Retinoic acid X receptor alpha activation effect molecule detection method based on fluorescence polarization technology

Abstract

The invention discloses a retinoic acid X receptor alpha activation effect molecule detection method based on a fluorescence polarization technology, and belongs to the technical field of biological analysis. According to the method, the change of a probe fluorescence polarization signal caused by the change of the binding state of the RXR alpha and the fluorescent nuclear receptor co-regulatory peptide is directly monitored, so that the rapid evaluation of the to-be-detected substance on the receptor activation effect is realized. According to the method, three core processes of background signal determination, to-be-detected substance signal detection and statistical analysis are completed on the molecular level by virtue of a conventional multifunctional microplate reader, so that the dependence on high-cost equipment or a complex cell model in the prior art is effectively avoided, and high efficiency, low cost and high flux of RXR alpha activator screening are realized. The method is suitable for early warning of toxicity of environmental compounds and efficient screening of RXRalpha targeted drugs, and a practical tool is provided for receptor function research and drug development.

Description

Technical Field

[0001] The present invention relates to a method for detecting retinoid X receptor alpha activation effect molecules based on fluorescence polarization technology, and belongs to the technical field of bioanalysis. Background Art

[0002] Retinoid X receptor α (RXRα) is a key member of the nuclear receptor superfamily. As a ligand-dependent transcription factor, it plays a crucial role in cellular development, metabolic regulation, and disease progression. In the absence of ligand binding, RXRα binds to co-repressors, maintaining the repression of gene transcription. Upon ligand binding, RXRα undergoes conformational changes, allowing it to bind to co-activators and promote transcriptional activation of target genes. RXRα not only independently regulates gene expression but also forms heterodimers with nuclear receptors such as retinoic acid receptors (RARs), thyroid hormone receptors (TRs), peroxisome proliferator-activated receptors (PPARs), and vitamin D receptors (VDRs), co-regulating these receptor-mediated gene expression. Due to its "universal chaperone" properties, RXRα occupies a central position in cellular signaling networks and has become an important drug target for a variety of diseases, including metabolic disorders, cancer, and neurodegenerative disorders. Due to its crucial regulatory role, screening for ligands targeting RXRα has become a key focus in drug development.

[0003] However, existing screening technologies face challenges such as low sensitivity, high cost, and strong equipment dependence. Therefore, the development of efficient and low-cost detection technology for RXRα activation effects will help promote its application in scientific research and clinical treatment.

[0004] Currently, detection technologies for RXRα activation effects are mainly based on the cellular and molecular levels, but both have significant limitations, which restrict the efficient screening of RXRα activators; cellular level detection technologies generally have problems of complex operation and indirect results. For example, luciferase reporter gene experiments require the construction of a receptor-reporter gene co-expression cell model, and go through multiple steps such as plasmid transfection, ligand stimulation, cell lysis and signal detection. The process is time-consuming and differences in transfection efficiency can easily lead to data fluctuations, affecting experimental reproducibility; another cellular level method, cell thermal shift assay (CETSA), is based on the principle of enhanced thermal stability of RXRα after ligand binding. It induces protein denaturation through temperature control and combines immunoblotting or mass spectrometry to quantify undegraded proteins; however, this method has low sensitivity and usually requires high concentrations of ligand to detect significant changes. At the same time, multi-step operations further limit detection throughput.

[0005] Although molecular-level detection technology can directly characterize the interaction between RXRα and ligands, it is difficult to apply on a large scale due to equipment dependence and cost issues. The fluorescence quenching method characterizes the activation effect by monitoring the changes in the spontaneous fluorescence of tryptophan residues in the RXRα ligand binding domain, but the fluorescence signal intensity is weak and the wavelength exceeds the detection range of conventional microplate readers, which limits its practicality. Differential scanning calorimetry (DSC) can provide fast and reliable single-shot measurement results by detecting changes in the thermal stability of the RXRα-ligand complex; however, this method requires multiple scans within a narrow temperature range to confirm the data, resulting in a longer overall detection cycle. At the same time, the high cost of equipment also limits its application in high-throughput screening; ultrafiltration mass spectrometry, as an important means of natural product screening, relies on ultrafiltration membranes to retain the RXRα-ligand complex and analyzes it through liquid chromatography-mass spectrometry (LC-MS). Although it is suitable for ligand screening, the multi-step tandem operation significantly reduces the detection efficiency; time-resolved fluorescence resonance energy transfer (TR-FRET) technology, such as LanthaScreen developed by Thermo Scientific, TM The TR-FRET RXRα detection kit can quantitatively measure the binding efficiency of coactivators to RXRα with high accuracy. However, this method relies on expensive specialized instruments and reagents, resulting in high experimental costs. Furthermore, common multi-functional microplate readers generally lack TR-FRET detection capabilities, further limiting its application.

[0006] The aforementioned methods each have advantages in specificity and sensitivity, but are generally limited by complex procedures, equipment dependence, insufficient throughput, or high costs. Therefore, developing a highly sensitive, low-equipment-dependant, high-throughput, and cost-effective detection method would facilitate efficient screening of novel RXRα ligands. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for detecting retinoid X receptor α activating effector molecules based on fluorescence polarization technology, wherein the method is specifically as follows:

[0008] (1) After the retinoic acid X receptor α solution and the fluorescent nuclear receptor coregulatory peptide solution are mixed and incubated, the fluorescence polarization signal is detected, which is the background system fluorescence polarization signal.

[0009] (2) The retinoic acid X receptor α solution, the fluorescent nuclear receptor coregulatory peptide solution and the solution of the substance to be tested are mixed and incubated, and then the fluorescence polarization signal is detected, which is the fluorescence polarization signal of the system containing the substance to be tested.

[0010] (3) The fluorescence polarization signal of the background system and the fluorescence polarization signal of the test substance system were analyzed by T-test for significant difference. If the p value was less than 0.05, it could be determined that the test substance had an activation effect on RXRα.

[0011] Preferably, in step (1) and step (2), a multifunctional microplate reader is used to detect the fluorescence polarization signal.

[0012] Preferably, in step (1), the volume ratio of the retinoic acid X receptor alpha solution to the fluorescent nuclear receptor coregulatory peptide solution is 1:(0.9-1.1), wherein the molar concentration of the retinoic acid X receptor alpha solution is 100-2000 nmol / L, and the concentration of the fluorescent nuclear receptor coregulatory peptide is 100-400 nmol / L.

[0013] Preferably, the incubation conditions in step (1) and step (2) are: incubation at room temperature for 5 to 30 minutes.

[0014] Preferably, in step (2), the volume ratio of the retinoic acid X receptor alpha solution, the fluorescent nuclear receptor coregulatory peptide solution, and the test substance solution is 1:(0.9-1.1):1, wherein the molar concentration of the retinoic acid X receptor alpha solution is 100-2000 nmol / L, the concentration of the fluorescent nuclear receptor coregulatory peptide is 100-400 nmol / L, and the concentration of the test substance solution is 2-20 μmol / L.

[0015] The fluorescent nuclear receptor coregulatory peptide was purchased from Thermo Scientific, with the catalog number PV4578.

[0016] The amino acid sequence of retinoid X receptor alpha described in the present invention is as shown in SEQ ID NO: 1. It may also be a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID NO: 1. It may also be a protein having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1.

[0017] Technical effects of the present invention:

[0018] (1) The method of the present invention only requires the use of RXRα (retinoic acid X receptor α) and fluorescent nuclear receptor coregulatory peptides, and the experimental cost is low.

[0019] (2) The method of the present invention performs signal detection based on the fluorescence polarization detection mode. Ordinary multifunctional microplate readers can be equipped with this function, making the method easy to promote and use.

[0020] (3) The method of the present invention can be used to perform detection on a 384-well plate using a multifunctional enzyme reader, thereby enabling high-throughput detection of multiple substances.

[0021] (4) The method of the present invention can detect the activation effect of a substance on RXRα at the molecular level and has the advantages of good specificity and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of Example 1 using fluorescent nuclear receptor coregulatory peptides to detect the activation effect of RXRα agonist LG100268 on RXRα.

[0023] Figure 2 This is the result of Example 2 using fluorescent nuclear receptor coregulatory peptide to detect the activation effect of 1,3,5-tri-tert-butylbenzene on RXRα. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0025] The present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] The experiments in the following examples were all repeated three times in parallel, and the results were averaged.

[0029] The composition of the 50 mmol / L potassium phosphate buffer in the following examples is: 50 mmol / L potassium phosphate, 150 mmol / L potassium chloride, 0.5 mmol / L EDTA, and the pH is 7.5.

[0030] Fluorescent nuclear receptor coregulator peptides were from Thermo Scientific (FL-SRC 1-2peptide coactivator probe, Catalog No. PV4578). The multifunctional microplate reader was a SpectraMax i3x from Molecular Devices, USA. The 384-well plate was from Corning (Catalog No. 4511).

[0031] The RXRα protein used in the examples was synthesized by Wuxi OriGene Biotechnology Co., Ltd. (i.e., OriGene China), and its sequence is shown in SEQ ID NO: 1.

[0032] Example 1

[0033] A method for detecting retinoic acid X receptor α activation effect molecules based on fluorescence polarization technology

[0034] In this example, LG100268 is a product of MedChemExpress (item number HY-15340), which is the substance to be detected in Example 1. The mother solution is prepared using dimethyl sulfoxide. LG100268 is an effective RXRα agonist and is a positive substance for the feasibility of the detection method.

[0035] The specific steps are as follows:

[0036] (1) Detection of background system fluorescence polarization signal: RXRα solution with a molar concentration of 800 nmol / L and fluorescent nuclear receptor coregulator peptide solution with a molar concentration of 200 nmol / L were prepared using 50 mmol / L potassium phosphate buffer. Then, 10 μL of the above RXRα protein solution and 10 μL of fluorescent nuclear receptor coregulator peptide solution and 10 μL of blank solvent were added to each well of a 384-well plate (the blank solvent in this experiment was potassium phosphate buffer solution containing 2% dimethyl sulfoxide). After incubation at room temperature for 5 minutes, the final concentration of RXRα was 400 nmol / L, and the final concentration of fluorescent nuclear receptor coregulator peptide was 100 nmol / L. Fluorescence polarization signal detection was performed using a SpectraMax i3x multi-function microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0037] (2) Detection of fluorescence polarization signals of the system containing the substance to be detected: RXRα solution with a molar concentration of 800 nmol / L and fluorescent nuclear receptor coregulator peptide solution with a molar concentration of 200 nmol / L were prepared using 50 mmol / L potassium phosphate buffer, and LG100268 solution with a molar concentration of 20 μmol / L (containing 2% dimethyl sulfoxide) was prepared using 50 mmol / L potassium phosphate buffer. Then, 10 μL of the above RXRα protein, 10 μL of fluorescent nuclear receptor coregulator peptide solution, and 10 μL of the above 20 μmol / L LG100268 solution were added to each well of a 384-well plate. After incubation at room temperature for 5 minutes, the final concentration of RXRα was 400 nmol / L, the final concentration of fluorescent nuclear receptor coregulator peptide was 100 nmol / L, and the concentration of LG100268 was 10 μmol / L. Fluorescence polarization signal detection was performed using a SpectraMax i3x multi-function microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0038] (3) Determine the activation effect of the test substance on RXRα: The fluorescence polarization signal of the background system and the fluorescence polarization signal of the test substance system are obtained by three parallel samples. The T-test is used to analyze the significant difference between the two sets of data, and the results are as follows: Figure 1 shown.

[0039] pass Figure 1It can be seen that p=0.0152, which is less than 0.05. Therefore, the fluorescence polarization signal of the system containing the substance to be detected is significantly increased compared with the fluorescence polarization signal of the background system. It can be determined that the substance to be detected LG100268 has an activation effect on RXRα.

[0040] Example 2

[0041] A method for detecting retinoid X receptor α activating effector molecules based on fluorescence polarization technology. In this embodiment, the substance to be detected is 1,3,5-tri-tert-butylbenzene (1,3,5-TTBB), a product of Merck Sigma-Aldrich (223778-10G).

[0042] The specific steps are as follows:

[0043] (1) Detection of background system fluorescence polarization signal: After preparing a 100 nmol / L RXRα solution and a 100 nmol / L fluorescent nuclear receptor coregulator peptide solution using 50 mmol / L potassium phosphate buffer, 10 μL of the above RXRα protein solution and 10 μL of fluorescent nuclear receptor coregulator peptide solution and 10 μL of blank solvent (the blank solvent in this experiment is a potassium phosphate buffer solution containing 2% dimethyl sulfoxide) were added to each well of a 384-well plate. After incubation at room temperature for 5 minutes, the final concentration of RXRα was 50 nmol / L, and the final concentration of fluorescent nuclear receptor coregulator peptide was 50 nmol / L. Fluorescence polarization signal detection was performed using a SpectraMax i3x multi-function microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0044] (2) Detection of fluorescence polarization signals of the system containing the substance to be detected: RXRα solution with a molar concentration of 100 nmol / L and fluorescent nuclear receptor coregulator peptide solution with a molar concentration of 400 nmol / L were prepared using 50 mmol / L potassium phosphate buffer, and 1,3,5-TTBB solution with a molar concentration of 2 μmol / L (containing 2% dimethyl sulfoxide) was prepared using 50 mmol / L potassium phosphate buffer. Subsequently, 10 μL of the above RXRα protein, 10 μL of fluorescent nuclear receptor coregulator peptide solution, and 10 μL of the above 2 μmol / L 1,3,5-TTBB solution were added to each well of a 384-well plate. After incubation at room temperature for 5 minutes, the final concentration of RXRα was 50 nmol / L, the final concentration of fluorescent nuclear receptor coregulator peptide was 200 nmol / L, and the concentration of LG100268 was 1 μmol / L. Fluorescence polarization signal detection was performed using a SpectraMax i3x multi-function microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0045] (3) Determine the activation effect of the test substance on RXRα: The fluorescence polarization signal of the background system and the fluorescence polarization signal of the test substance system are obtained by three parallel samples. The T-test is used to analyze the significant difference between the two sets of data, and the results are as follows: Figure 2 shown.

[0046] pass Figure 2 It can be seen that p = 0.0134, which is less than 0.05. Therefore, the fluorescence polarization signal of the system containing the test substance 1,3,5-TTBB in this embodiment is significantly increased compared with the fluorescence polarization signal of the background system, which can be judged that the test substance 1,3,5-TTBB has an activation effect on RXRα.

[0047] Example 3

[0048] In this example, the detection method is the same as that in Example 1, except that: in this example, a 2000 nmol / L RXRα solution and a 400 nmol / L fluorescent nuclear receptor coregulatory peptide solution are prepared using 50 mmol / L potassium phosphate buffer; a 10 μmol / L LG100268 solution (containing 2% dimethyl sulfoxide) is prepared using 50 mmol / L potassium phosphate buffer; after incubation at room temperature for 30 minutes, the final concentration of RXRα is 1000 nmol / L, the final concentration of the fluorescent nuclear receptor coregulatory peptide is 200 nmol / L, and the concentration of LG100268 is 5 μmol / L.

[0049] The experimental results of this embodiment are similar to those of embodiment 1.

[0050] In summary, the method of the present invention can be used to detect the activation effect of retinoid X receptor α using an enzyme marker.

Claims

1. A method for detecting retinoid X receptor α activating effector molecules based on fluorescence polarization technology, characterized by: The specific steps are as follows: (1) Mixing the retinoic acid X receptor α solution and the fluorescent nuclear receptor coregulatory peptide solution and incubating them, and then detecting the fluorescence polarization signal, which is the background system fluorescence polarization signal; (2) mixing the retinoic acid X receptor α solution, the fluorescent nuclear receptor coregulatory peptide solution, and the test substance solution and incubating them, and then detecting the fluorescence polarization signal, which is the fluorescence polarization signal of the test substance system; (3) The fluorescence polarization signal of the background system and the fluorescence polarization signal of the test substance system were analyzed by T-test for significant difference. If the p value was less than 0.05, it could be determined that the test substance had an activation effect on RXRα.

2. The method for detecting retinoid X receptor alpha activating effector molecules based on fluorescence polarization technology according to claim 1, characterized in that: In step (1), the volume ratio of the retinoic acid X receptor alpha solution to the fluorescent nuclear receptor coregulatory peptide solution is 1:(0.9-1.1), wherein the molar concentration of the retinoic acid X receptor alpha solution is 100-2000 nmol / L, and the concentration of the fluorescent nuclear receptor coregulatory peptide is 100-400 nmol / L.

3. The method for detecting retinoid X receptor alpha activating effector molecules based on fluorescence polarization technology according to claim 1, characterized in that: The incubation conditions in step (1) and step (2) are: incubation at room temperature for 5 to 30 minutes.

4. The method for detecting retinoid X receptor alpha activating effector molecules based on fluorescence polarization technology according to claim 1, characterized in that: In step (2), the volume ratio of the retinoic acid X receptor alpha solution, the fluorescent nuclear receptor coregulatory peptide solution, and the test substance solution is 1:(0.9-1.1):1, wherein the molar concentration of the retinoic acid X receptor alpha solution is 100-2000 nmol / L, the concentration of the fluorescent nuclear receptor coregulatory peptide is 100-400 nmol / L, and the concentration of the test substance solution is 2-20 μmol / L.

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