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