degradation.
PPARγ-Competitor Assay Kit	Thermo Fisher Scientific	PV6136	https://www.thermofisher.com/order/catalog/product/PV6136
PPARγ-LBD Ligand Screening Assay Kit	Cayman	600616	https://www.caymanchem.com/product/600616
Rosiglitazone (Rosi)	aladdin, China	122320-73-4	The agonists of PPARγ
Shaker	ZHICHENG, China	ZWY-211C	Bacterial culture expansion and induction of protein expression
Triphenyl phosphate (TPHP)	Macklin, China	T819317	The detected environmental pollutants
Tris	Solarbio, China	T8230	Prepare buffers for the protein purification process.
Tryptone	OXOID Limited, China	LP0042B	Prepare Lysogeny Broth (LB) medium.
Ultrasonic Cleaner	Kimberly, China	LHO-1	Disrupt the bacteria to achieve complete lysis
Urea	Solarbio, China	U8020	Prepare buffers for the protein purification process.
Yeast extract	OXOID Limited, China	LP0021B	Prepare Lysogeny Broth (LB) medium.

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