GPCR-based ultramicro drug screening method and application thereof in screening of antipruritic traditional Chinese medicines

Abstract

The invention belongs to the field of biological pharmacy, and discloses a GPCR-based ultramicro drug screening method and application thereof in screening of antipruritic traditional Chinese medicines. According to the invention, an Echo ultrasonic pipetting system is combined with a GPCR function experiment (beta-arrest recruitment) to establish the ultramicro screening method for screening the GPCR ligand. The Echo 550 system utilizes ultrasonic waves to beat liquid into 2.5 nL liquid drops and then transfer the liquid drops into a 384 or 1536 pore plate, rapid and accurate sample adding of at least 2.5 nL (1 / 400 mu L) can be achieved, ultra-micro screening and high-throughput screening can be achieved at the same time, and the method is particularly suitable for screening of itching relieving drugs.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and specifically relates to a GPCR-based method for screening ultra-micro drugs and its application in screening traditional Chinese medicine for relieving itching. Background Technology

[0002] G protein-coupled receptors (GPCRs) are the largest family of receptors in the human body, participating in the regulation of various physiological functions and the development of diseases. The GPCR family comprises over 800 members, and the GRAFS classification system divides GPCRs into five classes: A (Rhodopsin), B (Secretin), C (Glutamate), F (Frizzled / Taste2), and Adhesion. Currently, over 33% of marketed drugs target GPCRs, making them the largest class of drug targets.

[0003] GPCR ligand types include agonists, antagonists, biased agonists, allosteric modulators, etc. Agonists are ligands that activate receptors to produce corresponding downstream signals; antagonists are ligands that inhibit the production of downstream signals caused by agonists. GPCRs primarily activate two signaling pathways: the G protein signaling pathway and the β-arrestin signaling pathway. For GPCRs, biased agonists are agonists that selectively activate either the G protein signaling pathway or the β-arrestin signaling pathway. Clinically, the use of biased agonists can effectively avoid drug side effects. For example, the marketed drug, the biased agonist of the μ-opioid receptor (TRV-130, oxalidin), selectively activates the G protein signaling pathway while having a weaker activation of the β-arrestin signaling pathway. Since the G protein signaling pathway mainly mediates analgesia, while the β-arrestin signaling pathway mainly mediates respiratory depression, nausea, vomiting, and other side effects, TRV-130 can effectively avoid drug side effects. Allosteric modulators are those that, when used in combination with agonists, can alter the effect and potency of agonists. Positive allosteric modulators, when used in combination with agonists, can increase the potency or efficacy of agonists, such as the positive allosteric modulator VU050601331 for the Y4R receptor.

[0004] Currently, commonly used GPCR screening methods include G protein signaling-based methods and calcium flux-based methods. These methods are widely used in screening various GPCR ligands; however, their universality, accuracy, and timeliness still need improvement. G proteins are mainly composed of three subunits: α, β, and γ. The α subunits can be broadly classified into Gαi, Gαs, Gαq, and G12 / 13. Different subclasses of the α subunit typically trigger different downstream signaling pathways. For example, activation of Gαi-coupled GPCRs can inhibit cAMP production; activation of Gαs-coupled GPCRs can promote cAMP production; and activation of Gαq-coupled GPCRs can promote calcium flux production. 2+ The GPCRs coupled with Gα12 / 13 are generated; activation of these GPCRs can mediate the Rho signaling pathway; and the Gβγ subunit can also induce Ca2+. 2+ The formation of G protein dissociation signaling pathways is a significant issue. For the same GPCR (such as MRGPRX1), it can couple to both Gαi and Gαq. Designing a screening system targeting the G protein dissociation process requires constructing screening systems for different α subunits, which is time-consuming and labor-intensive. Since calcium ions are the second messenger in the human body, many physiological processes involve calcium. 2+ Therefore, the specificity of screening systems based on calcium ion detection needs to be improved.

[0005] The scarcity of precious Chinese medicinal herbs, difficult-to-isolate and purify herb monomers, and difficult-to-synthesize compounds significantly increases screening costs and limits their application in screening. Currently, there are few nanoscale screening systems for GPCRs. Therefore, establishing ultra-micro screening methods is of significant economic importance for screening these difficult-to-obtain Chinese medicinal herb monomers or compounds. Summary of the Invention

[0006] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a GPCR-based method for ultra-micro drug screening.

[0007] Another objective of this invention is to provide the application of the above-mentioned GPCR-based ultra-micro drug screening method in the screening of antipruritic traditional Chinese medicine.

[0008] The objective of this invention is achieved through the following technical solution: A GPCR-based method for screening ultra-micro drugs includes the following steps: (1) Construct expression vector A, which can express GPCR and smBiT fusion protein; (2) Construct expression vector B, which can express β-arrestin and LgBiT fusion protein; (3) Mix expression vector A and expression vector B, transfect them into animal cells and culture them to obtain cells for detection; (4) When screening agonists, the cells used for detection are digested and resuspended in buffer, then the substrate is added, and the resulting cell mixture is aliquoted into 384 or 1536-well plates for incubation. Then the compound to be screened is added using the Echo sonic pipetting system, and the intensity of the light is immediately detected using the ultrasensitive luminescence mode of the Envision microplate reader. (5) When screening antagonists, the cells used for detection are digested and resuspended in buffer, aliquoted into 384 or 1536-well plates, the compound to be screened is added using an Echo sonic pipetting system and incubated, and then an agonist solution containing the substrate is added using an Echo sonic pipetting system. The intensity of the light is immediately detected using the ultrasensitive luminescence mode of an Envision microplate reader.

[0009] The vector framework of expression vector A in step (1) is preferably a pcDNA series vector; more preferably a pcDNA3.1 vector.

[0010] In step (1), smBiT in the fusion protein can be located at the C-terminus or N-terminus of the GPCR; preferably, it is located at the C-terminus of the GPCR.

[0011] The fusion protein mentioned in step (1) is preferably obtained by sequentially linking GPCR, linker1 and smBiT.

[0012] The amino acid sequence of linker1 is as follows: GSSGGGGSGGGGSSG.

[0013] The vector framework of expression vector B mentioned in step (2) is preferably a pcDNA series vector; more preferably a pcDNA3.1 vector.

[0014] In step (2), LgBiT in the fusion protein can be located at the C-terminus or N-terminus of the β-repressor protein; preferably, it is located at the N-terminus of the β-repressor protein.

[0015] The fusion protein mentioned in step (2) is preferably obtained by sequentially linking LgBiT, linker2 and β-repressor protein.

[0016] The amino acid sequence of linker2 is as follows: GGSGGGGSGGSSSGG.

[0017] The β-repressor protein mentioned in step (2) is either β-repressor protein 1 or β-repressor protein 2.

[0018] In step (3), the expression vector A and expression vector B are mixed in a mass ratio of 1:0.1 to 1:10; more preferably, they are mixed in a mass ratio of 1:0.5 to 1:4.

[0019] The animal cells mentioned in step (3) are normal cells or cancer cells; preferably HEK 293T cells, HEK 293 cells or HeLa cells.

[0020] The preferred culture time in step (3) is 24-48 h.

[0021] The buffer solution described in steps (4) and (5) is preferably PBS or HBSS.

[0022] The substrates described in steps (4) and (5) are preferably coelenterazine or furimazine.

[0023] The concentration of the substrate in the cell mixture in step (4) is preferably 5 to 50 μM; more preferably 20 to 30 μM.

[0024] The volume of cells in each well is 15–30 μL, more preferably 20 μL, as described in steps (4) and (5).

[0025] The number of cells in each well as described in steps (4) and (5) is 10. 4 ~10 6 10 cells; more preferably 6 × 10 cells per well. 4 ~10 5 Each cell.

[0026] The incubation time in step (4) is preferably 15 to 20 minutes.

[0027] The preferred addition volume of the compound to be screened in step (4) is 2.5 nL / well.

[0028] The preferred addition volume of the compound to be screened in step (5) is 2.5 nL / well.

[0029] The incubation conditions described in step (5) are preferably 37 °C for 0.5 to 2 h; more preferably 37 °C for 1 h.

[0030] The preferred addition volume of the substrate-containing agonist solution in step (5) is 2.5 nL / well.

[0031] The concentration of the substrate in the agonist solution containing the substrate described in step (5) is preferably 5 to 100 μM; more preferably 10 to 50 μM; and most preferably 20 to 30 μM.

[0032] The concentration of the agonist in the substrate-containing agonist solution described in step (5) is preferably 0.1 to 50 mM; more preferably 1 to 20 mM.

[0033] The above-mentioned GPCR-based ultra-micro drug screening method is applied to the screening of antipruritic traditional Chinese medicine.

[0034] The principle of this invention is as follows: In the NanoBiT system, the large fragment LgBiT and the small fragment SmBiT combine to form a complete luciferase. After the GPCR is activated by an agonist, it binds to β-arrestin. At this point, the luciferase formed by the combination of LgBiT and SmBiT acts on the substrate, producing cold light.

[0035] The present invention has the following advantages and effects compared with the prior art: (1) Currently, over 33% of marketed drugs target GPCRs, making GPCRs the largest class of drug targets. GPCR activation triggers numerous signaling pathways, making it difficult to rapidly and accurately screen for drugs acting on GPCRs using a single, universal method. For example, screening based on G protein signaling pathways is challenging because of the vast number of G proteins; a single G protein cannot be used to screen for drugs acting on numerous GPCRs. Calcium ions, as second messengers, are involved in many physiological responses, resulting in low specificity and accuracy in calcium flux-based screening. However, the β-arrestin-based screening method described in this application is designed because almost all GPCR activation triggers β-arrestin recruitment, offering good universality, high specificity, and high accuracy.

[0036] (2) In the prior art, the substrate incubation time for screening based on G protein signals is as long as 1-2 hours (Inoue A, et al. Illuminating G-protein-coupling selectivity of GPCRs[J]. Cell, 2019, 177(7): 1933-1947. e25.), and the screening method based on calcium flow requires an incubation time of about 1 hour for calcium ion dyes, while the screening method involved in this application only requires about 15 minutes.

[0037] (3) Conventional screening methods require a large amount of drugs and are not suitable for precious Chinese medicines, Chinese medicine monomers that are difficult to separate and purify, and compounds that are difficult to synthesize. The screening method provided by this invention only requires nanoliters (nL) and is an ultra-micro screening method.

[0038] In summary, because calcium ions are the body's second messenger, many physiological processes involve calcium. 2+The process involves the recruitment of β-arrestin after activation of most GPCRs. Therefore, this application utilizes the Echo ultrasonic pipetting system combined with GPCR functional experiments (β-arrestin recruitment) to establish an ultra-micro screening method for screening GPCR ligands. The Echo 550 system uses ultrasound to break up liquid into 2.5 nL droplets and then transfers them to 384 or 1536-well plates, enabling rapid and accurate sample loading of at least 2.5 nL (1 / 400 μL), and simultaneously achieving ultra-micro screening and high-throughput screening. Attached Figure Description

[0039] Figure 1 This is a comparison of the cold light signals of fMLF, an agonist of formyl peptide receptor 1 (FPR1) in the GPCR family, at different cell concentrations, using an ultra-micro screening method.

[0040] Figure 2 This is a graph showing the results of screening for the agonist fMLF and antagonist boc2 of the formyl peptide receptor 1 (FPR1) family of GPCRs using the ultra-micro screening method.

[0041] Figure 3 This is a graph showing the results of screening compound 48 / 80, an agonist of MRGPRX2 in the GPCR family, using an ultra-micro screening method.

[0042] Figure 4 This is a graph showing the results of screening the agonist chemokine C9 peptide (YFPGQFAFS) of CMKLR1 in the GPCR family using an ultra-micro screening method.

[0043] Figure 5 This is a graph showing the results of screening MRGPRX2 agonists from a variety of compounds using an ultra-micro screening method. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0045] The cDNA sequence of the smBiT gene is as follows: GTGACCGGCTACCGGCTGTTCGAGGAGATTCTG.

[0046] The LgBiT gene sequence is referenced in addgene: Plasmid #234738 CMV-LgBit.

[0047] The cDNA sequence of Linker1 is as follows: GGATCGAGCGGTGGTGGCGGGAGCGGAGGTGGAGGGTCGTCAGGT.

[0048] The cDNA sequence of Linker2 is as follows: GGAGGTTCGGGAGGCGGTGGCTCGGGCGGATCTTCAAGCGGCGGG.

[0049] linker1-smBiT is obtained by connecting linker1 and smBiT as described above, and is synthesized by the company.

[0050] LgBiT-linker2 is obtained by connecting the aforementioned LgBiT and linker2, and is synthesized by the company.

[0051] The FPR1 gene sequence was obtained by synthesizing the FPR1 gene, which was obtained by referring to positions 128-1180 in GenBank accession number NM_001193306.2.

[0052] The sequence reference number for the β-arrestin2 gene is GenBank EU883572.1, and the β-arrestin2 gene was synthesized by the company.

[0053] The MRGPRX2 gene sequence was obtained by synthesizing the gene from position 399 to position 1391 in GenBank accession number NM_001303615.2.

[0054] The CMKLR1 gene sequence was obtained by synthesizing the gene from position 572 to position 1693 in GenBank accession number NM_001142343.2.

[0055] Example 1: Comparison of signal intensity at different cell concentrations (1) Using pcDNA3.1(+) as a vector, linker1-smBiT was inserted into the multiple cloning site using molecular cloning technology. EcoR I and Xho Between I, FPR1 from the GPCR member is inserted into the multiple cloning site. BamH I and EcoR Between I and II, the pcDNA3.1FPR1-smBiT plasmid was constructed. Using pcDNA3.1(+) as a vector, LgBiT-linker2 was inserted into the multiple cloning site using molecular cloning technology. Nhe I and EcoR Between I, β-arrestin2 is inserted into the multiple cloning site. EcoR I and EcoR Between V, the pcDNA3.1β-arrestin2-LgBiT plasmid was constructed. The correctness of the plasmid sequence was confirmed by gene sequencing.

[0056] (2) Human cervical cancer HeLa cells were transfected with pcDNA3.1 FPR1-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid at a mass ratio of 1:1 using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific). After culturing for 1 day, the cells were digested and resuspended in HBSS solution, and the substrate coelenterazine (final concentration 20 μM) was added. The cells were then transferred to 384-well plates with a cell volume of 20 μL / well. Experimental groups with different cell numbers were set up, including 0.5 × 10⁶ cells / well. 4 Cells / well, 2×10 4 1 cell / well and 6 × 10 4 Cells / well were incubated at room temperature for 15 min, and then 2.5 nL of a 10 mM formylpeptide receptor 1 agonist fMLF solution was added using an Echo sonic pipetting system. The intensity of the cold light was immediately detected using the ultrasensitive luminescence mode of an Envision microplate reader. A control group was also set up, the only difference between the control group and the experimental group being that the formylpeptide receptor 1 agonist fMLF was not added.

[0057] The results are as follows Figure 1 As shown, the higher the cell count, the stronger the cold light signal (6×10⁻⁶). 4 1 cell / well is optimal.

[0058] Example 2 FPR1 is closely associated with inflammatory diseases such as acute lung injury, and it has also been reported to play an important role in the course of pruritus. This invention is of great significance for the discovery of therapeutic drugs for the above-mentioned diseases.

[0059] (1) Same as step (1) in Example 1.

[0060] (2) Basically the same as step (2) in Example 1, except that the number of cells per well is 6 × 10 4 indivual.

[0061] (3) Human cervical cancer HeLa cells were transfected with pcDNA3.1 FPR1-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid at a mass ratio of 1:1 using Lipofectamine™ 3000 transfection reagent. After culturing for 1 day, the cells were digested and resuspended in HBSS solution and transferred to 384-well plates with 6 × 10⁶ cells per well. 4Cell culture was administered at a volume of 20 μL per well. Then, using an Echo sonic pipetting system, 2.5 nL of a 10 mM FPR1 antagonist, {Boc}-FLFLF (Boc2), was added to each well containing cells. After incubation at 37°C for 1 h, 2.5 nL of a 10 mM Coelenterazineh agonist fMLF solution was added. The agonist concentration was immediately measured using the ultrasensitive luminescence mode of an Envision microplate reader. A control group was also included; the only difference between the control and experimental groups was the absence of both the antagonist and agonist.

[0062] The results are as follows Figure 2 As shown, the method provided by the present invention can effectively test the antagonist of fMLF, and the detection effect is not affected by the simultaneous addition of substrate and agonist.

[0063] Example 3 MRGPRX2 is closely associated with diseases accompanied by itching, such as chronic urticaria and atopic dermatitis. This invention is of great significance for the discovery of therapeutic drugs for these diseases.

[0064] (1) Using pcDNA3.1(+) as a vector, linker1-smBiT was inserted into the multiple cloning site using molecular cloning technology. EcoR I and Xho Between I and II, the Mas-associated G protein-coupled receptor X2 (MRGPRX2) from the GPCR member is inserted into the multiple cloning site. Hind III and EcoR Between steps I and II, the pcDNA3.1 MRGPRX2-smBiT plasmid was constructed. The construction of the pcDNA3.1 β-arrestin2-LgBiT plasmid was the same as in Example 1. The correctness of the plasmid sequence was confirmed by gene sequencing.

[0065] (2) The procedure is basically the same as in Example 1 (2), except that: pcDNA3.1 MRGPRX2-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid are transfected into human embryonic kidney HEK 293T cells at a mass ratio of 1:4. After culturing for 1 day, the cells are digested and resuspended in HBSS solution and the substrate fumarazine (final concentration of 10 μM) is added. The cells are then transferred to 384-well plates with 1 × 10⁶ cells per well. 5Cells were incubated at 20 μL / well for 15 min at room temperature. Then, 2.5 nL of 10 mM MRGPRX2 agonist compound 48 / 80 solution was added using an Echo sonic pipetting system. The intensity of the luminescence was immediately detected using an Envision microplate reader in ultrasensitive luminescence mode. A control group was also set up, the only difference between the control and experimental groups being that no agonist was added.

[0066] The results are as follows Figure 3 As shown, the method provided by this invention can effectively detect the MRGPRX2 agonist compound 48 / 80, and moreover, it is compatible with... Figure 2 Compared to (maximum signal strength approximately 3.5 × 10⁻⁶), 5 The signal in this embodiment is stronger (maximum signal strength approximately 1.2 × 10⁻⁶). 7 This indicates that the use of Furimazine substrate can effectively improve signal strength.

[0067] Example 4 CMKLR1 is a G protein-coupled receptor that is widely involved in the regulation of inflammation, itching, immunity, metabolism, and the nervous system. This invention is of great significance for the discovery of therapeutic drugs for the aforementioned diseases.

[0068] (1) Using pcDNA3.1(+) as a vector, linker1-smBiT was inserted into the multiple cloning site using molecular cloning technology. EcoR I and Xho Between I, insert CMKLR1 from the GPCR member into the multiple cloning site. BamH I and EcoR I. Construct the pcDNA3.1CMKLR1-smBiT plasmid. The construction of the pcDNA3.1β-arrestin2-LgBiT plasmid was the same as in Example 1. The correctness of the plasmid sequence was confirmed by gene sequencing.

[0069] (2) The procedure is basically the same as in Example 1 (2), except that: pcDNA3.1 CMKLR1-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid are transfected into human embryonic kidney HEK 293T cells at a mass ratio of 2:1. After culturing for 1 day, the cells are digested and resuspended in HBSS solution, and the substrate coelenterazine (final concentration 30 μM) is added. The cells are then transferred to 384-well plates with 1×10⁻⁶ cells per well. 5Cells were incubated at 20 μL / well for 15 min at room temperature. Then, 2.5 nL of a 10 mM CMKLR1 agonist chemokine C9 peptide (YFPGQFAFS) solution was added using an Echo sonic pipetting system. The intensity of the cold light was immediately detected using the ultrasensitive luminescence mode of an Envision microplate reader. A control group was also set up, the only difference between the control and experimental groups being that the agonist was not added.

[0070] The results are as follows Figure 4 As shown, the method provided by the present invention can effectively detect the agonist chemokine C9 peptide of CMKLR1, and the signal differentiation is obvious, which can be used to screen ligands of CMKLR1.

[0071] Example 5: Screening for MRGPRX2 agonists from a variety of compounds (1) The construction of the pcDNA3.1 MRGPRX2-smBiT plasmid was the same as in Example 3. The construction of the pcDNA3.1 β-arrestin2-LgBiT plasmid was the same as in Example 1. The correctness of the plasmid sequence was confirmed by gene sequencing.

[0072] (2) The procedure is basically the same as in Example 1 (2), except that: pcDNA3.1 MRGPRX2-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid are transfected into human embryonic kidney HEK 293T cells at a mass ratio of 1:4. After culturing for 1 day, the cells are digested and resuspended in HBSS solution, and the substrate coelenterazine (final concentration 20 μM) is added. The cells are then transferred to 384-well plates with 1×10⁻⁶ cells per well. 5 Cells were incubated at room temperature for 15 min using 20 μL of cell slurry per well. Then, 2.5 nL of various compounds (matrine, osthol, bergamot lactone, quercetin, emodin-8-O-β-D-glucoside, gallic acid, rutin, chlorogenic acid, vanillic acid, compoud 48 / 80, and kaempferol) at a concentration of 10 mM were added using an Echo sonic pipetting system. The intensity of the cold light was immediately detected using the ultrasensitive luminescence mode of an Envision microplate reader. A control group was also included; the only difference between the control and experimental groups was that no compounds were added.

[0073] The results are as follows Figure 5 As shown, among the many compounds, the signal intensity of the MRGPRX2 agonist is significantly higher than that of the solvent control and other compounds, indicating that the method involved in this application can successfully screen for MRGPRX2 agonists.

[0074] Comparative Example 1 Sampling in 96-well plates cannot be done using an Echo ultrasonic pipetting system because the plate must be inverted with the wells facing down; if the wells are upside down, the liquid will spill out. The 96-well plate procedure below uses a pipette, which cannot achieve micro-volume addition, resulting in relatively poor high-throughput screening performance. As seen in the antagonist screening process below (an experiment optimized by the applicant), the drug addition volume for a 96-well plate is 33 μL. Adding such a large volume of liquid to 90 μL of cell culture will cause cell motility, leading to instability in the entire system. However, adding too small a volume of drug will result in inaccurate loading. This is not very advantageous for screening valuable samples.

[0075] Agonist screening: Human cervical cancer HeLa cells were transfected with pcDNA3.1 FPR1-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid at a 1:1 ratio using Lipofectamine™ 3000 transfection reagent. After 1 day of culture, the cells were digested and transferred to 96-well plates, with 2 × 10⁻⁶ cells per well. 5 The cell suspension volume was 90 μL, and then 10 μL of 200 μM coelenterazine was added. The mixture was incubated at room temperature for 15 min, and then 10 μL of 10 μM formyl peptide receptor 1 agonist fMLF was added. The intensity of the cold light was immediately detected using the ultrasensitive cold light (luminescence) mode of the Envision microplate reader.

[0076] Antagonist screening: Human cervical cancer HeLa cells were transfected with pcDNA3.1 FPR1-smBiT plasmid and pcDNA3.1 β-arrestin2-LgBiT plasmid at a 1:1 ratio using Lipofectamine™ 3000 transfection reagent. After 1 day of culture, the cells were digested and transferred to 96-well plates, with 2 × 10⁻⁶ cells per well. 5 The cell suspension volume was 90 μL. 10 μL of 10 μM MPR1 antagonist was added. After incubation at 37°C for 1 h, 11 μL of 200 μM coelenterazine was added. After incubation at room temperature for 15 min, 12 μL of 10 μM formyl peptide receptor 1 agonist fMLF was added. The intensity of the cold light was immediately detected using the ultrasensitive cold light (luminescence) mode of the Envision microplate reader.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for screening ultra-micro drugs based on GPCR, characterized in that... Includes the following steps: (1) Construct expression vector A, which can express GPCR and smBiT fusion protein; (2) Construct expression vector B, which can express β-repressor protein and LgBiT fusion protein; (3) Mix expression vector A and expression vector B, transfect them into animal cells and culture them to obtain cells for detection; (4) When screening agonists, the cells used for detection are digested and resuspended in buffer, then the substrate is added, and the resulting cell mixture is dispensed into 384 or 1536-well plates for incubation. Then the compound to be screened is added using the Echo sonic pipetting system, and the intensity of the cold light is immediately detected using the ultrasensitive cold light mode of the Envision microplate reader. (5) When screening antagonists, the cells used for detection are digested and resuspended in buffer, aliquoted into 384 or 1536-well plates, the compound to be screened is added using an Echo sonic pipetting system and incubated, and then an agonist solution containing the substrate is added using an Echo sonic pipetting system. The intensity of the cold light is immediately detected using the ultrasensitive cold light mode of the Envision microplate reader.

2. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: The vector framework of expression vector A mentioned in step (1) is selected from the pcDNA series vectors; The vector framework of expression vector B described in step (2) is selected from the pcDNA series vectors.

3. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: In step (1), smBiT is located at the C-terminus of the GPCR in the fusion protein. In step (2), LgBiT is located at the N-terminus of the fusion protein.

4. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: The fusion protein mentioned in step (1) is obtained by sequentially linking GPCR, linker1 and smBiT; The amino acid sequence of linker1 is shown below: GSSGGGGSGGGGSSG; The fusion protein mentioned in step (2) is obtained by sequentially linking LgBiT, linker2, and β-repressor protein; The amino acid sequence of linker2 is as follows: GGSGGGGSGGSSSGG.

5. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: The animal cells mentioned in step (3) are HEK 293T cells, HEK 293 cells, or HeLa cells; The buffer solution mentioned in steps (4) and (5) is PBS or HBSS; The substrates mentioned in steps (4) and (5) are coelenterin h or formazan.

6. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: In step (3), expression vector A and expression vector B are mixed in a mass ratio of 1:0.1 to 1:

10. The concentration of the substrate in the cell mixture described in step (4) is 5–50 μM; The number of cells in each well as described in steps (4) and (5) is 10. 4 ~10 6 One cell; The concentration of the substrate in the agonist solution containing the substrate described in step (5) is 5–100 μM; The concentration of the agonist in the substrate-containing agonist solution described in step (5) is 0.1–50 mM.

7. The GPCR-based ultra-micro drug screening method according to claim 6, characterized in that: In step (3), expression vector A and expression vector B are mixed in a mass ratio of 1:0.5 to 1:

4. The concentration of the substrate in the cell mixture described in step (4) is 20–30 μM; The number of cells in each well as described in steps (4) and (5) is 6 × 10. 4 ~10 5 One cell; The concentration of the substrate in the agonist solution containing the substrate described in step (5) is 10–50 μM; The concentration of the agonist in the substrate-containing agonist solution described in step (5) is 1–20 mM.

8. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: The incubation time described in step (3) is 24-48 h; The incubation time described in step (4) is 15 to 20 minutes; The incubation conditions described in step (5) are 37 °C for 0.5 to 2 h.

9. The GPCR-based ultra-micro drug screening method according to claim 1, characterized in that: The volume of cells in each well is 15–30 μL, as described in steps (4) and (5). The volume of the compound to be screened added in step (4) is 2.5 nL / well; The volume of the compound to be screened added in step (5) is 2.5 nL / well; The volume of the substrate-containing agonist solution added in step (5) is 2.5 nL / well.

10. The application of the GPCR-based ultra-micro drug screening method according to any one of claims 1 to 9 in the screening of antipruritic traditional Chinese medicine.

Images