PIEZO2 protein mutant as well as preparation and application thereof

Abstract

The invention provides a PIEZO2 protein mutant as well as preparation and application thereof. Wherein the amino acid sequence of the mutants of the rats has mutation at one or two of the following sites relative to the amino acid sequence of the wild type PIEZO2 protein: R2788, E2797, R2788, R2788, R2788, R2788, R2788, R2788, R2788, R2788 The amino acid sequence of the human mutant has mutation at the following site relative to the amino acid sequence of the wild type PIEZO2 protein: R2718. Based on the PIEZO2 mutant found in the invention, screening of small molecular agonists and inhibitors of PIEZO2 can be carried out in combination with a high-throughput fluorescent calcium imaging system.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a PIEZO2 protein mutant and a preparation and application thereof. Background Art

[0002] The mechanically gated PIEZO2 ion channel has been shown to mediate basic life activities in mammals, including touch, pain, proprioception, visceral sensation (mechanical perception such as breathing, blood pressure, and bladder filling). Gain-Of-Function (GOF) mutations in PIEZO2 cause distal joint contractures, while Loss-Of-Function (LOF) mutations can lead to symptoms such as abnormal touch, pain, proprioception, and urinary control. These research findings directly confirm the important physiological and pathological functions of the mechanical sensing mechanism mediated by the PIEZO2 channel in the human body and its clinical application prospects as an important drug target. For example, humans carrying PIEZO2 loss-of-function mutations lose the mechanical hypersensitive pain perception under pathological conditions.

[0003] Therefore, inhibitors of the PIEZO2 channel can be used to develop new non-opioid analgesics for the treatment of mechanical hypersensitivity or allodynia associated with diseases such as cancer and arthritis, as well as genetic diseases caused by GOF mutations of PIEZO2, such as distal arthrogryposis. However, no small molecule agonists or specific inhibitors of PIEZO2 have been reported so far.

[0004] The only reported way to activate PIEZO2 is by applying mechanical force to cells expressing it and recording the cationic current generated by it using patch clamp electrophysiology. Since it is difficult to apply mechanical force and perform electrophysiological recording in a high-throughput manner, this method is not suitable for high-throughput drug screening of PIEZO2.

[0005] Since the activation of PIEZO2 can cause calcium ions to flow into cells, the increase in intracellular calcium ion concentration caused by PIEZO2 activation can be detected by a high-throughput fluorescence imaging system (Fluorescent Imaging Plate Reader, FLIPR). Using FLIPR for cellular calcium signal detection, a previous study screened a library of more than 3 million small molecule compounds, but failed to successfully obtain a small molecule agonist for PIEZO2. Summary of the invention

[0006] One object of the present invention is to provide a PIEZO2 protein mutant;

[0007] Another object of the present invention is to provide a method for preparing the PIEZO2 protein mutant;

[0008] Another object of the present invention is to provide the application of the PIEZO2 protein mutant.

[0009] To achieve the above-mentioned purpose, on the one hand, the present invention provides a PIEZO2 protein mutant (slow-inactivation mutant), wherein the amino acid sequence of the mutant has a mutation at one or two of the following sites relative to the amino acid sequence of the wild-type mouse PIEZO2 protein: R2788, E2797; or, relative to the amino acid sequence of the wild-type human PIEZO2 protein, has a mutation at the following site: R2718.

[0010] In view of the ion channel characteristic of PIEZO2 that it has rapid inactivation (inactivation constant is less than 10 milliseconds), the present invention proposes to reduce its inactivation rate by mutating PIEZO2, thereby increasing the calcium ions flowing into the cell to a level that can be used for cellular calcium signal detection by a high-throughput fluorescence imaging system, thereby carrying out high-throughput screening of PIEZO2 small molecule agonists and inhibitors.

[0011] Based on this design idea, the present invention conducted mutation screening on PIEZO2 and found that the inactivation rate of the mutant PIEZO2 was reduced. It was also identified for the first time that it could respond to the Yoda1 small molecule to increase the intracellular calcium ion concentration, proving the feasibility of this design idea.

[0012] Based on the PIEZO2 mutant discovered in the present invention, the screening of small molecule agonists and inhibitors of PIEZO2 can be carried out in combination with a high-throughput fluorescent calcium imaging system.

[0013] According to some specific embodiments of the present invention, the amino acid sequence of the mutant has mutations relative to the amino acid sequence of the wild-type mouse PIEZO2 protein: R2788A and / or ΔE 2797; or, the amino acid sequence of the mutant has mutations at the following sites relative to the amino acid sequence of the wild-type human PIEZO2 protein: R2718Q, R2718A, R2718G, R2718I, R2718L, R2718P, R2718V, R2718F, R2718W, R2718Y, R2718D, R2718E, R2718H, R2718K, R2718S, R2718T, R2718C, R2718M or R2718N.

[0014] The mutation sites of the present invention are shown in the following table:

[0015]

[0016] According to some specific embodiments of the present invention, the amino acid sequence of the wild-type mouse PIEZO2 protein is shown as SEQ ID NO:1; the amino acid sequence of the wild-type human PIEZO2 protein is shown as SEQ ID NO:2.

[0017] On the other hand, the present invention also provides a method for preparing the PIEZO2 protein mutant, which comprises: performing site-directed mutagenesis by genetic engineering or gene synthesis to obtain the PIEZO2 protein mutant.

[0018] According to some specific embodiments of the present invention, the method comprises using pCDNA3.1 as a vector to introduce the mutation site into PIEZO2.

[0019] According to some specific embodiments of the present invention, the method comprises introducing the mutation site into PIEZO2 using pCDNA3.1-PIEZO2-Flag-mRubby2 and / or pCDNA3.1-PIEZO2-pp-GST-IRES-GFP.

[0020] In yet another aspect, the present invention further provides a cell having a PIEZO2 channel, and the PIEZO2 channel protein has the amino acid sequence of the PIEZO2 protein mutant described in the present invention.

[0021] In yet another aspect, the present invention further provides a polynucleotide for encoding the PIEZO2 protein mutant described in the present invention.

[0022] In yet another aspect, the present invention further provides a vector comprising the polynucleotide of the present invention.

[0023] In yet another aspect, the present invention further provides a cell, wherein the cell contains the vector of the present invention or expresses the PIEZO2 protein mutant of the present invention.

[0024] In yet another aspect, the present invention further provides a kit comprising the PIEZO2 protein mutant, polynucleotide, vector, or cell described in the present invention.

[0025] In yet another aspect, the present invention also provides the use of the PIEZO2 protein mutant or the cell in drug screening.

[0026] According to some specific embodiments of the present invention, the drug is a PIEZO2 inhibitor or a PIEZO2 agonist.

[0027] In yet another aspect, the present invention further provides a method for drug screening, wherein the method comprises using the PIEZO2 protein mutant described in the present invention to perform drug screening.

[0028] According to some specific embodiments of the present invention, the method includes performing drug screening based on the PIEZO2 protein mutant and using a high-throughput fluorescence analysis system.

[0029] According to some specific embodiments of the present invention, the method comprises expressing the PIEZO2 protein mutant of the present invention in cells, and then performing drug screening using a high-throughput fluorescence analysis system.

[0030] According to some specific embodiments of the present invention, the method includes transfecting cells with a plasmid encoding the PIEZO2 protein mutant described in the present invention, setting a calcium indicator in the cells, and then using a high-throughput fluorescence analysis system to record the changes in intracellular calcium signals caused by the drug to be screened as indicated by the calcium indicator.

[0031] According to some specific embodiments of the present invention, the cell is HEK293T cell, HEK293 cell, HeLa cell, MDCK cell or CHO cell.

[0032] According to some specific embodiments of the present invention, the calcium indicator is a calcium indicator protein, or other calcium fluorescent dyes.

[0033] According to some specific embodiments of the present invention, wherein the setting of the calcium indicator in the cell comprises co-transfection of a plasmid encoding the calcium indicator protein in HEK293T cells. According to some specific embodiments of the present invention, wherein the calcium indicator protein is GCaMP6 or GCaMP6f.

[0034] According to some specific embodiments of the present invention, the other calcium fluorescent dye is selected from one or more combinations of Fura-2, Fluo-3, Fluo-4 and Calcium 6.

[0035] According to some specific embodiments of the present invention, the method comprises the following steps:

[0036] (1) In HEK293T cells, plasmids encoding PIEZO2-R2788A and mRuby were co-transfected with a plasmid encoding the calcium indicator GCaMP6;

[0037] (2) Replace the cell culture medium with the experimental buffer: 1xHBSS, 10 mM HEPES, pH 7.35;

[0038] (3) Use FLIPR to transfer the drugs to be screened to the cell culture plate and record the changes in calcium signals indicated by GCAMp6 in real time.

[0039] According to some specific embodiments of the present invention, the method comprises the following steps:

[0040] (1) In a 384-well cell culture plate in which HEK293T cells were cultured, a plasmid encoding PIEZO2-R2788A was co-transfected with a plasmid encoding the calcium indicator GCaMP6;

[0041] (2) Two days after transfection, the cell culture medium was replaced with the experimental buffer: 1xHBSS, 10 mM HEPES, pH 7.35 using an ELx406 CW plate washer (BioTek, Winooski, VT);

[0042] (3) Use FLIPR to transfer the drugs to be screened to the cell culture plate and record the changes in calcium signals indicated by GCAMp6 in real time.

[0043] According to some specific embodiments of the present invention, the drug is a PIEZO2 inhibitor or a PIEZO2 agonist.

[0044] According to some specific embodiments of the present invention, when the drug is a PIEZO2 inhibitor, the method further comprises first activating the PIEZO2 channel with a PIEZO2 agonist, and then using the activated PIEZO2 channel to screen the PIEZO2 inhibitor.

[0045] According to some specific embodiments of the present invention, when the drug is a PIEZO2 inhibitor, the method further comprises first activating the PIEZO2 channel using Yoda1, and then using the activated PIEZO2 channel to screen the PIEZO2 inhibitor.

[0046] According to some specific embodiments of the present invention, when the drug is a PIEZO2 inhibitor, it also includes transfecting cells with a plasmid encoding the PIEZO2 protein mutant of the present invention, setting a calcium indicator in the cell, then activating the PIEZO2 channel with a PIEZO2 agonist, and then using a high-throughput fluorescence analysis system to screen the inhibitors.

[0047] In another aspect, the present invention also provides the use of a drug screened by the drug screening method of the present invention as a PIEZO2 inhibitor or a PIEZO2 agonist.

[0048] In another aspect, the present invention also provides the use of drugs screened by the drug screening method of the present invention in the preparation of analgesic drugs.

[0049] According to some specific embodiments of the present invention, the PIEZO2 agonist is Yodal; and the PIEZO2 inhibitor is Pranlukast or Zafirlukast.

[0050] In yet another aspect, the present invention also provides use of Pranlukast or Zafirlukas as a PIEZO2 inhibitor.

[0051] In yet another aspect, the present invention also provides use of a PIEZO2 inhibitor in the preparation of analgesics, wherein the PIEZO2 inhibitor is Pranlukast or Zafirlukas.

[0052] In summary, the present invention provides a PIEZO2 protein mutant and its preparation and application. The technical solution of the present invention has the following advantages:

[0053] In view of the ion channel characteristic of PIEZO2 that it has rapid inactivation (inactivation constant is less than 10 milliseconds), the present invention proposes to reduce its inactivation rate by mutating PIEZO2, thereby increasing the calcium ions flowing into the cell to a level that can be used for cellular calcium signal detection by a high-throughput fluorescence imaging system, thereby carrying out high-throughput screening of PIEZO2 small molecule agonists and inhibitors.

[0054] Based on the PIEZO2 mutant discovered in the present invention, the screening of small molecule agonists and inhibitors of PIEZO2 can be carried out in combination with a high-throughput fluorescent calcium imaging system.

[0055] The present invention overcomes the following existing problems: 1) it is difficult to activate PIEZO2 by applying mechanical force stimulation in a high-throughput manner; 2) it is difficult to detect calcium signals mediated by wild-type PIEZO2. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1A Schematic diagram of the mechanosensitive current of the PIEZO2 mutant in the whole-cell recording mode of Example 2.

[0057] Figure 1B Schematic diagram of the inactivation time of mutants in the whole-cell recording mode of PIEZO2 in Example 2.

[0058] Figure 1C for Figure 1A and Figure 1B Representative whole-cell mechanosensitive current traces in Figure 2.

[0059] Figure 1D The graph shows the increase in calcium signals of different channels induced by Yoda1 in Example 3.

[0060] Figure 1EThis is a graph showing the effects of the presence and absence of 10 μM Moda1 on the size of the mechanically sensitive current (left) and the inactivation constant (right) in the whole-cell recording mode of Example 3.

[0061] Figure 1F This is a representative time curve diagram of Example 3 in the whole-cell recording mode in the presence and absence of 10 μM Yoda1.

[0062] Figure 2 These are the concentration-dependent efficacy curves of Yoda1 in Example 4 for activating PIEZO2-R2788A.

[0063] Figure 3A The graph shows the structural formula of Pranlukast and the numbered curves of calcium signals induced in cells expressing Vector (left), mPIEZO1 (middle), and mPIEZO2-R2788A (right).

[0064] Figure 3B The concentration-dependent inhibitory efficacy curve of Pranlukast.

[0065] Figure 4 Dose response curve of NeZha1 for FLIPR analysis of calcium response to PIEZO2-R2788A Yoda1.

[0066] Figure 5 Scatter plots of the maximum mechanically activated current of HEK cells transfected with wild-type PIEZO2 treated with Nezha1 and Nezha2, respectively;

[0067] Figure 6 Scatter plot of maximum mechanically activated current and statistical graph of inactivation time constant tau value of wild-type human PIEZO2 HEK cells transfected with Nezha1.

[0068] Figure 7 Mechanosensitive currents recorded from cultured wild-type DRG neurons treated with Nezha1.

[0069] Figure 8 and Fig. 9 It is a graph of the paw retraction percentage for different gram weights of von Frey.

[0070] Fig.10 and Fig.11 It is a graph showing the percentage of von Frey response before and after CFA treatment and intraplantar injection.

[0071] Fig.12 and Fig.13 This is a graph showing the von Frey response curve after intragastric administration of Vehicle or Nezha1 to mice.

[0072] Fig.14 and Fig.15 It is a graph showing the percentage of response before and after CFA treatment and after oral administration. DETAILED DESCRIPTION

[0073] The implementation process of the present invention and the beneficial effects produced are described in detail below through specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0074] Example 1

[0075] This example provides a method for screening small molecule agonists of wild-type PIEZO-2 (PIEZO2-WT) based on the FLIPR high-throughput fluorescent calcium imaging system:

[0076] (1) HEK293T cells were cultured in a 384-well cell culture plate, and the plasmid encoding PIEZO2-WT was co-transfected with the plasmid encoding the calcium indicator GCaMP6s or GCaMP6f. The amount of plasmids transfected was PIEZO1-WT (55 ng / well), GCaMP6f / s (25 ng / well), PIEZO2-WT (90 ng / well), PEI was 2 μl / well, the amount of cells was 15,000-20,000 / well, 40 μl / well, and the transfection system was 20 μl / well.

[0077] (2) Two days after transfection, the cell culture medium was replaced with the experimental buffer: 1x HBSS, 10 mM HEPES, pH 7.35 using an ELx406 CW plate washer (BioTek, Winooski, VT).

[0078] (3) Use FLIPR to transfer compounds to cell culture plates and record changes in calcium ion signals in real time.

[0079] (4) A total of 63,000 small molecule compounds were screened.

[0080] (5) Cells expressing PIEZO1 / GCAMP6 responded to the PIEZO agonist Yoda1, with a significant increase in calcium signals indicated by GCAMAMP6.

[0081] (6) No small molecule drugs were screened that could specifically increase calcium signals in PIEZO2 / GCAMP6-expressing cells.

[0082] Example 2

[0083] This example provides the construction and screening of the PIEZO2 slow inactivation mutant of the present invention:

[0084] Since more than 3 million compounds were screened in the literature, the present invention screened more than 60,000 compounds in Example 1, and no agonist of PIEZO2-WT was identified using the FLIPR high-throughput calcium imaging system. Based on this, it is speculated that since the expression level of PIEZO2 is only about 1 / 10 of that of PIEZO1, and the inactivation is faster than PIEZO1, the increase in calcium signal mediated by PIEZO2-WT may not be sufficient to be detected by the FLIPR system. Therefore, this example constructs and screens mutants that slow down the inactivation rate of PIEZO2 for screening of PIEZO2 agonists based on calcium signals.

[0085] This example was constructed on the vectors pCDNA3.1-PIEZO2-Flag-mRubby2 and pCDNA3.1-PIEZO2-pp-GST-IR ES-GFP (according to ClonExpress II One Step Cloning Kit (Novozyme) was used to design primers and construct the corresponding sites of PIEZO2 (R2788A and ΔE2797) (Table 1); the construction methods of pCDNA3.1-PIEZO2-Flag-mRubby2 and pCDNA3.1-PIEZO2-pp-GST-IRES-GFP were as follows: Piezo2 was subcloned into the pcDNA3.1 expression vector, which encodes a C-terminal glutathione S-transferase (GST) tag with a precise protease cleavage site in the middle, followed by a GFP coding sequence driven by an internal ribosomal entry site (IRES) (for monitoring the efficiency of transient transfection and protein expression). We call this expression plasmid PIEZO2-pp-GST-IRES-GFP. The C-terminal GST and IRES-GFP were replaced by the fusion-expressed m Rubby2-Flag tag, namely pCDNA3.1-PIEZO2-Flag-mRubby2. All mutants were subcloned using the One Step Cloning Kit (Vazyme Biotech) (see Structure and mechanogating of the mammalian tactile channel PIEZO2).

[0086] Table 1

[0087]

[0088]

[0089] PIEZO2 wild type (WT) and mutants were expressed in HEK293T cells, and the current induced by mechanical stimulation was recorded using the electrophysiological patch clamp method. It was found that the current size of PIEZO2 mutants PIEZO2-R2788A and PIEZO2-ΔE2797 did not change significantly, but the inactivation was significantly slowed down ( Figures 1A to 1C ,in Figure 1A and Figure 1B They are statistical graphs of maximum current and inactivation time, respectively, and each point represents a cell; Figure 1C This indicates that the inactivation of the mutant is significantly slowed down after mutation), and the inactivation of PIEZO2-R2788A is more obvious than that of PIEZO2-ΔE2797.

[0090] Example 3

[0091] This example provides a method for discovering a small molecule agonist of PIEZO2, Yoda1, using the slow-inactivation mutant PIEZO2-R2788A:

[0092] PIEZO1-WT, PIEZO2-WT, slow-inactivation mutants PIEZO2-R2788A and PIEZO2-ΔE2797 were co-expressed with GCAMPI6 in HEK293T cells, and FLIPR was used to detect changes in calcium signals indicated by GCAMPI6. It was found that PIEZO1-WT and PIEZO2-R2788A could respond to Yoda1 to cause an increase in calcium signals, while PIEZO2-WT and PIEZO2-ΔE2797 did not respond to Yoda1 to cause an increase in calcium signals ( Figure 1D ).

[0093] The above results suggest that Yoda1 can activate PIEZO2, but the calcium signal caused by the rapid inactivation of PIEZO2 cannot be detected (although the inactivation rate of PIEZO2-ΔE2797 is slowed down, Yoda1 is not enough to cause detectable changes in calcium signals). To prove this hypothesis, the present invention expressed PIEZO2 in HEK293T cells, first recorded a stable mechanosensitive current in the cells, then added 10 μM Yoda1 and gave the same mechanical force stimulation, and found that Yoda1 can enhance the mechanosensitive current mediated by PIEZO2, but does not affect its inactivation rate ( Figure 1E and Figure 1F ). Therefore, the present invention discovered the small molecule agonist Yoda1 of PIEZO2 by using the slow inactivation mutant PIEZO2-R2788A and calcium signal detection.

[0094] Example 4

[0095] This example provides a method for high-throughput screening of small molecule agonists of PIEZO2 using the slow-inactivation mutant PIEZO2-R2788A and FLIPR:

[0096] (1) Plasmids encoding PIEZO1-WT, PIEZO2-R2788A, and mRuby were co-transfected with a plasmid encoding the calcium indicator GCaMP6 in a 384-well cell culture plate for culturing HEK293T cells.

[0097] (2) Two days after transfection, the cell culture medium was replaced with the experimental buffer: 1x HBSS, 10 mM HEPES, pH 7.35 using an ELx406 CW plate washer (BioTek, Winooski, VT).

[0098] (3) Use FLIPR to transfer compounds to cell culture plates and record changes in calcium signals indicated by GCAMp6 in real time.

[0099] After two rounds of screening, 5274 compounds were screened, and Yoda1, an agonist of PIEZO2-R2788A, was identified.

[0100] EC of PIEZO2-R2788A activated by -Yoda1 detected by FLIPR 50 1.5μM ( Figure 2 , which shows the concentration-dependent efficacy curve of Yoda1 activating PIEZO2-R2788A and the EC obtained by fitting. 50 ).

[0101] Example 5

[0102] This example provides a high-throughput screening of small molecule inhibitors of PIEZO2 using the slow-inactivation mutant PIEZO2-R2788A, the agonist Yoda1, and FLIPR:

[0103] (1) In a 384-well cell culture plate in which HEK293T cells were cultured, a plasmid encoding PIEZO2-R2788A was co-transfected with a plasmid encoding the calcium indicator GCaMP6;

[0104] (2) Two days after transfection, the cell culture medium was replaced with the experimental buffer: 1xHBSS, 10 mM HEPES, pH 7.35 using an ELx406 CW plate washer (BioTek, Winooski, VT);

[0105] (3) 3000 small molecule compounds in the Selleck drug library were transferred to the cell culture plate using FLIPR, and the calcium signal indicated by GCaMP6 was recorded in real time for 200 seconds, followed by the addition of 6 mM Yoda1 to activate the expressed PIEZO2-R2788A;

[0106] (4) By comparing with the control group with DMSO, three drugs were screened that have inhibitory effects on Yoda1-induced PIEZO2-R2788A-mediated calcium signaling: Pranlukast (CAS103177-37-3) ( Figure 3A , which shows the structural formula of Pranlukast and the numbered curves of calcium signals induced in cells expressing Vector (left), mPIEZO1 (middle), and mPIEZO2-R2788A (right). ATP was added to cells expressing Vector after 200 seconds, while 20 μM Yoda1 was added to cells expressing PIEZO1 or PIEZO2-R2788A);

[0107] (5) IC of Pranlukast on Yoda1-induced PIEZO2-R2788A-mediated calcium signal inhibition was obtained using FLIPR assay 50 The concentrations of Pranlukast and PIEZO1-WT were 6mM, 1mM, and 9mM, respectively, but Pranlukast had no inhibitory effect on the calcium signal mediated by Yoda1-induced PIEZO1-WT or slowly inactivated PIEZO1-R2482A, indicating that Pranlukast specifically inhibits the calcium response of Yoda1-induced PIEZO2-R2788A ( Figure 3B , which shows the concentration-dependent inhibitory efficacy curve of Pranlukast and the IC50 obtained by fitting).

[0108] The present invention named the screened small molecule inhibitors of PIEZO2 as Nezha series: Nezha1 (Pranlukast).

[0109] Example 6

[0110] NeZha1 (Pranlukast) inhibits Yoda1-induced PIEZO2-R2788A-mediated calcium response experiment

[0111]

[0112] Experimental materials: 293T cells, mPiezo1 plasmid, Piezo2-R2788A plasmid, Gcamp6s plasmid.

[0113] Experimental methods:

[0114] (1) In a 384-well cell culture plate in which HEK293T cells were cultured, a plasmid encoding PIEZO2-R2788A or a plasmid encoding PIEZO1 was co-transfected with a plasmid encoding the calcium indicator GCaMP6;

[0115] (2) Two days after transfection, the cell culture medium was replaced with 20 μl of the experimental buffer: 1x HBSS, 10 mM HEPES, pH 7.35 using an ELx406 CW plate washer (BioTek, Winooski, VT);

[0116] (3) Using FLIPR, 20 μl of 100 μM, 33.33 μM, 11.11 μM, 3.7 μM, 1.23 μM, 0.4 μM, and 0.14 μM NeZha1 were transferred to the cell culture plate, and the calcium signal indicated by GCaMP6 was recorded in real time for 200 s. Subsequently, 20 μl of 60 μM Yoda1 was added to activate the expressed PIEZO2-R2788A; the final concentrations of NeZha1 were 33.33 μM, 11.11 μM, 3.7 μM, 1.23 μM, 0.4 μM, 0.14 μM, and 0.046 μM, respectively, and the final concentration of Yoda1 was 22.22 μM.

[0117] Experimental results:

[0118] The results are as follows Figure 4 As shown, Figure 4 Figure 3 is the dose-response curve of NeZha1 on the calcium response of PIEZO2-R2788A Yoda1 analyzed by FLIPR. The curve was fitted with the Boltzmann equation and each data point represents the mean ± SEM. A is the IC of Nezha1 for inhibiting the Yoda1-induced PIEZO2-R2788A-mediated calcium response. 50 0.67 μM; Figure 4 B is the calcium response curve of the control compound (NeZha6 montelukast) inhibiting Yoda1-induced PIEZO2-R2788A-mediated.

[0119] The structure of the control compound is:

[0120]

[0121] Example 7

[0122] Inhibition of mechanosensitive currents mediated by wild-type mouse and human PIEZO2 by Nezha1 and compound NeZha2 (Zafirlukas)

[0123]

[0124] Experimental materials: HEK293T cells, Axopatch 200B amplifier, Narishige micromanipulation system, BF150-86-10 glass electrode, MF830 electrode polisher, heka patch clamp electrophysiology system.

[0125] Experimental methods: wild-type mouse or human PIEZO2 plasmids were transfected into HEK293 cells, and electrophysiological patch clamp techniques were used to test the inhibitory effect of Nezha1 on the mechanosensitive current mediated by wild-type mouse or human PIEZO2. The details are as follows:

[0126] HEK293T cells were cultured, transiently transfected, and patch-clamp experiments were performed using an Axopatch 200B amplifier (Axon Instruments). For whole-cell patch-clamp recordings, the resistance of the recording electrode was 2–3 MΩ when filled with an intracellular solution consisting of (in mM) 133 CsCl, 1 CaCl2, 1 MgCl2, 5 EGTA, 10 HEPES (pH 7.3 with CsOH), 4 MgATP, and 0.4 Na2GTP. The extracellular solution consisted of (in mM) 133 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES (pH 7.3 with NaOH), and 10 glucose. All experiments were performed at room temperature. Currents were sampled at 20 kHz and filtered at 2 kHz using Clampex 10.4 software (Axon Instruments).

[0127] During patch clamp recordings, mechanical stimulation was applied to the cells at an angle of 80° using a fire-polished glass pipette (tip diameter 3–4 μm). The downward movement of the probe to the cell was controlled by a clampex-controlled piezoelectric crystal micromanipulator (E625LVPZT controller / amplifier; Physics Instruments). The probe up and down movement speed was 1 μm / ms, and the stimulation was maintained for 150 ms. A series of mechanical steps of 1 μm increments were applied every 20 s, and the current was recorded at a holding potential of −70 mV. In step-poking experiments, the cell was ruptured at the end of the poking. In agonist stimulation experiments, after a mechanical force stimulation was applied to the cell to generate a stable mechanosensitive current, DMSO or Yoda1 was added to the external solution, and the cell was stimulated with a mechanical force of the same step size, and the current was recorded.

[0128] Experimental results:

[0129] like Figure 5 As shown, Figure 5Scatter plots of the maximum mechanically activated current recorded in HEK cells transfected with wild-type mouse PIEZO2 treated with Nezha1 and Nezha2, respectively. Statistical significance was assessed by one-way analysis of variance (ANOVA) compared with PIEZO2 without drug treatment, *p<0.05. Figure 5 It showed that Nezha1 and compound NeZha 2 could significantly inhibit the mechanosensitive current mediated by wild-type PIEZO2.

[0130] like Figure 6 As shown, Figure 6 The maximum mechanically activated current scatter plot and inactivation time constant tau value statistical graph of Nezha1-treated wild-type human PIEZO2 HEK cells were recorded. One-way analysis of variance (ANOVA) was used to evaluate the statistical significance compared with PIEZO2 without drug treatment, *p<0.05. Figure 6 It showed that Nezha1 could significantly inhibit the mechanosensitive current mediated by wild-type human PIEZO2, but did not affect the inactivation time constant.

[0131] Example 8

[0132] Nezha1 inhibits PIEZO2 at the cellular and animal levels

[0133] Experimental materials: B6 wild-type mice.

[0134] Experimental method: The present invention cultured primary wild-type mouse dorsal root ganglion cells (DRG) in vitro, collected DRG from B6 wild-type male mice, digested with 1.25% collagenase and papain, centrifuged, transferred to polyD-laminin slides, and cultured in DMEM / F-12+NGF+GDNF medium for 24 hours. PIEZO2 was highly expressed on DRG. Using patch clamp technology, it was found that the mechanosensitive current of DRG neurons treated with 20μM Nezha1 was reduced compared with the mechanosensitive current of DRG neurons treated with the same amount of solvent in the control group, and this inhibitory effect could be reversed by washing away the compound.

[0135] Experimental results:

[0136] like Figure 7 As shown, it is shown that Nezha1 can inhibit endogenously expressed PIEZO2. The control group is an equal amount of solvent (electrophysiological external solution + equal volume of DMSO).

[0137] Example 9

[0138] Mouse animal model experiment

[0139] Experimental materials: B6 wild-type male mice.

[0140] 4.1 Administration by injection

[0141] Experimental methods: This experiment tested the PIEZO2-mediated tactile sensation and CFA-induced touch pain in mice by intraplantar injection of 4 mM Nezha1 and von Frey test 15 minutes later, and compared with injection of Vehicle (PBS+1% DMSO+0.5% Tween80).

[0142] Experimental results:

[0143] like Figures 8 to 11 As shown, Figure 8 and Fig. 9 is the percentage of paw withdrawal to different gram weights of von Frey (6 tests) [Vehicle, n = 25; NeZha6 n = 8; NeZha1, n = 26]; Fig.10 and Fig.11 The percentage of von Frey responses before CFA treatment (baseline, n = 52), 24 hours after CFA treatment, and intraplantar injection of Vehicle (n = 24), 4mM Nezha6 (n = 4), or 4mM NeZha1 (n = 21) (6 trials). Compared with Vehicle injection, Nezha1 injection significantly inhibited the tactile sensation of mice ( Figure 8 and Fig. 9 ) and CFA-induced pain responses ( Fig.10 and Fig.11 ). In addition, the experiment found that 1mM Nezha1 also had the same inhibitory effect.

[0144] The components of the vehicle are: 1% DMSO and 0.5% Tween80 in PBS solution.

[0145] 4.2 Oral administration

[0146] Experimental methods: This experiment tested the PIEZO2-mediated tactile sensation and CFA-induced allodynia in mice. The CFA-induced inflammation model was induced by intraplantar injection of 10 μl of Freund's complete adjuvant, and behavioral tests were performed 24 hours later. Nezha1 was administered by gavage at 100 mg / kg, and the von Frey test was performed 1 hour later, and the mice were compared with mice gavaged with normal saline.

[0147] Experimental results:

[0148] like Figures 12 to 15 As shown, Fig.12 and Fig.13The mice were intragastrically administered with Vehicle (NaOH aqueous solution with equimolar concentration), 10 mg / kg or 100 mg / kg NeZha1, and the von Frey reaction was tested at 1 h after administration [Vehicle, n = 23; 10 mg / kg NeZha, n = 6; 100 mg / kg NeZha1, n = 15]; Fig.14 and Fig.15 The percentage of von Frey responses before CFA treatment (baseline, n=23), 24 h after CFA treatment, and after intragastric administration of Vehicle (n=14), 10 mg / kg Nezha6 (n=4), or 100 mg / kg NeZha1 (n=10) (6 experiments). Figures 12 to 15 It shows that the mouse's sense of touch ( Fig.12 and Fig.13 ) and CFA-induced allodynia ( Fig.14 and Fig.15 ) showed a significant inhibitory effect.

[0149] Efficacy experiments at the animal level further demonstrated that Nezha1 can effectively inhibit the action of Piezo2 and may be used as a potential analgesic drug.

Claims

1. A PIEZO2 protein mutant, wherein: The amino acid sequence of the mutant has mutations at one or two of the following sites relative to the amino acid sequence of the wild-type mouse PIEZO2 protein: R2788, E2797, or, relative to the amino acid sequence of the wild-type human PIEZO2 protein, has mutations at the following sites: R2718.

2. The PIEZO2 protein mutant according to claim 1, wherein The amino acid sequence of the mutant has mutations relative to the amino acid sequence of the wild-type mouse PIEZO2 protein: R2788A and / or ΔE 2797; or, the amino acid sequence of the mutant has mutations at the following sites relative to the amino acid sequence of the wild-type human PIEZO2 protein: R2718Q, R2718A, R2718G, R2718I, R2718L, R2718P, R2718V, R2718F, R2718W, R2718Y, R2718D, R2718E, R2718H, R2718K, R2718S, R2718T, R2718C, R2718M or R2718N.

3. The PIEZO2 protein mutant according to claim 1 or 2, wherein: The amino acid sequence of the wild-type mouse PIEZO2 protein is shown in SEQ ID NO:1; the amino acid sequence of the wild-type human PIEZO2 protein is shown in SEQ ID NO:

2.

4. A method for preparing a PIEZO2 protein mutant according to any one of claims 1 to 3, comprising: The PIEZO2 protein mutant is obtained by performing site-directed mutagenesis through genetic engineering or gene synthesis.

5. The method according to claim 4, wherein: The method comprises using pCDNA3.1 as a vector to introduce the mutation site into PIEZO2; preferably using pCDNA3.1-PIEZO2-Flag-mRubby2 and / or pCDNA3.1-PIEZO2-pp-GST-IRES-GFP to introduce the mutation site into PIEZO2.

6. A polynucleotide encoding the PIEZO2 protein mutant according to any one of claims 1 to 3. A vector comprising the polynucleotide according to claim 6.

8. A cell, comprising the vector according to claim 7 or expressing the PIEZO2 protein mutant according to any one of claims 1 to 3. 9 . A kit comprising the PIEZO2 protein mutant according to any one of claims 1 to 3 , the polynucleotide according to claim 6 , the vector according to claim 7 , or the cell according to claim 8 .

10. Use of the PIEZO2 protein mutant according to any one of claims 1 to 3 or the cell according to claim 8 in drug screening.

11. The use according to claim 10, wherein: The drug is a PIEZO2 inhibitor or a PIEZO2 agonist.

12. A drug screening method, wherein: The method comprises using the PIEZO2 protein mutant described in any one of claims 1 to 3 to perform drug screening.

13. The drug screening method according to claim 12, wherein: The method comprises performing drug screening based on the PIEZO2 protein mutant and utilizing a high-throughput fluorescence analysis system.

14. The drug screening method according to claim 13, wherein: The method comprises expressing the PIEZO2 protein mutant according to any one of claims 1 to 3 in cells (preferably HEK293T cells, HEK293 cells, HeLa cells, MDCK cells or CHO cells), and then performing drug screening using a high-throughput fluorescence analysis system.

15. The drug screening method according to claim 14, wherein: The method comprises transfecting cells with a plasmid encoding the PIEZO2 protein mutant according to claims 1 to 3, and setting a calcium indicator in the cells (preferably co-transfecting HEK293T cells with a plasmid encoding the calcium indicator), and then using a high-throughput fluorescence analysis system to record the changes in intracellular calcium signals caused by the drug to be screened indicated by the calcium indicator.

16. The drug screening method according to any one of claims 12 to 15, wherein: The drug is a PIEZO2 inhibitor or a PIEZO2 agonist.

17. The drug screening method according to claim 16, wherein: When the drug is a PIEZO2 inhibitor, the method further comprises first activating the PIEZO2 channel using a PIEZO2 agonist (preferably Yoda1), and then using the activated PIEZO2 channel to screen the PIEZO2 inhibitor (preferably using a plasmid encoding the PIEZO2 protein mutant described in claims 1 to 3 to transfect cells, and setting a calcium indicator in the cells, then activating the PIEZO2 channel using a PIEZO2 agonist, and then using a high-throughput fluorescence analysis system to screen the inhibitors).

18. Use of a drug screened by the drug screening method according to any one of claims 12 to 16 as a PIEZO2 inhibitor or PIEZO2 agonist.

19. Use of a drug screened by the drug screening method according to any one of claims 12 to 16 in the preparation of analgesic drugs.

20. The use according to claim 18 or 19, wherein: The PIEZO2 agonist is Yodal; the PIEZO2 inhibitor is Pranlukast or Zafirlukas.

21. Use of Pranlukast or Zafirlukas as a PIEZO2 inhibitor.

22. Use of a PIEZO2 inhibitor in the preparation of analgesics, wherein the PIEZO2 inhibitor is Pranlukast or Zafirlukas.

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