Specific inhibition polypeptide of lactoacylated S100A9 protein and application of specific inhibition polypeptide in improvement of viral myocarditis and cardiac dysfunction

Abstract

The invention discloses a specific inhibition polypeptide of milk acylation S100A9 protein and application of the specific inhibition polypeptide in improvement of viral myocarditis and cardiac dysfunction. The invention starts from the pathogenesis of myocarditis, and provides a therapeutic drug CCP-K26 for cardiac dysfunction caused by viral myocarditis based on a milk acylation modified target protein-S100A9K26la identified by utilizing proteomics and modification omics in an animal model, and the CPP-K26 polypeptide is formed by connecting 13 amino acids (HQYSRKEGHPDTLC) of S100A9K26la with a cell-penetrating peptide (HLYVSPWGG). Experiments prove that the polypeptide CCP-K26 can relieve the inflammation level and cardiac dysfunction of mice with myocarditis, so that the polypeptide CCP-K26 can be used as a medicine for treating viral myocarditis, and an effective technical means is provided for precise treatment of patients with myocarditis.

Description

Technical Field

[0001] The present invention relates to a specific inhibitory polypeptide of lactylated S100A9 protein, and also relates to the application of the polypeptide in improving cardiac dysfunction in viral myocarditis. The present invention belongs to the field of pharmaceutical technology. Background Art

[0002] Myocarditis is a non-ischemic inflammatory heart disease among cardiovascular diseases, characterized by inflammatory infiltration and myocardial damage. Myocarditis is usually diagnosed by endomyocardial biopsy and cardiac magnetic resonance imaging (CMRI) analysis. The annual incidence worldwide is approximately 1.8 million cases, with an incidence rate of (1.0 - 2.2) / 1 million. The number of patients with myocarditis is increasing year by year, and it has become one of the public health problems that need to be solved urgently. Myocarditis is usually caused by cardiotropic virus infections, especially Coxsackievirus (CVB3), followed by active inflammatory damage to the myocardium. Viral myocarditis (VMC) remains the main cause of heart failure and sudden cardiac death in young people, and the 5-year mortality rate of heart failure is >60% (Ponikowski et al., 2014). VMC is not only characterized by cardiomyocyte death, but also by the parallel activation of innate and adaptive immune responses and the production of inflammatory factors.

[0003] The onset symptoms of viral myocarditis are relatively concealed and not easily diagnosed. Patients often show non-specific systemic symptoms in the early stage of the disease, including fever, muscle soreness, respiratory and gastrointestinal symptoms. The overall burden of myocarditis in China is still relatively high. At present, the treatment of viral myocarditis mainly focuses on supportive treatment. Patients with hemodynamic failure may require mechanical support. There is no specific treatment plan for viral myocarditis, and the treatment mainly lies in reducing the cardiac load, improving myocardial metabolism and cardiac function, and promoting myocardial repair. Large doses of vitamin C and energy mixture can be intravenously infused, and adrenal cortical hormones are used in critically ill patients. Since the pathogenesis of obvious myocardial dysfunction in myocarditis is the result of maladaptive hyperimmune responses triggered by viral infections, treatment targeting the regulation of immune responses is considered potentially beneficial, and some progress has been made in the research of myocarditis. However, there is currently no specific drug treatment for viral myocarditis, and exploring more treatment strategies for myocarditis is an important clinical problem that needs to be solved urgently.

[0004] Lactyl modification is a novel epigenetic modification with lactic acid as the substrate, which can regulate gene expression and protein function at the same time. More and more protein lactyl modifications have been identified and proven to play a key role in the progression of immune inflammation. In viral myocarditis, the lactic acid level is significantly increased and is related to the prognosis of patients, which suggests that lactyl modification may be an important pathogenic mechanism of viral myocarditis. However, the role and targets of lactyl modification in immune cells are still blank. Therefore, it is very necessary to deeply explore the molecular mechanism of viral myocarditis based on lactyl modification, provide beneficial management and treatment clues for the treatment of cardiomyopathy patients, and identify new and reliable therapeutic drugs. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a target closely related to the occurrence of viral myocarditis and its application in the preparation of drugs for treating or improving cardiac dysfunction in viral myocarditis.

[0006] Another purpose of the present invention is to provide a specific inhibitory polypeptide of the above target and its application in improving cardiac dysfunction in viral myocarditis.

[0007] In order to achieve the above purposes, the present invention adopts the following technical means:

[0008] The present invention identified the lactyl-modified protein S100A9 that was significantly upregulated in viral myocarditis mice through proteomics and modification omics (S100A9 is a calcium-binding protein, mainly restrictively expressed in mononuclear-macrophage lineage, neutrophils, and keratinocytes under specific pathological conditions, and is involved in the inflammatory response and tumor development process. Reference: Hessian PA, Edgeworth J, Hogg N. MRP-8and MRP-14, two abundant Ca 2+-binding proteins of neutrophils and monocytes[J]. jLeu-koc Biol, 1993, 53(2): 197-204. Markowitz J, Carson WE. Review of S100A9 biology and its role in cancer[J]. Biochim Biophys Acta, 2013, 1835(1): 100-109), and it was determined that the lactylation modification site was at the 26th amino acid (26K) of its sequence. Therefore, this protein was named S100A9K26la. Based on the S100A9K26la protein, the present invention developed a polypeptide CPP-K26 that can enter cells and inhibit S100A9K26la intracellularly. The CPP-K26 polypeptide can enter various immune cells, specifically inhibit S100A9K26la, and thereby improve the cardiac dysfunction in myocarditis. We intraperitoneally injected this polypeptide into viral myocarditis mice for five consecutive days and found that the levels of inflammatory factors such as IL-6 and IL-1β in viral myocarditis mice decreased, the inflammatory infiltration and cardiac fibrosis decreased, the cardiac output increased, and the end-diastolic volume of the heart decreased, indicating that CPP-K26 can improve the cardiac dysfunction caused by myocarditis.

[0009] Based on the above research, the present invention first proposed the application of the lactylation-modified S100A9 protein as a target closely related to the occurrence of viral myocarditis in the preparation of drugs for treating or improving cardiac dysfunction in viral myocarditis. The amino acid sequence of the S100A9 protein is shown as SEQ ID NO.1, wherein the lactylation modification site is at the 26th amino acid (26K) of the sequence shown as SEQ ID NO.1.

[0010] Secondly, the present invention also proposed the application of an inhibitor for inhibiting the lactylation-modified S100A9 protein in the preparation of drugs for treating or improving cardiac dysfunction in viral myocarditis.

[0011] Thirdly, the present invention also proposed a specific inhibitory polypeptide for the lactylation S100A9 protein, named CPP-K26. The amino acid sequence of the polypeptide is shown as SEQ ID NO.6.

[0012] Finally, the present invention also proposed the application of the specific inhibitory polypeptide in the preparation of drugs for treating or improving cardiac dysfunction in viral myocarditis.

[0013] Preferably, the specific inhibitory polypeptide can reduce the level of inflammatory factors in patients with viral myocarditis, reduce inflammatory infiltration and cardiac fibrosis, increase cardiac output, and reduce the end-diastolic volume of the heart, so as to achieve the purpose of treating or improving cardiac dysfunction caused by viral myocarditis.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] At present, due to the lack of specific drugs for treating myocarditis in clinical practice, the severity and mortality rate are relatively high, resulting in a heavy medical cost for patients and a social and economic burden. Starting from the pathogenesis of myocarditis, the present invention is based on the lactylation modification target protein - S100A9K26la identified by proteomics and modification omics in an animal model, and proposes a therapeutic drug CCP-K26 for cardiac dysfunction caused by viral myocarditis. The CPP-K26 polypeptide is composed of a cell-penetrating peptide (HLYVSPWGG) linked to 13 amino acids (HQYSRKEGHPDTLC) of S100A9K26la. Experiments have shown that the polypeptide CCP-K26 can reduce the inflammatory level and cardiac dysfunction in mice with myocarditis, so it can be used as a new therapeutic drug for viral myocarditis. In summary, the present invention provides a new treatment plan and an effective technical means for the precise treatment of patients with myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Representative immunoblotting and quantitative results of the lactylation level of S100A9K26 in neutrophils in peripheral blood of mice with viral myocarditis at different time points;

[0017] Figure 2 First-level mass spectrometry diagram of CPP-K26;

[0018] Figure 3 First-level mass spectrometry diagram of CPP-K26R;

[0019] Figure 4 Representative immunoblotting and quantitative results of the lactylation level of S100A9K26 in neutrophils of mice with myocarditis after injection of CCP-K26 and CPP-K26R polypeptides;

[0020] Figure 5 PCR quantitative results of the expression levels of cardiac inflammatory factors in mice with myocarditis after injection of CCP-K26 and CPP-K26R polypeptides;

[0021] Figure 6 Representative M-mode echocardiogram and quantitative results of cardiac function in mice with myocarditis after injection of CCP-K26 and CPP-K26R polypeptides. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0023] Example 1 Screening of the lactylation-modified target protein - S100A9K26la

[0024] 1.1 Construction of a viral myocarditis mouse model

[0025] The virus used in this example is CVB3 (Woodruff strain). Before infection, the virus titer was regularly evaluated by measuring the 50% tissue culture infectious dose (TCID50) of monolayer HeLa cells. 1×10 5 Plaque forming unit (PFU) of CVB3 was dissolved in 100 μl of phosphate buffered solution (PBS) and injected into the abdominal cavity of C57BL / 6 mice to establish a viral myocarditis mouse model.

[0026] 1.2 Echocardiography of mice

[0027] After the mice were anesthetized and depilated, they were fixed in the supine position on a heating pad to keep their body temperature at 37°C. A FUJIFILM VisualSonics Vevo 3100LT small animal ultrasonic imaging instrument was used to perform transthoracic echocardiography on the mice. Two-dimensional and M-mode echocardiogram images were obtained in the parasternal long-axis view of the left ventricle. Cardiac function indexes of more than 3 cardiac cycles were measured, including left ventricular ejection fraction (EF) and shortening fraction (FS).

[0028] 1.3 Screening and identification of neutrophil lactylation-modified proteins in viral myocarditis

[0029] Peripheral blood neutrophils of mice with viral myocarditis for 7 days were isolated, and PANKLA antibody was used for proteomics and lactylation modification proteomics to screen differentially expressed proteins and analyze their biological functions. The specific methods are as follows:

[0030] ① Protein extraction

[0031] The samples were taken out from -80°C and 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitor, 3 μM TSA, 50 mM NAM) were added respectively, followed by ultrasonic lysis. Centrifuge at 12,000 g for 10 min at 4°C to remove cell debris. Transfer the supernatant to a new centrifuge tube and measure the protein concentration using a BCA kit.

[0032] ② Trypsin digestion

[0033] According to the measured protein concentration, an equal amount of protein was taken from each sample, and the samples in different groups were diluted and adjusted to the same concentration and volume with the lysis solution. DTT was added to the above protein solution to make the final concentration of DTT 4.5 mM, mixed well, and incubated at 55°C for 30 min. Cool on ice until reaching room temperature. (Note: When feeling with hands, the solution should not be too cold or too hot.) Add the corresponding volume of iodoacetamide to make the final concentration 9 mM, mix well, and place in the dark at room temperature for 15 min. Add 6 volumes of acetone to precipitate the protein, and place at -20°C for more than four hours or overnight. Centrifuge at 8,000×g for 10 min at 4°C to collect the precipitate, and evaporate acetone for 2 - 3 min. Add 100 μL TEAB2 to redissolve the precipitate, add 1 mg / ml trypsin Trypsin-TPCK at 1 / 50 of the sample mass, and digest overnight at 37°C. Adjust the pH value to about 3 with phosphoric acid to terminate the enzymatic reaction.

[0034] ③ Modification enrichment

[0035] Dissolve the peptide segments in IP buffer solution (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0), transfer the supernatant to the pre-washed lactylation resin (antibody resin product number PTM-1401RM, sourced from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio), place on a rotary shaker at 4°C, gently shake and incubate overnight. After incubation, wash the resin 4 times with IP buffer solution and twice with deionized water in sequence. Finally, use 0.1% trifluoroacetic acid eluent to elute the peptide segments bound to the resin, elute three times in total, collect the eluate and vacuum freeze-dry. After drying, desalt according to the C18 ZipTips instruction manual, and vacuum freeze-dry for liquid chromatography-tandem mass spectrometry analysis.

[0036] ④ LC-MS / MS high-resolution mass spectrometry detection

[0037] The peptide segments were dissolved in mobile phase A of liquid chromatography and separated using a NanoElute ultra-high performance liquid system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: 0 - 42 min, 7% - 24% B; 42 - 54 min, 24% - 32% B; 54 - 57 min, 32% - 80% B; 57 - 60 min, 80% B, and the flow rate was maintained at 450 nL / min. After being separated by the ultra-high performance liquid system, the peptide segments were injected into a Capillary ion source for ionization and then analyzed by a timsTOF Pro mass spectrometer. The ion source voltage was set at 1.6 kV, and both the peptide segment parent ions and their secondary fragments were detected and analyzed using a high-resolution TOF. The secondary mass spectrometry scan range was set at 400 - 1500 m / z. The data acquisition mode used the parallel accumulation serial fragmentation (PASEF) mode. After one primary mass spectrometry acquisition, 10 PASEF mode acquisitions were performed to obtain secondary spectra of parent ion charge numbers in the range of 0 - 5. The dynamic exclusion time for tandem mass spectrometry scanning was set at 30 s to avoid repeated scanning of parent ions.

[0038] ⑤ Bioinformatics analysis

[0039] During the GO enrichment analysis, proteins were classified into three major categories: biological process, cellular component, and molecular function through GO annotation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway enrichment analysis, and finally, these pathways were classified according to the pathway hierarchical classification method on the KEGG website (http: / / www.genome.jp / kegg / ). The InterPro (http: / / www.ebi.ac.uk / interpro / ) database was used to analyze the enrichment of functional domains of differentially expressed proteins. In the three functional enrichment analyses (GO, KEGG pathways, and protein domains), Fisher's exact two-tailed test was used to compare the enrichment of differentially expressed proteins with all proteins, and the standard FDR control method was used to correct multiple hypothesis tests. A corrected P < 0.05 was considered significantly enriched.

[0040] The results are as Figure 1 shown in the figure, which is the level of S100A9 lactylation in neutrophils in the peripheral blood of mice with viral myocarditis at different time points (con / 3 days / 7 days / 14 days) detected by immunoblotting. The results show that after infection with the myocarditis virus, the lactylation level of neutrophils in the peripheral blood of mice gradually increased over time, reached a peak on the seventh day, and then gradually decreased, returning to the baseline level on the 14th day. This proves that the lactylation of S100A9 in mice significantly increased after viral myocarditis infection.

[0041] Protein S100A9 was selected as a target closely related to the pathogenesis of viral myocarditis, and its amino acid sequence is:

[0042] MANKAPSQMERSITTIIDTFHQYSR-(lactyl)K-EGHPDTLSKKEFRQMVEAQLATFMKKEKRNEALINDIMEDLDTNQDNQLSFEECMMLMAKLIFACHEKLHENNPRGHGHSHGKGCGK (as shown in SEQ ID NO.1), wherein the lactylation modification site was determined to be at the 26th amino acid (K26) of the sequence shown in SEQ ID NO.1, and the S100A9 protein containing the K26 lactylation site was named S100A9K26la.

[0043] Example 2 Antibody Development

[0044] According to the sequence and modification type of the S100A9K26la protein, 2 modified antigen polypeptides (Table 1) were designed and synthesized for animal immunization, purification and detection; at the same time, 1 unmodified control polypeptide (Table 1) was designed and synthesized for purification and detection. Immunogenic mass spectrometry detection was carried out, and the difference between the measured mass and the theoretical mass of the 3 peptide segments was within 10 ppm, and the sequence was correct. After multiple immunizations of 6 SPF experimental-grade New Zealand white rabbits, a small amount of serum was taken for ELISA detection respectively to preliminarily evaluate the titer and specificity of the antiserum. Sufficient rabbit serum was taken for affinity purification on Protein A and immunogenic polypeptide columns. The purified antibodies of R2, R3, R5, and R6 were labeled as Ab2, Ab3, Ab5, and Ab6 respectively. The purified antibodies were first subjected to ELISA and DotBlot detection, and the antibodies that passed the detection were subjected to subsequent Western detection. Based on the above antibody detection and analysis, Ab2 was selected as the specific antibody against anti-S100A9K26la.

[0045] The results of Ab2 antibody were as follows: ELISA positive (OD450>1.0), antibody dilution greater than 54,000, and 10 times stronger than the signal recognizing the unmodified polypeptide. The positive detection limit of Dotblot reached 4 ng, and it was more than 10 times stronger than the signal recognizing the unmodified polypeptide.

[0046] The results of other applications showed that: Ab2 antibody could recognize endogenous S100A9K26la by WB, and the band position was correct. The polypeptide information is shown in Table 1. The amino acid sequence of Ab2 antibody is:

[0047] MANKAPSQMERSITTIIDTFHQYSR-(lactyl)K-EGHPDTLSKKEFRQMVEAQLATFMKKEKRNEALINDIMEDLDTNQDNQLSFEECMMLMAKLIFACHEKLHENNPRGHGHSHGKGCGK (shown in SEQ ID NO.2).

[0048] Table 1 Modified and unmodified polypeptides designed and synthesized

[0049]

[0050] Example 3 Development of CCP-K26 polypeptide and injection into mice

[0051] The polypeptide consists of a cell-penetrating peptide (HLYVSPWGG, abbreviated as CPP) and a short peptide of S100A9 K26 (HQYSRKEGHPDTLC) or a short peptide of S100A9 K26R (HQYSRREGHPDTLC). It is named CPP-K26 or CPP-K26R and is capped at both ends during synthesis. The synthetic peptide is purified to >95% purity by high performance liquid chromatography for in vitro and in vivo use. The amino acids of the peptide for in vivo use are all D-isotypes. For in vitro experiments, the polypeptide is dissolved in PBS to produce a 10 mM stock solution. For in vivo use, CPP-K26 and CPP-K26R are dissolved in PBS and kept on ice until injection. Before injection, the solution is placed at room temperature. The polypeptide information is shown in Table 2, and the N-terminus and C-terminus of the polypeptide are capped by acetylation (AC) and amidation (NH2) respectively. Figure 2 Is the first-order mass spectrum of CPP-K26, Figure 3 Is the first-order mass spectrum of CPP-K26R.

[0052] C57BL / 6 mice started to receive intraperitoneal injection of 5 mg / kg polypeptide once a day for five consecutive days on the 2nd day after receiving CVB3 intraperitoneally. On the 7th day, their cardiac function was detected by ultrasound and the expression level of cardiac inflammatory factors was detected by PCR.

[0053] Table 2 Therapeutic polypeptides designed and synthesized

[0054]

[0055] The results are as Figure 4As shown, this figure shows the level of S100A9 K26 lactylation in neutrophils of mice with viral myocarditis after injection of the CCP-K26 polypeptide. Compared with the control polypeptide CPP-K26R, the continuous injection of the CPP-K26 polypeptide significantly inhibited the level of S100A9 K26la in neutrophils of mice with myocarditis, indicating that the CCP-K26 polypeptide is an effective inhibitor of S100A9 K26la.

[0056] The results are as Figure 5 shown. This figure shows the expression levels of cardiac inflammatory factors in mice with viral myocarditis after injection of the CCP-K26 polypeptide. Compared with the control polypeptide CPP-K26R, the continuous injection of the CPP-K26 polypeptide significantly reduced the expression of the pro-inflammatory cytokines IL-1β and IL-6 mRNA in the hearts of mice with viral myocarditis, indicating that the CCP-K26 polypeptide can effectively reduce the production of pro-inflammatory cytokines in the hearts of mice with viral myocarditis.

[0057] The results are as Figure 6 shown. This figure shows the cardiac function of mice with viral myocarditis after injection of the CCP-K26 polypeptide. Compared with the control polypeptide CPP-K26R, the left ventricular ejection fraction (Ejection fraction, EF) and shortening fraction (Shortening fraction, FS) of the hearts of mice with viral myocarditis after continuous injection of the CPP-K26 polypeptide. This indicates that the CCP-K26 polypeptide can effectively improve cardiac dysfunction in mice with viral myocarditis.

Claims

1. Use of lactylated S100A9 protein as a target closely related to viral myocarditis in the preparation of a drug for treating or improving cardiac dysfunction caused by viral myocarditis, wherein the amino acid sequence of the lactylated S100A9 protein is shown in SEQ ID NO.1, wherein: The lactoylation modification site is located at the 26th amino acid in the sequence shown in SEQ ID NO.

1.

2. Use of an inhibitor for inhibiting lactylated S100A9 protein in the preparation of a drug for treating or improving cardiac dysfunction caused by viral myocarditis, wherein the amino acid sequence of the lactylated S100A9 protein is shown in SEQ ID NO.1, wherein: The lactoylation modification site is located at the 26th amino acid in the sequence shown in SEQ ID NO.

1.

3. A specific inhibitory polypeptide of lactoylated S100A9 protein, named CPP-K26, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

6.

4. Use of the specific inhibitory polypeptide according to claim 3 in the preparation of a drug for treating or improving cardiac dysfunction caused by viral myocarditis.

5. The use according to claim 4, characterized in that The specific inhibitory polypeptide can reduce the level of inflammatory factors in patients with viral myocarditis, reduce inflammatory infiltration of cardiac fibrosis, increase cardiac output and reduce the end-diastolic volume of the heart, thereby achieving the purpose of treating or improving cardiac dysfunction caused by viral myocarditis.

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