A serine protease inhibitor and its application

Abstract

A serine protease inhibitor and its application, including Fc-serpinE3-404 and Fc-serpinE3-424. The amino acid sequence of Fc-serpinE3-404 is shown in SEQ ID NO.1, and the amino acid sequence of Fc-serpinE3-424 is shown in SEQ ID NO.2. The serine protease inhibitors Fc-serpinE3-404 and Fc-serpinE3-424 of the present invention can bind to TMPRSS11E and effectively inhibit the activity of TMPRSS11E. Therefore, the serine protease inhibitor of the present invention can effectively inhibit the inflammatory response by inhibiting the activity of TMPRSS11E. A yeast strain stably expressing this fusion protein is obtained, with high expression level and low cost. In terms of purification, the affinity chromatography step in the purification process has a very high purification efficiency and is easy to scale up production.

Description

Technical Field

[0001] The present invention belongs to the field of protease inhibitors, and particularly relates to a serine protease inhibitor and its application. Background Art

[0002] Abnormal serine protease activity or imbalance between protease and protease inhibitor can lead to protease-mediated inflammatory responses. Therefore, there is a need for therapeutic agents targeting abnormal serine protease activity or the imbalance between protease and protease inhibitor. The serpinE3 fusion protein of the present invention has the potential to inhibit the activity of inflammatory cytokines.

[0003] Serine protease inhibitor (serpins) protein family is a protease inhibitor family with numerous members and wide distribution. It can inhibit serine protease activity and participate in regulating various physiological reactions in organisms, playing an important role in processes such as blood coagulation, immune response, fibrinolysis, inflammation, and tumor suppression in organisms. The functions of members of the serpins protein family are significantly different. Serpin inactivates the target protease by forming a covalent complex with it. The reactive centre loop (RCL) of Serpin is mainly composed of about 20 amino acid residues located at the C-terminus, exposed outside the main body of the Serpin protein, and carrying sites that can be specifically recognized by the target enzyme. The action mechanism of Serpin is different from the interaction between conventional enzymes and substrates. It completely inactivates the target enzyme by disrupting the structure of the target enzyme. Serpin first forms a covalent ester bond with the target enzyme, that is, forms an acyl-enzyme intermediate. It is explained that: RCL pulls the target enzyme to the other side of Serpin, thus causing conformational changes of the target enzyme. Therefore, Serpin-type inhibitors are also called suicide inhibitors.

[0004] In the human body, serine protease inhibitor Serpin can be divided into 9 subfamilies A-I, and the SerpinE family is one of them, including three members: SerpinE1, SerpinE2, and SerpinE3. Among them, SerpinE3 has been rarely studied. SerpinE3 has two coding forms, namely the full-length 404-amino acid form and the 424-amino acid form. Currently, there is no research on the preparation and functional application of SerpinE3. Summary of the Invention

[0005] Technical problem to be solved: The present invention provides a serine protease inhibitor and its application. The provided fusion protein shows inhibitory activity. Specifically, the genes encoding the full-length 404 amino acids and 424 amino acids of human serpinE3 are cloned, linked with Fc, and the genes are transformed into yeast. The Fc-serpinE3-404 and Fc-serpinE3-424 fusion proteins are purified. They have strong inhibitory activity against the serine protease TMPRSS11E. The serine protease TMPRSS11E is elevated in diseases such as lung cancer and inflammatory infections. Inhibiting TMPRSS11E can reduce inflammation and inhibit tumor progression.

[0006] The Fc-serpinE3-404 and Fc-serpinE3-424 proteins provided by the present invention can act as negative regulators to inhibit the activity of TMPRSS11E. The serine protease inhibitor fusion proteins described herein are expected to be used to reduce inflammatory symptoms including lung infections.

[0007] Technical solution: A serine protease inhibitor, including Fc-serpinE3-404 and Fc-serpinE3-424. The amino acid sequence of Fc-serpinE3-404 is shown in SEQ ID NO.1, and the amino acid sequence of Fc-serpinE3-424 is shown in SEQ ID NO.2.

[0008] The nucleotide sequence of the above-mentioned Fc-serpinE3-404 is shown in SEQ ID NO.3.

[0009] The nucleotide sequence of the above-mentioned Fc-serpinE3-424 is shown in SEQ ID NO.4.

[0010] A plasmid containing the above nucleotide.

[0011] A vector containing the above plasmid.

[0012] The above vector is yeast, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells or COS cells.

[0013] The application of the above serine protease inhibitor in the preparation of drugs for treating infectious inflammatory diseases such as bacterial and viral infections.

[0014] A drug for treating inflammations such as acute respiratory distress syndrome (ARDS), viral infections or sepsis, containing the above serine protease inhibitor.

[0015] Beneficial effects: The human serine protease inhibitors provided by the present invention include two forms, namely Fc-serpinE3-404 and Fc-serpinE3-424. TMPRSS11E can lead to enhanced inflammation. The serine protease inhibitors Fc-serpinE3-404 and Fc-serpinE3-424 of the present invention can bind to TMPRSS11E and effectively inhibit the activity of TMPRSS11E. Therefore, the serine protease inhibitor of the present invention can effectively inhibit the inflammatory response by inhibiting the activity of TMPRSS11E. A yeast strain stably expressing this fusion protein is obtained, with high expression level and low cost. In terms of purification, the affinity chromatography step in the purification process has a very high purification efficiency and is easy to scale up production. Description of the Drawings

[0016] Figure 1 Showing that serpinE3 can interact with TMPRSS11E protein.

[0017] Figure 2 Showing the construction of Fc-serpinE3-404 plasmid and Fc-serpinE3-424 plasmid.

[0018] Figure 3 Showing the SDS-PAGE electrophoresis results of the purified proteins of Fc-serpinE3-404 and Fc-serpinE3-424.

[0019] Figure 4 Showing the SDS-PAGE electrophoresis results of the purified protein of TMPRSS11E.

[0020] Figure 5 Showing the cleavage of the PAR1 substrate peptide by the purified protein of TMPRSS11E. Among them, A. Schematic diagram of the N-terminal site of PAR1 cleaved by TMPRSS11E. B. The fluorescent peptide Abz-LDPRSFLL-Y(3-NO2)(100 μM) was incubated with the purified TMPRSS11E-CD protein or thrombin as substrates respectively, and the light absorption value was detected. C. Determination of the kinetic parameters of TMPRSS11E-CD cleaving the substrate Abz-LDPRSFLL-Y(3-NO2).

[0021] Figure 6Fc-serpinE3-404 and Fc-serpinE3-424 were shown to inhibit the cleavage of PAR1 fluorogenic peptide by TMPRSS11E. Inhibitors at different concentrations (0 - 30 nM) were incubated with a fixed concentration of enzyme (10 nM) at 37 °C in a buffer containing 100 mM Tris / HCl (pH 8.5) with 500 μg / mL BSA, and then 100 μM of the PAR1 fluorogenic peptide substrate Abz-LDPRSFLL-Y(3-NO2) was added. The total volume of the system was 100 μL, and the fluorescence value at 420 nm was recorded by a multimode microplate reader to determine the residual enzyme activity.

[0022] Figure 7 It was shown that TMPRSS11E was induced to be upregulated in the lung tissues of a mouse model of LPS-induced acute lung injury and was mainly expressed in lung epithelial cells and macrophages. (A) qPCR was used to detect the mRNA level of TMPRSS11E in mouse lung tissues. (B) Western blot was used to verify the protein expression of TMPRSS11E in mouse lung tissues. (C) qPCR was used to detect the mRNA levels of TNF-α, IL-1β, and IL-6 in mouse lung tissues. (D) Immunohistochemistry was used to analyze the expression of TMPRSS11E. Red arrows represent macrophages; black arrows represent epithelial cells; scale bar: 100 μm.

[0023] Figure 8 .Knockdown of TMPRSS11E inhibited lung tissue injury in a mouse model induced by LPS. (8A) qPCR and western blot were used to detect the expression of TMPRSS11E. Left: qPCR analysis; Right: western blot. (8B) H&E staining was used to analyze the pathological changes in mouse lung tissues. (8C) TMPRSS11E was knocked down in vivo, and qPCR was used to detect the mRNA levels of TNF-α, IL-1β, and IL-6 in mouse lung tissues.

[0024] Figure 9 Fc-serpinE3-404 and Fc-serpinE3-424 proteins significantly inhibited the secretion of inflammatory factors such as TNF-α, IL-1β, and IL-6 by LPS-stimulated macrophages. Detailed implementation manners

[0025] Example 1

[0026] To search for serpin inhibitors of TMPRSS11E, we constructed eukaryotic expression plasmids of SerpinE1, serpinE2, SerpinE3-404, and SerpinE3-424 (pCMV3-SERPINE1-MYC, pCMV3-SERPINE2-MYC, pCMV3-SERPINE3-404-MYC, pCMV3-SERPINE3-424-MYC), and simultaneously constructed a eukaryotic expression plasmid of TMPRSS11E, pCMV3-11E-FLAG. Then, by co-transfecting 293T cells with plasmids and performing co-immunoprecipitation, it was found that only SerpinE3-404 and SerpinE3-424 could interact with TMPRSS11E. The results are shown in Figure 1 。

[0027] Example 2

[0028] Construct recombinant expression vectors by fusing serpinE3-404 or serpinE3-424 to the Fc portion of IgG and inserting them into the pPICZ(alpha)C backbone plasmid. The plasmid construction diagrams are shown in Figure 2 。The specific construction steps are divided into two steps. The first step is to insert Fc, and the second step is to insert serpinE3-404 or serpinE3-424.

[0029] When inserting Fc in the first step, the restriction enzyme sites used are as follows: the N-terminal restriction enzyme site is EcoR I, and the C-terminal restriction enzyme site is Not I.

[0030] In the second step, insert serpinE3-404 or serpinE3-424. Construct pPICZa-Fc-serpinE3-404 and pPICZa-Fc-serpinE3-424 plasmids.

[0031] When constructing the pPICZa-Fc-serpinE3-404 plasmid and inserting serpinE3-404, the restriction enzyme sites are as follows: the N-terminal restriction enzyme site is Sal I, and the C-terminal restriction enzyme site is Age I.

[0032] The PCR amplification primers are as follows:

[0033] F: 5-TCGACCACCTCCGTGAAGGAA-3

[0034] R: 5-CCGGTTTAGTCTAGGGGATTT-3

[0035] Construct the pPICZa-Fc-serpinE3-424 plasmid. When inserting serpinE3-424, the restriction enzyme sites are as follows: the N-terminal restriction enzyme site is Sal I, and the C-terminal restriction enzyme site is AgeI.

[0036] The PCR amplification primers are as follows:

[0037] F: 5-TCGACCACCTCCGTGAAGGAA-3

[0038] R: 5-CCGGTCTAGAAAGAATGCTTA-3

[0039] Example 3: Yeast expression and purification to obtain Fc-serpinE3-404 and Fc-serpinE3-424 proteins.

[0040] Transform the constructed pPICZa-Fc-serpinE3-404 or pPICZa-Fc-serpinE3-424 recombinant plasmid into the competent cells of Pichia pastoris X-33. Coat it on a solid YPD medium plate containing 100 μg / mL bleomycin and culture at 28 °C for 48 - 72 h. Pick monoclonal colonies and culture them overnight in YPD liquid medium. Extract genomic DNA and perform PCR verification to obtain an amplification product with the correct size, and screen and identify positive strains. It is initially proved that recombinant Pichia pastoris containing the Fc-serpinE3 gene is successfully obtained. Culture the transformed bacteria, induce expression by methanol, and obtain a bacterial solution secreting Fc-serpinE3. Purify it using a protein A column to successfully obtain a protein with a relatively high purity. Then, detect the purified protein by polyacrylamide gel electrophoresis.

[0041] The specific steps are as follows:

[0042] 1. Prepare the plasmid for electroporation: To transfer the recombinant plasmid into Pichia pastoris X33, the recombinant plasmid needs to be linearized. Digest the correctly sequenced recombinant plasmids pPICZa-Fc-serpinE3-404 and pPICZa-Fc-serpinE3-424 with PmeI alone. Reaction conditions: 37 °C, 5 h. Then, recover the digested fragments by agarose gel electrophoresis and store them at -20 °C for later use.

[0043] 2. Prepare the yeast for electroporation and prepare competent cells

[0044] 3. Electroporation:

[0045] 4. Identify positive recombinant transformants by PCR and screen Fc-serpinE3-404 and Fc-serpinE3-424 / X-33 yeast expression strains.

[0046] Separate monoclonal colonies were inoculated into 3 mL of YPD liquid medium and cultured overnight at 30 °C with shaking at 230 rpm; 1 mL of the bacterial solution was taken, centrifuged at 12,000 rpm for 2 min at room temperature, the supernatant was discarded, and the precipitate was washed with 500 μL of PBS; centrifuged for another 2 min, the supernatant was discarded, and the precipitate was dissolved in 100 μL of ddH2O; boiled in a water bath for 10 min; frozen at -80 °C for 30 min; boiled in a water bath for 10 min; centrifuged for 2 min again, and 1 μL of the supernatant was taken as a template for PCR. The identification primers are as follows:

[0047] Forward primer: 5'-GACTGGTTCCAATTGACAAGC-3';

[0048] Reverse primer: 5'-GCAAATGGCATTCTGACATCC-3';

[0049] The cloned strains identified as positive by PCR were used for the induced expression of proteins.

[0050] 5. Expression of Fc-serpinE3-404 and Fc-serpinE3-424 fusion proteins in Pichia pastoris.

[0051] (1) The selected positive recombinant yeast strains were inoculated into 25 mL of BMGY (250 mL conical flask) medium and cultured at 30 °C with shaking at 220 rpm until OD600 = 2 - 6. In a 50 mL centrifuge tube, centrifuged at 3000 g for 5 min, and the supernatant was discarded.

[0052] (2) The cells were resuspended with BMMY and transferred to a 1 L conical flask, 100 mL of BMMY medium was added, and cultured at 30 °C with shaking at 220 rpm for methanol-induced expression;

[0053] (3) Every 24 h, 100% methanol was added to a final concentration of 1%, and the medium was collected after 72 h of induction.

[0054] (4) The target protein was purified using a protein A column. SDS-PAGE gel electrophoresis was used for protein analysis. The results are as Figure 3 shown.

[0055] Example 4 Prokaryotic expression and protein purification of TMPRSS11E-CD

[0056] TMPRSS11E, also known as DESC1, belongs to the HAT / DESC subfamily of type II transmembrane serine proteases. It has been reported in the literature that TMPRSS11E is highly expressed in various tumors including lung cancer, promoting tumor proliferation and invasion. Recent reports have shown that TMPRSS11E is upregulated in patients with ARDS and rosacea, a chronic inflammatory skin disease. The most typical feature of type II transmembrane serine proteases (TTSPs) is the catalytic domain (CD) containing a serine, histidine, aspartic acid triad catalytic active center, which degrades various substrates through the active center of its catalytic domain and participates in various pathological processes such as virus activation, tumor occurrence and development, inflammation, embryonic development, and tissue remodeling. We constructed a prokaryotic expression plasmid pET28a-TMPRSS11E-CD for the catalytic domain of TMPRSS11E and transformed the successfully constructed plasmid into Escherichia coli BL21 cells. Induce expression with 0.1 mM / L IPTG. Centrifuge the induced bacterial solution and discard the supernatant. Sonicate to break the cells and lyse the bacteria. Then centrifuge again and discard the supernatant to collect the precipitate. At this time, the precipitate is mainly protein inclusion bodies. After washing, dissolving, and refolding the inclusion bodies, finally purify the TMPRSS11E-CD protein using a nickel column. Analyze the purified protein by SDS-PAGE. Figure 4 Showing the purified TMPRSS11E-CD protein.

[0057] Example 5 Cleavage of PAR1 fluorogenic peptide by TMPRSS11E-CD protein.

[0058] Protease-activated receptors (PARs) are a highly conserved family of G protein-coupled receptors (GPCRs). Their classical activation mode is through irreversible cleavage by proteases at the N-terminal active site of PARs to form a new N-terminus. The newly formed N-terminus acts as a tethered ligand to bind to the second extracellular loop structure of the PAR transmembrane protein, thereby triggering an intracellular signal cascade amplification reaction. We designed and synthesized a PAR1 amino-terminal internally quenched (FRET) substrate peptide (Abz-LDPR↓SFLL-Y(3-NO2)), and this sequence contains a cleavage site activated by protease, as indicated by the arrow. The Tyr(NO2) of this substrate peptide can quench Abz, so no fluorescence can be detected. When this substrate is cleaved by protease, Abz is no longer quenched by Tyr(NO2), and fluorescence can be detected immediately. In previous studies, we found that TMPRSS11E can cleave the fluorescent peptide of PAR1 and emit fluorescence. The specific procedure is as follows: First, synthesize the PAR1 fluorescent substrate peptide: Abz-LDPRSFLL-Y(3-NO2); prepare a 100 mM Tris / HCl (pH 8.5) buffer containing 500 μg / mL BSA; then co-incubate the substrate and the enzyme: incubate the PAR1 substrate peptide at a fixed concentration (100 μM) with 2 nM, 5 nM, 10 nM of TMPRSS11E-CD or 10 nM of thrombin respectively; the incubation temperature is 37 °C, and the total volume of the system is 100 μL. A multifunctional microplate reader records the fluorescence value at 420 nm. The results are as Figure 5 shown that TMPRSS11E can effectively cleave the PAR1 fluorescent peptide, and with the increase of the concentration, the cleavage effect is more significant.

[0059] Example 6 Inhibitory function of Fc-serpinE3-404 and Fc-serpinE3-424 on the activity of TMPRSS11E.

[0060] Finally, the inhibitory activity of Fc-myc-serpinE3 was identified. The applicant found that the recombinant proteins Fc-serpinE3-404 and Fc-serpinE3-424 have a strong inhibitory effect on TMPRSS11E. The serine protease inhibitor activity assay is based on the protease activity assay method, and the activity of the inhibitor is calculated by measuring the residual activity of the enzyme after inhibition. By adding TMPRSS11E and the fluorescent peptide substrate, the change value of the generated fluorescence is monitored to measure the enzyme activity. The effectiveness of the protease inhibitor can be monitored by the change in the fluorescence intensity of the TMPRSS11E cleavage of the fluorescent peptide substrate.

[0061] The results are as Figure 6Show: The recombinant proteins Fc-serpinE3-424 and Fc-serpinE3-404 obtained by the Pichia pastoris expression system used in the present invention have a strong inhibitory effect on TMPRSS11E.

[0062] Example 7 uses the LPS tracheal instillation method to simulate an acute lung injury mouse model caused by pulmonary bacterial infection.

[0063] At different time points after LPS stimulation, mouse lung tissues were taken to detect the expression level of TMPRSS11E. As Figure 7 shown in A, compared with the control group, the mRNA level of TMPRSS11E showed a significant increase from 3 hours to 12 hours after LPS stimulation. Relative to the control group, the protein level of TMPRSS11E showed a significant up-regulation at 6 hours and 12 hours after LPS stimulation ( Figure 7 B). At the same time, the mRNA levels of TNF-α, IL-1β, and IL-6 also increased significantly with the extension of time after LPS stimulation ( Figure 7 C). The above in vivo experimental results indicate that TMPRSS11E is induced to be up-regulated in the lung tissues of mice with LPS-induced acute lung injury. Immunohistochemical analysis was further used to explore the expression distribution of the induced TMPRSS11E in mouse lung tissues ( Figure 7 D). The results showed that in the lung tissues of mice with LPS-induced acute lung injury, TMPRSS11E was expressed in lung epithelial cells, and strong positive staining of TMPRSS11E also appeared in macrophages, suggesting that TMPRSS11E is co-expressed in macrophages and participates in the inflammatory response.

[0064] Example 8 Knockdown of TMPRSS11E inhibits lung tissue injury in a mouse model induced by LPS.

[0065] To verify the effect of TMPRSS11E on the inflammatory process, first, the plasmids pLKO.1-shcontrol, pLKO.1-shTMPRSS11E-1, and pLKO.1-shTMPRSS11E-2 were transfected into C57BL / 6 mice by nasal instillation. 48 hours after transfection, LPS (2 mg / kg) was instilled into the trachea, and mouse lung tissues were taken 12 hours after instillation. As Figure 8As shown in Figure A, compared with the group transfected with the pLKO.1-shcontrol plasmid, both the groups transfected with the pLKO.1-shTMPRSS11E-1 and pLKO.1-shTMPRSS11E-2 plasmids could effectively interfere with the expression of TMPRSS11E, and the interference effect of shTMPRSS11E-1 was more significant. At the same time, the results of H&E staining showed that compared with the shcontrol+LPS group, the lung tissue injury was significantly reduced after interfering with the expression of TMPRSS11E, manifested as partial inflammatory cell infiltration in the lung interstitium, thinning of the alveolar wall, and slight edema in the lung septum (as shown in Figure 8 Figure B). In addition, we also detected the effect of TMPRSS11E knockdown on pro-inflammatory factors. As shown in Figure 8 Figure C, compared with the shcontrol+LPS group, the mRNA levels of TNF-α, IL-1β, and IL-6 in the group with TMPRSS11E interference were significantly down-regulated. The above in-vivo experimental results showed that interfering with the expression of TMPRSS11E in vivo could significantly reduce the lung tissue injury induced by LPS in the mouse model and simultaneously down-regulate the mRNA levels of TNF-α, IL-1β, and IL-6 in the mouse lung tissue.

[0066] Example 9

[0067] Macrophages RAW264.7 were cultured in a six-well culture plate. The cell culture medium was incubated with Fc-serpinE3-424 and Fc-serpinE3-404 proteins, and then LPS with a final concentration of 10 μg / mL was added for continuous treatment for 3 hours, and the cell supernatant was collected. The protein levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 were detected by ELISA. As shown in Figure 9 it can be seen that Fc-serpinE3-424 and Fc-serpinE3-404 proteins significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 inflammatory factors.

[0068] There are many methods and ways to specifically implement this technical solution of the present invention. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by the prior art.

Claims

1. Use of serine protease inhibitors in the preparation of a medicament for treating a disease by inhibiting the activity of TMPRSS11E, wherein the disease is an inflammation caused by a lung infection, and the serine protease inhibitors include Fc-serpinE3-404 and Fc-serpinE3-424, the amino acid sequence of Fc-serpinE3-404 is shown as SEQ ID NO.1, and the amino acid sequence of Fc-serpinE3-424 is shown as SEQ ID NO.3.

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