Polypeptide membrane anchoring high-throughput screening method for gpcrs

Abstract

The polypeptide membrane anchoring high-throughput screening method for GPCR is that the C terminal of GPCR is connected with tetracycline-controlled transcriptional activator (tTA) through tobacco etch virus protease substrate sequence (TEV-Substrate). When GPCR is combined with the membrane anchoring polypeptide, the TEV protease cuts the substrate to release tTA, activates the expression of luciferase reporter gene (Luc), and the cell expressing luciferase can detect the production amount of oxidized luciferin by using an enzyme label instrument, so that the accurate screening of the targeted polypeptide compound is realized. The screening method provided by the application can quickly screen the polypeptide combined with GPCR, is not limited to the screening of only a certain signal path in the traditional GPCR function detection, and can realize the high-throughput screening of the polypeptide and GPCR target by combining the luciferase reporter gene.

Description

Technical Field

[0001] This invention relates to the field of peptide drug screening, and more specifically to a high-throughput screening method for peptide membrane anchoring targeting GPCRs. Background Technology

[0002] Currently, approximately 40% of FDA-approved drugs target GPCR receptors, making research on GPCR receptors and their peptide ligands crucial for drug development. GPCR receptors possess specific downstream signaling pathways (multiple G proteins / β-arrestin 1 / 2). Current methods for detecting GPCR binding to ligands primarily rely on detecting primary signals (G proteins / β-arrestin) or secondary signals (cAMP, IP1, etc.). Typically, each method can only detect one signaling pathway, significantly limiting the detection of ligand-receptor binding. Furthermore, current methods for detecting peptide binding to GPCRs require the synthesis of physical peptides, which is costly and time-consuming, further hindering the identification of targets for peptide compounds.

[0003] Examples of existing technical solutions: a. One-GO biosensor detection: can only detect 10 downstream signaling pathways of GPCRs, and requires the synthesis of physical peptides; b. Nanobit / BRET and other detection methods: require the synthesis of solid peptides; only detect a specific signaling pathway; c. Salsa screening system: requires the synthesis of solid polypeptides; the C-terminus of the GPCR receptor is modified, making it easy to bind to β-arrestin1 / 2 protein, resulting in a high false positive rate; and it can only detect GPCR receptors that can recruit β-arrestin1 / 2.

[0004] Existing technologies for GPCR peptide ligand screening suffer from drawbacks such as difficulty in synthesizing solid peptides, long synthesis cycles, high costs, and unsuitability for long-term storage. This screening method only requires the synthesis of peptide membrane-anchored plasmids. Plasmid synthesis is relatively inexpensive, and the plasmids can be continuously regenerated through expression in *E. coli*, thus saving significant research costs.

[0005] Existing high-throughput screening methods for GPCRs and peptide ligands can only screen for specific downstream pathways, resulting in significant limitations. This screening method does not target any single downstream pathway of a GPCR; as long as the ligand binds to the GPCR, it can be detected based on the relevant detection signal. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a high-throughput screening method for GPCR-based peptide membrane anchoring.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solution: A high-throughput screening method for GPCR-based peptide membrane anchoring, the high-throughput screening method comprising the following steps: Synthesis of S1 GPCR plasmid, membrane-anchored peptide plasmid, and luciferase reporter plasmid: Based on the PDGFR sequence shown in SEQ ID No. 1, a plasmid capable of fusion expression of peptide, PDGFR, and TEV protease was synthesized. The peptide sequence was linked to PDGFR with a flexible linker, and PDGFR was linked to the TEV protease sequence with a 3*FLAG sequence. The plasmid vector was plvx-puro. The optimal linking method between the above protein amino acid sequences was selected after screening, and the sequence did not affect the peptide activity. A GPCR plasmid with its C-terminus linked to tTA via TEV-Substrate was synthesized. This plasmid vector was pcDNA3.1. A luciferase reporter plasmid TRE-Luc (TRE stands for transcriptional response element) was synthesized. The plasmid vector was plvx-puro. S2 Cell Plating: Take healthy HEK 293T cells from a T25 flask, digest with 1 mL of trypsin for 1 min, add 1 mL of complete culture medium to stop digestion, transfer to a centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of complete culture medium for reselection, count, and dilute to 3.5*10⁻⁶. 5 Seeds were seeded at 96-well white plates with cells / mL and cultured in a cell culture incubator; S3 Transfection: Observe cell growth in 96-well plates. When cells reach approximately 80% confluence (18-20 h), transfect plasmids using Lipofectamine 3000. Prepare transfection solutions (per well in a 96-well plate) according to the following ratio: Solution A (Opti-MEM 5 μL + Lipofectamine 3000 0.2 μL), Solution B (Opti-MEM 5 μL + p3000 0.2 μL + pcDNA3.1-repector 40 ng + TRE-Luc plasmid 40 ng + membrane-anchored peptide plasmid 40 ng). Mix solutions A and B thoroughly and incubate at room temperature for 15 min. Add the mixed transfection solution to the 96-well white-background plates. Incubate in a cell culture incubator (37℃, 5% CO2) for 20-24 h after transfection.

[0008] S4 assay: Discard the culture medium, wash twice with PBS, add incubation solution (containing fluorescein), 100 μL / well, and directly test on the instrument. Transfer to a BioTek microplate reader to detect Luciferase activity. S5 demonstrates the feasibility of the membrane-anchored peptide method through experiments S2, S3, and S4, enabling high-throughput screening of peptides and GPCR targets.

[0009] Furthermore, this method does not require the synthesis of physical peptides; it only requires the synthesis of membrane-anchored peptide plasmids to achieve high-throughput screening of peptides and GPCR targets. Compared to the synthesis of physical peptides, plasmid synthesis is relatively inexpensive and can be continuously regenerated through expression in E. coli, which can save a lot of research costs. As long as there is binding between GPCR and peptide ligand, the peptide can be screened out.

[0010] Furthermore, the amino acid sequence of the peptide, taking GLP-1 as an example, is shown in SEQ ID No. 2.

[0011] Furthermore, the amino acid sequence of the flexible linker sequence linking the polypeptide amino acid sequence to the PDGFR amino acid sequence is shown in SEQ ID No. 3.

[0012] Furthermore, the amino acid sequence of the 3*FLAG sequence is shown in SEQ ID No. 4.

[0013] This invention provides a high-throughput screening method for GPCR-based peptide membrane anchoring, which has the following advantages compared to existing technologies: (1) This invention does not require the synthesis of physical peptides, but only the synthesis of membrane-anchored peptide plasmids to achieve high-throughput screening of peptides and GPCR targets. Compared with the synthesis of physical peptides, plasmid synthesis is relatively inexpensive and can be continuously regenerated through expression in E. coli, which can save a lot of scientific research costs.

[0014] (2) This invention is not limited to screening only a certain signaling pathway in traditional GPCR functional detection. As long as GPCR and peptide ligand are bound, they can be screened. Attached Figure Description

[0015] Figure 1 To experimentally verify the results of membrane-anchored peptide activity; Figure 1 a is a validation diagram of the activity of various membrane-anchored peptides, including multiple receptors of the GPCR Class A and Class B1 families, and the peptide ligands are all corresponding endogenous ligands; Figure 1 b is the dose-dependent curve of membrane-anchored GLP-1 peptide (tGLP-1); Figure 2 This is a feasibility test diagram for the TRE-LUC system; Figure 3 This diagram illustrates the specificity of different membrane-anchored peptides for the GLP-1R target. Figure 4This is a schematic diagram of a high-throughput screening method for peptide membrane anchoring targeting GPCRs. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments: like Figure 1-4 As shown, Based on the PDGFR sequence shown in SEQ ID No. 1, a plasmid for fusion expression of polypeptide, PDGFR, and TEV protease was synthesized; a GPCR plasmid C-terminus linked to tTA via TEV-Substrate was synthesized; a luciferase reporter plasmid for TRE transcription initiation was synthesized; and a reporter plasmid for cAMP detection, CRE-LUC, was synthesized. Cell plating: Take HEK 293T cells in good growth condition from T25 flasks, digest with 1 mL of trypsin for 1 min, add 1 mL of complete culture medium to stop digestion, transfer to centrifuge tubes, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of complete culture medium for reselection, count, dilute to 3.5*105 cells / mL, plate in 96-well white plates, and culture in a cell culture incubator.

[0017] Transfection: Observe cell growth in 96-well plates. When cells reach approximately 80% confluence (18-20 h), transfect plasmids using Lipofectamine 3000. Prepare transfection solutions (per well in a 96-well plate) according to the following ratio: Solution A (Opti-MEM 5 μL + Lipofectamine 3000 0.2 μL), Solution B (Opti-MEM 5 μL + p3000 0.2 μL + pcDNA3.1-repector 40 ng + CRE-LUC plasmid 40 ng + membrane-anchored peptide plasmid 40 ng). Mix solutions A and B thoroughly and incubate at room temperature for 15 min. Add the mixed transfection solution to the 96-well white-background plates. Incubate in a cell culture incubator (37℃, 5% CO2) for 20-24 h after transfection.

[0018] The activity of membrane-anchored peptides was verified by cAMP accumulation (cAMP signaling is a secondary signal generated by the binding of Gs protein after GPCR activation, and is often used as an indicator of ligand activation ability of GPCRs; the CRE-LUC reporter plasmid method has been reported in many top SCI papers and can specifically detect the amount of cAMP accumulation). Receptors selected included Class A and Class B1 of the GPCR family. The results are as follows... Figure 1a. We selected 12 GPCRs (including Class A and Class B1) and their endogenous polypeptide ligands (synthetic membrane-anchored peptides) and used the CRE-LUC reporter plasmid detection method to verify the activation ability of membrane-anchored peptides on receptors. The results showed that 8 of the 12 membrane-anchored peptides could activate receptors, indicating that for the vast majority of peptides, anchoring them to the cell membrane via PDGFR does not affect their activity. Figure 1 Taking tGLP-1 as an example, a dose-response curve was plotted to show that membrane-anchored peptides, similar to solid peptides, have an EC50 and a dose plateau (the amount of transfected membrane-anchored plasmid), thus excluding toxic reactions.

[0019] Based on the above cell plating and transfection methods, the applicability of the membrane anchoring method to the TRE-LUC system was verified. Taking tGLP-1 and tVIP as examples, the results are as follows: Figure 2 After tGLP-1 and tVIP activate their corresponding receptors, TEV enzymes can cleave tTA transcription factors. tTA then binds to TRE, promoting Luciferase translation and expression. Our results demonstrate the feasibility of this system and its dose-dependent nature. This system is no longer limited to detecting cAMP signals and can be applied to the detection of a wider range of GPCRs. It is no longer limited to a single signaling pathway; as long as the peptide binds to the GPCR, a signal can be generated and detected.

[0020] like Figure 3 As shown, based on the above cell plating and transfection methods, tGLP-1R was co-transfected with membrane-anchored peptide plasmids such as tGLP-1, tCRH, and tCGRP, and the specificity of the membrane-anchored peptides was detected (GLP-1 is an endogenous ligand of GLP-1R). The results showed that tGLP-1 and tGLP-1R were significantly specific. Compared with other membrane-anchored peptides, tGLP-1 exhibited the strongest activation ability and could clearly distinguish the positive group from other control groups. Regarding the specificity of membrane-anchored peptides against GPCR targets, in GLP-1R stably transfected cell lines, seroconversion to tGLP-1, tGIP, and other peptide membrane-anchored plasmids showed that the tGLP-1 group exhibited the highest Luciferase signal, demonstrating the best activation ability. The above detection results validated the feasibility of the high-throughput screening method for G protein-coupled receptor peptide membrane anchoring from various aspects. It not only verified that the membrane-anchored peptides possess activity similar to solid peptides and exhibit a plateau phase, but also validated the feasibility of the TRE-LUC reporter system, which can be used to detect the ability of membrane-anchored peptides to activate tGPCRs; and further validated the specificity of the membrane-anchored peptides in this method. In summary, the experiments, based on the activity of the membrane-anchored peptides, the feasibility of the reporter system, and the specificity of the membrane-anchored peptides, collectively demonstrate the feasibility of the method.

[0021] The amino acid sequence of the polypeptide, taking GLP-1 as an example, is shown in SEQ ID No. 2.

[0022] The amino acid sequence of the flexible linker sequence that links the polypeptide amino acid sequence to the PDGFR amino acid sequence is shown in SEQ ID No. 3.

[0023] The amino acid sequence of the 3*FLAG sequence is shown in SEQ ID No. 4.

[0024] Principle: This invention relates to a high-throughput screening method for peptide membrane anchoring targeting G protein-coupled receptors (GPCRs). The C-terminus of the GPCR is linked to a transcription activator (tTA) controlled by the artificial transcription factor tetracycline via a tobacco erosion virus protease substrate sequence (TEV-Substrate). When the GPCR binds to the membrane-anchored peptide, the TEV protease cleaves the substrate, releasing tTA and activating the expression of the luciferase reporter gene (Luc). Cells expressing luciferase can be monitored using an ELISA reader to detect the amount of oxidative luciferin produced, enabling precise screening of targeted peptide drugs. The screening method provided by this invention can rapidly screen for peptides that bind to GPCRs, going beyond the limitations of traditional GPCR functional assays that target only a single signaling pathway; furthermore, the combination with the luciferase reporter gene allows for high-throughput screening of both peptides and GPCR targets.

[0025] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A high-throughput screening method for GPCR-based peptide membrane anchoring, characterized in that, Includes the following steps: Synthesis of S1 GPCR plasmid, membrane-anchored peptide plasmid, and luciferase reporter plasmid: Based on the PDGFR sequence shown in SEQ ID No. 1, a plasmid (membrane-anchored peptide plasmid) capable of fusion expression of peptide, PDGFR, and TEV protease was synthesized. The peptide amino acid sequence was linked to the PDGFR amino acid sequence with a flexible linker, and the PDGFR amino acid sequence was linked to the TEV protease amino acid sequence with a 3*FLAG sequence. The plasmid vector was plvx-puro. The linkage methods between the above protein amino acid sequences were screened to select sequences that did not affect peptide activity. A GPCR plasmid with its C-terminus linked to tTA via TEV-Substrate was synthesized. This plasmid vector was pcDNA3.

1. A luciferase reporter plasmid TRE-Luc was synthesized, where TRE is a transcriptional reaction element, and the plasmid vector was plvx-puro. S2 Cell Plating: Take healthy HEK 293T cells from a T25 flask, digest with 1 mL of trypsin for 1 min, add 1 mL of complete culture medium to stop digestion, transfer to a centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of complete culture medium for reselection, count, and dilute to 3.5*10⁻⁶. 5 Seeds were seeded at 96-well white plates with cells / mL and cultured in a cell culture incubator; S3 transfection: Observe the cell growth in 96-well plates. When the cells reach a confluence of about 80%, transfect the GPCR plasmid, membrane-anchored peptide plasmid, and luciferase reporter plasmid with Lipofectamine 3000. Culture in a cell culture incubator at 37°C and 5% CO2 for 20-24 h. S4 assay: Discard the culture medium, wash twice with PBS, add 100 μL of fluorescein incubation solution per well, and directly test on the instrument. Transfer to a BioTek microplate reader to detect Luciferase activity. S5 demonstrates the feasibility of the membrane-anchored peptide method through experiments S2, S3, and S4, enabling high-throughput screening of peptides and GPCR targets.

2. The method according to claim 1, characterized in that, This method does not require the synthesis of physical peptides; it only requires the synthesis of membrane-anchored peptide plasmids to achieve high-throughput screening of peptides and GPCR targets. Compared with the synthesis of physical peptides, plasmid synthesis is relatively inexpensive and can be continuously regenerated through expression in E. coli, which can save a lot of research costs. As long as there is binding between GPCR and peptide ligand, the peptide can be screened.

3. The method according to claim 1, characterized in that, The amino acid sequence of the polypeptide, taking GLP-1 as an example, is shown in SEQ ID No.

2.

4. The method according to claim 1, characterized in that, The amino acid sequence of the flexible linker sequence that links the polypeptide amino acid sequence to the PDGFR amino acid sequence is shown in SEQ ID No.

3.

5. The method according to claim 1, characterized in that, The amino acid sequence of the 3*FLAG sequence is shown in SEQ ID No. 4.

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