Selenium-substituted polymer, preparation method and application thereof

Abstract

The present invention discloses a selenopolymer, a preparation method and an application thereof. The selenopolymer is obtained by ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride; preferably, the selenopolymer is obtained by ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride and an amino acid containing N-carboxylic anhydride. The biocompatible selenopolymer provided by the present invention is applied to site-specifically bind it to a polypeptide or protein containing free cysteine, and the biological inhibitory activity of the polypeptide or protein can be completely retained, while enhancing its antioxidant capacity. When the polypeptide or protein is used as a drug, the serum half-life is extended, and a lasting protective activity is provided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a selenopolymer, a preparation method and application thereof. Background Art

[0002] Human α-1-antitrypsin (A1AT) is a member of the serine protease inhibitor superfamily and one of the most abundant endogenous serine protease inhibitors in human plasma. It is mainly secreted by hepatocytes into the circulatory system to prevent proteases, especially neutrophil elastase (NE), from damaging tissues under inflammatory conditions (Travis, J., et al, Am J Med 1988, 84(6A), 37-42; Carrell, RW, et al, N Engl J Med 2002, 346(1), 45-53; Kolarich, D., et al, Proteomics 2006, 6(11), 3369-80; Hunt, JM et al. Curr Mol Med 2012, 12(7), 827-835). After the protruding active center loop (RCL, position 357-366) on the surface of A1AT reacts with the active serine on the protease, RCL breaks and changes its conformation, thereby forming an inactive covalent serine protease inhibitor complex (Huntington, JA, et al. Nature 2000, 407 (6806), 923-926).

[0003] A1AT deficiency (A1ATD) is a common genetic disease that affects approximately 3.4 million people worldwide (de Serres, FJ, Chest 2002, 122(5), 1818-1829; Kelly E, Respir Med 2010, 104(6), 763-772). Currently, the only treatment for A1ATD is weekly intravenous injection (60 mg / kg body weight) of natural A1AT (nA1AT) derived from human plasma to increase its concentration in the blood and lungs (Silverman, EK, et al, N Engl J Med 2009, 360(26), 2749-2957; Hubbard, RC et al, Am J Med 1988, 84(6A), 52-62). However, the nA1AT in this treatment regimen is expensive and its supply is insufficient to meet clinical needs (Wewers, MD et al, COPD 2013, 10 Suppl 1, 64-67). Another disadvantage of nA1AT is its short half-life in vivo due to its instability, proteolysis and oxidative inactivation. It is reported that smokers with moderate A1ATD have a greatly increased risk of developing COPD (chronic obstructive pulmonary disease) (Stoller, JK et al, Am J Respir Crit Care Med 2012, 185 (3), 246-259); when exposed to tobacco smoke, the Met358 residue at the RCL of A1AT is easily oxidized to methionine sulfoxide, resulting in protein inactivation; in addition, oxidized A1AT can also be detected in patients with bronchiectasis and rheumatism (Sepper, R. et al, J Clin Immunol 1995, 15 (1), 27-34; Chidwick, K. et al, Ann Rheum Dis 1991, 50 (12), 915-916). Therefore, protecting A1AT from oxidation can significantly improve its therapeutic effect.Although fusion methods such as pegylation or including Fc, HSA, PAS, XTEN or CDR loops can prolong the circulation time of protein drugs in vivo, they are unable to effectively protect proteins from oxidative inactivation (Zhang, Y. et al, Angew Chem Int Ed Engl 2013, 52(32), 8295-8298; Ko, J Het al, Chem Soc Rev 2018, 47(24), 8998-9014; Schellenberger, V. et al, Nat Biotechnol 2009, 27(12), 1186-1190; Schlapschy, M. et al, Protein Eng Des Sel 2013, 26(8), 489-501; Kontos, S. et al, Chem Soc Rev 2012, 41(7), 2686-95; Chen, C. et al, Progress in Polymer Science 2020, 105, 101241; Ekladious, I. et al, Nature Reviews Drug Discovery 2019, 18(4), 273-294).

[0004] Selenium is an essential trace element highly related to human health and various diseases. Compared with sulfur, selenium has a larger atomic radius and has higher activity in many chemical reactions such as nucleophilic substitution, redox reaction and free radical reaction. These unique chemical reactions possessed by selenium and its important role in biology have made selenopolymers developed for research such as drug delivery, adaptive materials and enzyme mimics (Xia, JHet al, Macromolecules 2018, 51 (19), 7435-7455; Xu, H. et al, Acc Chem Res 2013, 46 (7), 1647-1658). However, up to now, there is no report that selenopolymers are used to protect proteins from oxidative damage. Summary of the invention

[0005] The present invention aims to provide a selenopolymer, a preparation method and application thereof, and adopts the selenopolymer to protect proteins from oxidative damage.

[0006] In order to achieve the above object, according to one aspect of the present invention, a selenopolymer is provided. The selenopolymer is obtained by ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride; preferably, the selenopolymer is obtained by ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride and an amino acid containing N-carboxylic anhydride.

[0007] Furthermore, the selenoamino acid containing N-carboxylic anhydride is selenomethionine NCA; the amino acid containing N-carboxylic anhydride is sarcosine NCA; preferably, the end of the selenopolymer has a maleimide residue; preferably, the selenoamino acid containing N-carboxylic anhydride also has a side chain, and the side chain is selected from one or more of the group consisting of oligoethylene glycol, oligoethylene glycol monomethyl ether, polyethylene glycol, polyethylene glycol monomethyl ether, N-methylethylamine, N,N-dimethylethylamine, N,N-diethylethylamine, ethyltrimethylammonium salt, propyltrimethylammonium salt, acetic acid and propionic acid.

[0008] Furthermore, the selenopolymer is P(SeMet) 3 -CO-(Sar) 105 or P(EG 4 -SeHC) 30 -b-(Sar) 15 .

[0009] According to another aspect of the present invention, there is provided a use of any of the above-mentioned selenopolymers in protecting proteins from oxidative damage; preferably, the protein contains free thiol groups; more preferably, the protein contains cysteine ​​residues.

[0010] Furthermore, the protein is an enzyme or an antibody; preferably, the enzyme is a protease or an antibody that is easily oxidized; more preferably, the protease is α-1-antitrypsin.

[0011] Furthermore, the selenopolymer is coupled to the protein; preferably, the selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue.

[0012] According to another aspect of the present invention, a protein for resisting oxidative damage is provided, wherein the protein is coupled with any of the above-mentioned selenopolymers.

[0013] Furthermore, the selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue; preferably, the protein is an enzyme; more preferably, the protein is a protease or antibody that is easily oxidized; more preferably, the protease is α-1-antitrypsin.

[0014] According to another aspect of the present invention, there is provided a use of any of the above proteins in the preparation of a drug.

[0015] Furthermore, when the protein is α-1-antitrypsin, the drug is a serine protease inhibitor; preferably, the selenopolymer is P(SeMet) 3 -CO-(Sar) 105 or P(EG 4 -SeHC) 30 -b-(Sar) 15, the application of proteins that resist oxidative damage in the preparation of drugs with protective effects on lung injury.

[0016] According to another aspect of the present invention, a method for preparing a selenopolymer is provided. The preparation method comprises ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride to obtain a selenopolymer; preferably, the selenoamino acid containing N-carboxylic anhydride and an amino acid containing carboxylic anhydride are ring-opening polymerized to obtain a selenopolymer; preferably, the selenoamino acid containing N-carboxylic anhydride is selenomethionine NCA; the amino acid containing carboxylic anhydride is sarcosine NCA; more preferably, the preparation method further comprises the step of adding a maleimide residue to the end of the selenopolymer.

[0017] Further, the method comprises the following steps: dissolving sarcosine NCA, selenomethionine NCA and benzylamine in anhydrous DMF respectively, and reacting to obtain P(Sar-ran-SeMet); dissolving P(Sar-ran-SeMet) in ddH 2 O, add NHS-C 3 -Mal and TEA were reacted in DMSO to prepare P(Sar-ran-SeMet)-Mal.

[0018] Further, the method comprises the following steps: dissolving EG4-SeHC NCA in anhydrous DMF, adding benzylamine, and stirring at room temperature to react to obtain a first reaction solution; adding sarcosine NCA to the first reaction solution to react and generate P(EG4-SeHC-co-Sar); dissolving P(EG4-SeHC-co-Sar) in anhydrous DMF, and adding NHS-C 3 -Mal and TEA were reacted in DMSO to prepare P(EG4-SeHC-co-Sar)-Mal.

[0019] According to one aspect of the present invention, a method for preparing the above-mentioned protein that resists oxidative damage is provided. The preparation method comprises coupling a selenopolymer with a protein; preferably, the selenopolymer is coupled with a free thiol group of the protein via a maleimide residue at the end; more preferably, the preparation method comprises the following steps: mixing the above-mentioned selenopolymer containing an equimolar amount of maleimide with the protein in a buffer and coupling at 4°C.

[0020] Furthermore, the buffer comprises 200 mM NaCl, 0.1 mM TCEP and 20 mM sodium phosphate buffer, and the buffer has a pH of 7.0; preferably, the molar ratio of the selenopolymer to the protein to be coupled is 4:1 to 6:1.

[0021] By using the biocompatible selenopolymer provided by the present invention and site-specifically binding it to a polypeptide or protein containing free cysteine, the biological inhibitory activity of the polypeptide or protein can be completely retained while enhancing its antioxidant capacity. When the polypeptide or protein is used as a drug, the serum half-life is prolonged, providing a lasting protective activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 The SDS-PAGE gel image of A1AT in Example 1 is shown;

[0024] Figure 2 shows the size exclusion chromatogram of A1AT in Example 1;

[0025] Figure 3 The NE inhibitor screening kit in Example 1 is used to detect the inhibitory effect of A1AT and oxidized A1AT on NE;

[0026] Figure 4 The structural diagram of selenopolymers SeP1 and SeP2 in Example 1 is shown;

[0027] Figure 5 The p(EG4-SeHC) in Example 1 is shown before and after being oxidized by NCS. 1 HNMR test results;

[0028] Figure 6 The figure shows the inhibition of NE activity after 10 nM A1AT or a mixture of 10 nM A1AT and different equivalents of selenopolymers were oxidized by 100 equivalents of NCS in Example 1;

[0029] Figure 7 The SDS-PAGE gel images of A1AT, A1AT-SeP1 and A1AT-SeP2 in Example 1 are shown;

[0030] Figure 8 The SEC analysis results of A1AT-SeP1 and A1AT-SeP2 in Example 1 are shown;

[0031] Figure 9a-9e The antioxidant effect of the selenopolymer in Example 1 is shown, wherein Figure 9a The inhibitory activities of A1AT, A1AT-SeP1 and A1AT-SeP2 against NE in Example 1 are shown; Figure 9bThe inhibitory activity of A1AT, A1AT-SeP1, and A1AT-SeP2 on NE after incubation with 100 equivalents of NCS oxidation at room temperature for 30 minutes in Example 1 is shown; Fig.9c The inhibitory activity of A1AT, A1AT-SeP1, and A1AT-SeP2 on NE after incubation with 200 equivalents of NCS at room temperature for 30 minutes in Example 1 is shown; Figure 9d The inhibitory activity of A1AT, A1AT-SeP1, and A1AT-SeP2 on NE after incubation with 200 equivalents of NCS at room temperature for 60 minutes in Example 1 is shown; Fig.9e The relative activity of A1AT, A1AT-SeP2 and 200 equivalents or 40 equivalents of NCS in Example 1 to inhibit NE at different time periods after incubation at room temperature is shown;

[0032] Fig.10 The inhibitory activity of 10 nM A1AT, a mixture of A1AT and an equivalent amount of SeP1 or SeP2, A1AT-SeP1, and A1AT-SeP2 on NE after oxidation by 200 equivalents of NCS in Example 1 is shown, where * indicates P ≤ 0.05; *** indicates P ≤ 0.001;

[0033] Fig.11 The activity of myeloperoxidase (MPO) in the bronchoalveolar lavage fluid of mice infused with different doses of porcine pancreatic elastase (PPE) in Example 1 is shown, where * indicates P≤0.05;

[0034] Fig.12a and Figure 12b The protective effects of A1AT and A1AT-SeP2 in Example 1 on PPE-induced lung injury in mice are shown; Fig.12a The activity of MPO in BALF of mice after 24 hours of pretreatment with A1AT and A1AT-SeP2 in Example 1 and after 24 hours of PPE treatment was shown; Figure 12b The activity of MPO in BALF of mice after intratracheal inhalation of PPE 72 hours after pretreatment with A1AT and A1AT-SeP2 in Example 1 and after PPE treatment 24 hours later is shown, where * indicates P ≤ 0.05; ** indicates P < 0.01; *** indicates P < 0.001;

[0035] Fig.13 The PK curves of A1AT and A1AT-SeP2 in Example 1 after tail vein injection in mice are shown;

[0036] Fig.14 The figure shows the changes in the body weight of mice at different times after intraperitoneal injection of p(EG4-SeHC) into mice in Example 1 (day 1, day 8, day 15, day 22). DETAILED DESCRIPTION

[0037] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0038] Glossary:

[0039] NCA is the abbreviation of N-Carboxyanhydride, that is, N-carboxylic anhydride.

[0040] SeP: refers to Se polypeptide in the present invention.

[0041] Human alpha-1-antitrypsin (A1AT) is a natural serine protease inhibitor that protects tissues from damage caused by excessive protease activity and is used as an enhanced therapy for the treatment of patients with A1AT deficiency. However, A1AT is sensitive to oxidative inactivation and has a short circulation half-life. Current methods are unable to effectively protect therapeutic proteins from oxidative damage in vivo. In response to this, the present invention provides a novel biocompatible selenopolymer and site-specifically binds it to a polypeptide or protein containing free cysteine, such as A1AT. A1AT bound to a selenopolymer completely retains its inhibitory activity against neutrophil elastase (NE), while enhancing its antioxidant capacity, prolonging its serum half-life, and providing lasting protective activity in a mouse model of acute lung injury.

[0042] According to a typical embodiment of the present invention, a selenopolymer is provided. The selenopolymer is obtained by ring-opening polymerization (ROP) of a selenoamino acid containing N-carboxylic anhydride (NCA), and preferably, the selenopolymer is obtained by ring-opening polymerization (ROP) of a selenoamino acid containing N-carboxylic anhydride (NCA) and an amino acid containing N-carboxylic anhydride (NCA).

[0043] In a typical embodiment of the present invention, the selenoamino acid containing N-carboxylic anhydride is selenomethionine NCA; the amino acid containing carboxylic anhydride is sarcosine NCA. In order to more conveniently couple with the protein, preferably, the end of the selenopolymer has a maleimide residue. In order to increase the selenium content in the copolymer and improve its water solubility, the selenoamino acid containing N-carboxylic anhydride also has a side chain, and the side chain is selected from one or more of the group consisting of oligoethylene glycol, oligoethylene glycol monomethyl ether, polyethylene glycol, polyethylene glycol monomethyl ether, N-methylethylamine, N,N-dimethylethylamine, N,N-diethylethylamine, ethyltrimethylammonium salt, propyltrimethylammonium salt, acetic acid and propionic acid.

[0044] More preferably, the selenopolymer is P(SeMet) 3 -CO-(Sar) 105 or P(EG4 -SeHC) 30 -b-(Sar) 15 . The examples of the present invention verify that A1AT coupled with the selenopolymer fully retains its inhibitory activity against neutrophil elastase (NE), while enhancing its antioxidant capacity, prolonging its serum half-life, and providing lasting protective activity in an acute lung injury mouse model. These results indicate that coupling A1AT with a designed selenopolymer is a feasible strategy to improve its pharmacological properties. This approach is applicable to a variety of oxidation-sensitive biotherapeutic drugs.

[0045] According to a typical embodiment of the present invention, there is provided an application of the above-mentioned selenopolymer in protecting proteins from oxidative damage. In one embodiment of the present invention, the protein contains a free thiol group, for example, the protein contains a cysteine ​​residue. Typically, the protein is an enzyme or an antibody; preferably, the enzyme contains a group that is easily oxidized (such as a cysteine ​​disability); preferably, the enzyme is α-1-antitrypsin. The biocompatible selenopolymer provided by the present invention is site-specifically bound to a polypeptide or protein containing free cysteine, which can completely retain the biological inhibitory activity of the polypeptide or protein, while enhancing its antioxidant capacity. When the polypeptide or protein is used as a drug, the serum half-life is prolonged, providing a lasting protective activity.

[0046] Typically, the selenopolymer is coupled to the protein; preferably, the selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue. In addition, the selenopolymer can also be non-specifically coupled to the amino group on the protein via the terminal activated ester, thereby achieving the purpose of protecting the protein.

[0047] According to a typical embodiment of the present invention, a protein for resisting oxidative damage is provided, wherein the protein is coupled with the selenopolymer of the present invention.

[0048] Typically, the selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue; preferably, the protein is an enzyme; more preferably, the enzyme is α-1-antitrypsin.

[0049] According to a typical embodiment of the present invention, there is provided a use of the above-mentioned protein for resisting oxidative damage in preparing a drug. For example, when the protein is α-1-antitrypsin, the drug is a serine protease inhibitor.

[0050] According to a typical embodiment of the present invention, a method for preparing the above-mentioned selenopolymer is provided, comprising ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride to obtain a selenopolymer; preferably, a selenoamino acid containing N-carboxylic anhydride and an amino acid containing carboxylic anhydride are ring-opening polymerized to obtain a selenopolymer; typically, the selenoamino acid containing N-carboxylic anhydride is selenomethionine NCA; the amino acid containing carboxylic anhydride is sarcosine NCA; more preferably, the preparation method further comprises the step of adding a maleimide residue to the end of the selenopolymer. In a typical embodiment of the present invention, the preparation method of the above-mentioned selenopolymer comprises the following steps: dissolving sarcosine NCA, selenomethionine NCA and benzylamine in anhydrous DMF respectively, reacting to obtain P(Sar-ran-SeMet); dissolving P(Sar-ran-SeMet) in ddH 2 O, add NHS-C 3 -Mal and TEA were reacted in DMSO to prepare P(Sar-ran-SeMet)-Mal.

[0051] In a typical embodiment of the present invention, the preparation method of the selenopolymer comprises the following steps: dissolving EG4-SeHC NCA in anhydrous DMF, adding benzylamine, stirring and reacting at room temperature to obtain a first reaction solution; adding sarcosine NCA to the first reaction solution to react and generate P(EG4-SeHC-co-Sar); dissolving P(EG4-SeHC-co-Sar) in anhydrous DMF, adding NHS-C 3 -Mal and TEA were reacted in DMSO to prepare P(EG4-SeHC-co-Sar)-Mal.

[0052] According to a typical embodiment of the present invention, a method for preparing the above-mentioned protein that resists oxidative damage is provided. The preparation method comprises the following steps: mixing the above-mentioned selenopolymer containing an equimolar amount of maleimide with the protein in a buffer solution, and coupling at 4°C.

[0053] Preferably, the buffer comprises 200 mM NaCl, 0.1 mM TCEP and 20 mM sodium phosphate buffer, and the buffer has a pH of 7.0; preferably, the molar ratio of the selenopolymer to the protein to be coupled is 4:1 to 6:1.

[0054] The beneficial effects of the present invention will be further described below in conjunction with embodiments.

[0055] Example 1

[0056] Experimental Materials and Methods

[0057] 1. Animals

[0058] Female C57BL mice and male Kunming mice were purchased from Charies River, Beijing, China.

[0059] 2. Chemical synthesis

[0060] 2.1 Synthesis of EG4-SeHC NCA and Sarcosine NCA

[0061] EG4-SeHC NCA was synthesized according to the procedure reported in the literature [Wu, GQ et al, Chem. Commun. 2019, 55(54), 7860-7863], and sarcosine NCA was synthesized according to the procedure reported in the literature [Macromolecules 2011, 44, 6746–6758].

[0062] 2.2 Synthesis of selenomethionine NCA

[0063] Selenomethionine (700 mg, 3.5 mmol) and triphosgene (530 mg, 1.8 mmol) were placed in a 50 ml THF (tetrahydrofuran) solution, propylene oxide (~2.5 mL, ~35 mmol) was added, and the mixture was stirred and mixed at room temperature for 2 hours to react to generate selenomethionine NCA. The reaction solution was removed and the product was purified by chromatography on silica gel (dried at 90 ° C for at least 4 hours before use) (mobile phase: ethyl acetate / petroleum ether). The purified and recovered selenomethionine NCA was a light yellow oil that gradually solidified in the refrigerator (124 mg, yield: 16%). The molecular weight detected by high-resolution electrospray mass spectrometry (HR-ESI-MS) was 245.9633, which was consistent with the theoretical value of 245.9640.

[0064] 2.3 Synthesis of P(Sar-ran-SeMet)-Mal

[0065] Preparation of P(Sar-ran-SeMet): In a glove box, sarcosine NCA (76 mg, 0.66 mmol) and selenomethionine NCA (16 mg, 0.072 mmol) were dissolved in 1.0 mL of anhydrous DMF, and 48 μL of 0.1 benzylamine (dissolved in DMF) was added to the solution and stirred for 24 hours. After the complete conversion of NCA was detected by Fourier transform infrared spectroscopy (FI-IR), the peptide was precipitated with 50 mL of ether and desalted with PD-10. After lyophilization, the recovered peptide P(Sar-ran-SeMet) was a white powder (38 mg, yield: 64%), which was analyzed by SEC as a single peak (results not shown).

[0066] Add Mal tail to P(Sar-ran-SeMet): 10.0 mg P(Sar-ran-SeMet) was dissolved in 500 μL ddHO2 O, 500 μL of DMSO containing NHS-C3-Mal (3.8 mg, 0.014 mmol) and TEA (1 μL) was added to the solution, and stirred at room temperature for 24 hours. After desalting and freeze-drying, the white powdery peptide (8.0 mg, yield: 80%) was recovered. 1 H NMR was used to analyze the composition of the polypeptide and the efficiency of adding the Mal tail to the terminal (wherein, initiator: selenomethionine: sarcosine = 1:3:105).

[0067] Synthesis of 2.4P(EG4-SeHC-co-Sar)-Mal

[0068] Preparation of P(EG4-SeHC-co-Sar): In a glove box, dissolve 100 mg of EG4-SeHC NCA in 2.0 mL of anhydrous DMF, add 62 μL of 0.1 M benzylamine (dissolved in DMF), and stir at room temperature for 24 hours. After Fourier transform infrared spectroscopy (FT-IR) confirmed the conversion of NCA, take 200 μL and dissolve it for SEC analysis. Add sarcosine NCA (12.8 mg, 0.112 mmol) to the above mixed solution and stir again for 24 hours. Precipitate the copolypeptide in ether and desalt it by PD-10. After freeze-drying, recover the fluffy powder (64 mg, 66%). SEC analysis showed a single peak (results not shown), 1 H NMR analysis of the composition of the peptide (initiator: EG4-SeHC: sarcosine = 1:30:15).

[0069] P(EG4-SeHC-co-Sar) was tailed with NHS-C3-Mal: 10.0 mg of P(EG4-SeHC-co-Sar) obtained above was dissolved in 100 μL of anhydrous DMF, NHS-C3-Mal (1.0 mg, 0.0037 mmol) and TEA (0.5 μL) were added to the solution, and stirred at room temperature for 24 hours. After desalting by PD-10 and freeze-drying, the white powdery peptide (10.0 mg, yield 99%) was recovered. 1HNMR confirmed that Mal was successfully added to the end of P(EG4-SeHC-co-Sar) (results not shown).

[0070] 3. Construction of A1AT expression plasmid

[0071] The A1AT gene (P01009 (uniprot)) was codon-optimized and synthesized by Sangon Biotech (Shanghai). In order to improve the affinity of the protein to Ni-NTA resin, a flexible linker GGGS was added to the C-terminus of the A1AT protein and fused to a 10xHis tag. The gene fragment was cloned into the NcoI and HindIII sites of the pET28a vector using the CloneExpress II one-step cloning kit (Vazyme Biotech, #C112), and the cloned sequence was further confirmed by DNA sequencing.

[0072] 4. Expression and purification of A1AT protein

[0073] 5 mL of overnight culture was inoculated into 500 ml of TB medium supplemented with kanamycin (100 μg / mL) and cultured at 37°C, 220 rpm until OD600 reached about 0.6 to 0.8. The culture was cooled to room temperature and induced at 25°C for 20 hours by adding 0.2 mM IPTG. The cells were collected by centrifugation and resuspended in a buffer (pH 8.0) containing 200 mM NaCl, 20 mM imidazole, 2 mM TCEP and 20 mM sodium phosphate buffer. The cells were sonicated for 30 seconds, placed on ice for 1 minute, and after 15 minutes, the lysate was centrifuged at 4°C, 12,000 × g for 20 minutes. After the supernatant was added to a Ni-NTA column for affinity purification, it was gradient eluted (from 40mM to 300mM) with an imidazole solution containing 20mM sodium phosphate, 2mM TCEP and 200mM NaCl at pH 8.0, and the eluate was collected for SDS / PAGE analysis. A size exclusion column (HiLoad 16 / 600 Superdex 75prep Grade, balanced with 200mM NaCl, 0.1mMTCEP (sigma) and 20mM sodium phosphate buffer at pH 7.0) was used to further separate the protein and perform buffer exchange. The protein concentration was determined by a BCA kit (Beijing Solaibao Biotechnology Co., Ltd., #PC0020).

[0074] 5.A1AT Activity Assay

[0075] The inhibition of neutrophil elastase (NE) by A1AT was determined using the NE inhibitor screening kit (BioVision). 12.5 μL of diluted sample (final concentration of A1AT or A1AT conjugate was 10 nM), inhibitor control, DPBS control and 25 μL of NE solution were mixed and incubated at 37°C for 5 minutes. The reaction solution containing the substrate was added to the wells containing the above mixture (12.5 μL / well) and mixed immediately. The fluorescence values ​​at the excitation light of 400 nm (Ex400) and the emission light of 505 nm (Em505) were measured; after incubation at 37°C in the dark for 30 minutes, the fluorescence values ​​at the excitation light of 400 nm (Ex400) and the emission light of 505 nm (Em505) were measured again. The ratio of Ex(400) / Em(505) at T1 time R1 and the ratio of Ex(400) / Em(505) at T2 time R2 were calculated respectively. The fluorescence RFU generated by substrate hydrolysis is ΔRFU = R2 – R1. R1 and R2 were selected within the linear range by kinetic readings. The ΔRFU of the blank control was set as 100% relative activity value, and the relative activity of each inhibitor was calculated as follows:

[0076]

[0077] 6. Oxidation of A1AT by N-chlorosuccinimide (NCS)

[0078] 1 μM A1AT was incubated with different concentrations of NCS (Innochem) in 100 mM Tris, pH 8.5 at room temperature for different times (Le, DT et al, Biochemistry 2008, 47(25), 6685-94).

[0079] 7. Coupling of A1AT with polymers

[0080] Different polymers containing equivalent amounts of maleimide were incubated with A1AT (final concentration of 0.5 mg / mL in 200 mM NaCl, 0.1 mM TCEP and 20 mM sodium phosphate buffer, pH 7.0) at 4 °C overnight and analyzed by SDS / PAGE. The optimal molar ratio of polymer to A1AT ranged from 4:1 to 6:1. The coupling product was purified from the reaction mixture by SEC (HiLoad 16 / 600 Superdex75 preparative grade).

[0081] 8.PK experiments

[0082] Female C57BL mice (6 weeks old, weighing ~20 g) were randomly divided into two groups (6 mice / group). 100 μg A1AT or A1AT-SeP2 was injected through the tail vein. Venous blood was collected from the tail vein at different time points (0.5 hours, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours). The concentration of A1AT in each sample was detected by ELISA using SerpinA1Matched ELISA Antibody Pair Set, Human (#SEK10306, SinoBiological), and the data were processed by GraphPad Prism.

[0083] 9. Protective effect of A1AT-SeP2 on lung injury induced by porcine pancreatic elastase (PPE)

[0084] Female C57BL mice (6 weeks old) (8 mice / group, 3 groups in total) were anesthetized, and 50 μL of sample (PBS, 130 μM A1AT, or 130 μM A1AT-SeP2) was instilled into the trachea and inhaled into the lungs via a pipette. After 24 hours or 72 hours, 50 μL of PPE (150 U / kg) (Shanghai Yuanye Biotechnology Co., Ltd., #S31655) was instilled in the same way. Mice were killed 24 hours later and the results were as reported in the literature. 44 Bronchoalveolar lavage fluid (BALF) was collected, centrifuged at 1000g, 4°C for 10 minutes to separate cells, and the remaining red blood cells were lysed by hypotonic lysis mechanism using distilled water for 5 seconds. The cells were washed with PBS and resuspended in PBS containing 300 μL of 0.1% Triton-X (Beijing Solaibao Biotechnology Co., Ltd., #T8200). 50 μL of the sample was mixed with an equal volume of TMB (Novoprotein, #PA005), incubated at 37°C for 5 minutes, and then 50 μL of 2M H2SO4 was added to terminate the reaction, and the absorbance was measured at 450 nm to measure the activity of myeloperoxidase (MPO).

[0085] 10.p(EG4-SeHC) safety test

[0086] Male Kunming mice (6 weeks old) were randomly divided into two groups (n=6). On the 1st day, 8th day, 15th day, and 22nd day, 1 mg / kg p(EG4-SeHC) or 10 mL / kg PBS as a control were injected (ip), respectively. Body weight was measured 3 times a week.

[0087] 11. Data processing

[0088] Data are presented as mean ± SEM and statistically analyzed using GraphPad Prism. Error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001

[0089] Results and Analysis:

[0090] The expression plasmid encoding the A1AT gene was transformed and overexpressed in Escherichia coli BL21 (DE3). The A1AT protein was purified from the supernatant of the lysed cells by nickel-nitrilotriacetic acid resin (Ni-NTA) and size exclusion chromatography (SEC). The yield of purified A1AT was about 16 mg / L. Its purity was analyzed by SDS-PAGE and SEC. SDS-PAGE ( Figure 1 ) showed that the purified A1AT band size was about 50 kDa and eluted from the Superdex 75 column as a single peak without aggregation ( Figure 2 ). MS analysis showed that the molecular weight of A1AT was 46144Da, which was consistent with the theoretical molecular weight of 46142Da. A1AT is a natural inhibitor of neutrophil elastase (NE). Crystallographic studies have shown that the binding of NE to A1AT will cut its reaction ring to form a covalent complex (Huntington, JA et al, Nature 2000, 407 (6806), 923-926). Methionine 358 located in the active ring of A1AT is easily oxidized, resulting in protein inactivation. It is reported that N-chlorosuccinimide (NCS) can selectively oxidize methionine residues exposed on the surface of the protein to methionine sulfoxide (Boudier, C. et al, Biochem J 1994, 303 (Pt 1), 61-68). Therefore, the inventors incubated A1AT with 100 equivalents of NCS at room temperature for 30 minutes, and then the NE inhibitor screening kit evaluated the inhibitory activity of A1AT on NE. In this assay, the fluorescence generated by the hydrolysis of a specific fluorescent substrate by NE is detected and quantified. In the presence of an inhibitor such as A1AT, the hydrolysis of the fluorescent substrate is inhibited and the fluorescent signal decreases. Figure 3 As shown, 10 nM A1AT can completely inhibit the activity of NE. After incubation of A1AT with NCS, NE activity was almost unaffected, indicating that the inhibitory activity of A1AT was lost due to oxidation. As a control, NE activity was hardly affected in oxidation buffer containing an equal amount of NCS.

[0091] Peptides or peptide-like peptides prepared by ring-opening polymerization (ROP) of amino acid N-carboxylic anhydride (NCA) are widely used in biomedical engineering due to their biodegradability and biocompatibility. For example, polysarcosine (PSar) is an unstructured peptide-like peptide with excellent water solubility and non-polluting properties. As an excellent alternative to PEG, it has been used for protein coupling (Hou, Y. et al, Bioconjugate Chem 2019, 30 (6), 1604-1616; Hu, Y. et al, Bioconjugate. Chem. 2018, 29 (7), 2232-2238.). The inventors prepared two selenopolymers (SeP1 and SeP2) by copolymerization. Figure 4 ). SeMet was introduced during the random copolymerization of sarcosine NCA (SarNCA) and selenomethionine NCA (SeMet NCA) to prepare SeP1. Due to the hydrophobicity of SeMet, the inventors found that the polypeptide formed is water-soluble only when the Sar / SeMet molar ratio is greater than 10:1. In order to increase the selenium content in the copolymer and improve its water solubility, the water-soluble selenopolypeptide P(EG4-SeHC) (Wu, G. et al, Chem Commun (Camb) 2019, 55(54), 7860-7863) was introduced into SeP2, and PSar was introduced at the N-terminus of P(EG4-SeHC) to prolong its circulation time in the body. The structural formulas of SeP1 and SeP2 are as follows Figure 4 As shown, they all have maleimide at the end for coupling with cysteine. To demonstrate the reactivity of selenopolymers to NCS, P(EG4-SeHC) was mixed with 5 equivalents of NCS and NaOD in aqueous solution. 1 H NMR in situ monitoring revealed that the α-, β-, and γ-hydrogen signals of P(EG4-SeHC) were significantly shifted ( Figure 5 ), and the chemical shifts were similar to those of seleno-peptides, indicating that the peptides could be oxidized by NCS.

[0092] To determine whether SePs can protect A1AT from oxidative inactivation, A1AT was incubated with 100 equivalents of NCS and different equivalents of SePs at room temperature for 30 min, and then the inhibitory activity against NE was measured to evaluate the content of unoxidized A1AT. Figure 6 As shown, in the absence of SePs, A1AT lost most of its activity under oxidative conditions. SeP1 had no obvious protective effect on A1AT, while SeP2 could protect the activity of A1AT in a dose-dependent manner, probably because of its higher selenium content compared with SeP1. When A1AT was mixed with 8 equivalents of SeP2, A1AT retained about 50% of its inhibitory activity.

[0093] SePs can protect the A1AT mixed with it from NCS oxidation. The inventors speculated that if SePs were coupled near Met358 of the A1AT active ring, antioxidant protection might be more effective. Based on the crystal structure of A1AT, Cys232 with a free thiol group is located on the surface of A1AT and close to Met358. The inventors incubated A1AT with 4-6 equivalents of SePs at 4°C overnight, and obtained A1AT-SePs conjugates (A1AT-SeP1 and A1AT-SeP2) by SEC purification. The SDS-PAGE structure showed that the band of the A1AT-SePs conjugate migrated from the position of the A1AT molecular weight to the position of a higher molecular weight, and the conjugates were all in monomeric form ( Figure 5 , Figure 6 ). Figure 7 Shown are SDS-PAGE gel images of A1AT, A1AT-SeP1, and A1AT-SeP2; Figure 8 The results of SEC analysis of A1AT-SeP1 and A1AT-SeP2 are shown.

[0094] Next, the inventors tested the inhibitory activity of A1AT-SeP1 and A1AT-SeP2 on NE. The results showed that, similar to A1AT, 10 nM A1AT-SeP1 or A1AT-SeP2 could completely inhibit the activity of NE ( Figure 9a ), indicating that polymer coupling at Cys232 does not interfere with the activity of A1AT;. Next, the inventors incubated a series of different concentrations of A1AT, A1AT-SeP1 or A1AT-SeP2 with 100 equivalents of NCS to quantify and compare their inhibitory activities against NE. The results showed that the inhibitory activity of A1AT against NE was significantly reduced (IC50 = 22.0 ± 7.1 nM). In contrast, the inhibitory activity of A1AT-SeP1 (IC50 = 7.7 ± 1.0 nM) and A1AT-SeP2 (IC50 = 5.7 ± 1.0 nM) against NE was largely retained ( Figure 9b When the inventors increased the NCS to 200 equivalents, the activities of A1AT-SeP2 (IC50 = 7.4 ± 1.4 nM) and A1AT-SeP1 (IC50 = 13.0 ± 1.8 nM) decreased, and A1AT-SeP2 was more resistant to the oxidation ability of high concentration oxidants ( Fig.9c ). In addition, the inventors studied the oxidation-induced inactivation over a time course. The inventors first extended the incubation time from 30 minutes to 1 hour. As expected, under 200 equivalents of NCS treatment, A1AT-SeP2 still retained most of its activity (IC50 = 12.5 ± 2.5 nM), while the activities of A1AT and A1AT-SeP1 dropped sharply ( Figure 9d), which may be due to the relatively low selenium content of SeP1; then the inventors incubated A1AT and A1AT-SeP2 with 200 equivalents of NCS for different time periods and found that the activity of A1AT was rapidly lost with the extension of incubation time ( Fig.9e ), while A1AT-SeP2 still retained more than 50% of its original activity at 40 minutes. Next, the inventors reduced the amount of NCS from 200 equivalents to 40 equivalents and extended the incubation time, and found that A1AT-SeP2 retained about half of its original activity after 10 hours. In order to explore the effect of NCS oxidation on the activity of A1AT and A1AT-SePs conjugates, the inventors incubated 200 equivalents of NCS with A1AT, A1AT with equivalent amounts of polymers (such as SeP1, SeP2), and A1AT-SePs conjugates for 30 minutes, respectively, and compared their inhibitory activities against NE. As the inventors hypothesized, A1AT almost lost its inhibitory activity against NE, while the A1AT-SePs conjugate showed stronger antioxidant capacity than the mixture of A1AT and an equal amount of SePs; consistent with the antioxidant capacity of the polymer, under the same oxidation conditions, A1AT-SeP2 maintained a higher inhibitory activity than A1AT-SeP1 ( Fig.10 ).

[0095] Based on the above results, the inventors selected A1AT-SeP2 for further efficacy evaluation. The lung is the organ with the highest oxygen content, and the surface of lung tissue has therefore become a highly oxidative environment. Drugs delivered through the lungs are more easily oxidized, so antioxidant capacity is an important parameter for drug development and formulation. To this end, the inventors used a mouse model of lung injury induced by porcine pancreatic elastase (PPE) to investigate the protective effects of A1AT and A1AT-SeP2 on lung injury, and instilled different doses of PPE by inhalation into the trachea to optimize the appropriate dose for inducing lung injury, and selected myeloperoxidase (MPO), the most abundant proinflammatory enzyme in the azure granules of neutrophils, as a biomarker of inflammation (Julius, S, et al, Archives of Biochemistry and Biophysics 1962, 96 (3), 465-467). As expected, the inflammatory response in mice induced by the administration of 100 μg PPE (150 U / kg) resulted in a 15-fold increase in MPO activity in bronchoalveolar lavage fluid (BALF) ( Fig.11 Mice treated with A1AT and A1AT-SeP2 24 hours before PPE administration showed significant protection ( Fig.12a However, when treated with A1AT or A1AT-SeP2 72 hours before PPE administration, A1AT was no longer able to provide adequate protection, while A1AT-SeP2 still effectively suppressed inflammation caused by lung injury ( Figure 12b), which suggests that the antioxidant capacity of A1AT-SeP2 can prolong the residence time of drugs in the lungs.

[0096] PEGylation of peptides can reduce renal clearance and increase their half-life and has been used to generate long-acting biotherapeutics. To evaluate whether SeP conjugation could produce similar effects, a single-dose pharmacokinetic study was performed in female C57BL mice injected via the tail vein. Fig.13 As shown in Figure 2, the half-life of A1AT-SeP2 was significantly prolonged compared with A1AT, suggesting the potential of A1AT-SeP2 in long-acting therapy. Similar to PEG, the number of units and molecular weight of SeP2 can be rationally designed by controlling the polymerization conditions to regulate the circulation half-life and antioxidant capacity of the coupled protein.

[0097] The inventors also conducted a preliminary toxicity assessment of SeP2. Mice were administered 1 mg / kg P(EG4-SeHC) (2.7 times higher than the dose used in the efficacy study) weekly for 4 weeks by intraperitoneal injection. No obvious toxicity was observed, and all mice in the polymer-administered group showed similar body weights to those in the PBS control group, indicating the potential biosafety of SeP2 ( Fig.14 ).

[0098] In summary, the inventors designed and produced a new class of selenium-containing peptides, whose powerful antioxidant capacity has great potential in long-acting protein therapy. The inventors optimized the expression and purification of A1AT in Escherichia coli and specifically coupled SePs at the Cys232 site of A1AT. The effect of coupling SePs at Cys232 of A1AT on its inhibition of NE activity was negligible, but it was able to effectively inhibit the activity of oxidants associated with inflammatory lung diseases (including COPD, IPF, ARDS and asthma). Experiments in vivo and on mouse models showed that SeP-A1AT conjugates had strong and persistent antioxidant activity. In addition, the coupling of SePs significantly prolonged the circulation half-life of A1AT, which is conducive to its development and application in long-acting therapy. In addition, P(EG4-SeHC) showed good safety in mice. In summary, the inventors' research reveals the potential of using antioxidant polymers as pharmacological ingredients to enhance oxidizable biotherapeutics.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A selenopolymer, characterized in that: The selenopolymer is obtained by ring-opening polymerization of a selenoamino acid containing N-carboxylic anhydride and an amino acid containing N-carboxylic anhydride; the selenoamino acid containing N-carboxylic anhydride is selenomethionine NCA; the amino acid containing N-carboxylic anhydride is sarcosine NCA; the end of the selenopolymer has a maleimide residue, and the selenopolymer is P(SeMet)3-CO-(Sar) 105 or P(EG4-SeHC) 30 - b -(Sar) 15 , the structural formula is as follows: 。 2. Use of the selenopolymer as claimed in claim 1 in the preparation of proteins that resist oxidative damage.

3. The use according to claim 2, characterized in that: The protein contains free thiol groups.

4. The use according to claim 3, characterized in that: The protein contains cysteine ​​residues.

5. The use according to claim 2, characterized in that: The protein is an enzyme or an antibody.

6. The use according to claim 5, characterized in that: The protein is a protease or an antibody that is susceptible to oxidation.

7. The use according to claim 6, characterized in that: The protease is alpha-1-antitrypsin.

8. The use according to claim 2, characterized in that: The selenopolymer is coupled to the protein.

9. The use according to claim 8, characterized in that: The selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue.

10. A protein that resists oxidative damage, characterized in that: The protein is coupled with the selenopolymer as described in claim 1.

11. The protein according to claim 10, characterized in that The selenopolymer is coupled to the free thiol group of the protein via the terminal maleimide residue.

12. The protein according to claim 11, characterized in that The protein is a protease or an antibody that is susceptible to oxidation.

13. The protein according to claim 12, characterized in that The protease is alpha-1-antitrypsin.

14. Use of the protein according to any one of claims 10 to 13 in the preparation of a medicament.

15. The use according to claim 14, characterized in that: When the protein is alpha-1-antitrypsin, the drug is a serine protease inhibitor.

16. The use according to claim 15, characterized in that The invention relates to an application of the protein that resists oxidative damage in preparing a medicine for protecting lung damage.

Images