Identification of novel small open reading frame coding peptide (SEPs) and application of novel small open reading frame coding peptide (SEPs) in inflammation

Abstract

The invention provides identification and preparation of a small open reading frame coding peptide (SEP) and application of the SEPs in inflammation regulation and control, belongs to the technical field of biomedical polypeptides, and particularly relates to enrichment of SEPs in an animal infection model sample, mass spectrometry and sample preparation as well as application of the SEPs in treatment of excessive inflammatory response. The SEPs screened in the invention have the advantages of small molecular weight, low production cost, good anti-inflammatory activity, and good anti-inflammatory treatment effect on an LPS-induced RAW264.7 cell inflammation model. The compound can be used for preparing a preparation for preventing or treating inflammatory infectious diseases, and inflammations include but not limited to acute and chronic inflammatory diseases caused by bacteria, immunity, drugs and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical peptide technology, and specifically relates to the identification of poorly annotated small open reading frame-encoded peptides (SEPs) by mass spectrometry analysis, and their application in the treatment of excessive inflammatory responses in cell models, especially to the sequence, preparation method and functional verification of SEPs-5 with significant anti-inflammatory activity. Background Art

[0002] Inflammation is the body's defensive response to injury or infection, mediated by the immune system, aimed at clearing harmful stimuli and initiating repair processes. Its core characteristics include redness, swelling, heat, pain, and functional impairment (caused by vasodilation, exudate accumulation, and immune cell infiltration). Major challenges with existing therapeutics include antibiotic dependency and misuse. Many patients mistakenly believe antibiotics are a panacea, leading to the rise of drug-resistant strains, and antibiotics are ineffective against viral or autoimmune inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) have limitations: ibuprofen, for example, can damage the gastrointestinal mucosa, and long-term use increases cardiovascular risk. Side effects of glucocorticoids: long-term use can lead to skin atrophy, osteoporosis, and immunosuppression, exacerbating the pathological process of chronic inflammation. Furthermore, existing drugs are limited in their targeting: most target single inflammatory mediators, making it difficult to modulate complex networks. To address these challenges, we aimed to selectively screen for small open reading frame-encoded peptides (SEPs) with anti-inflammatory activity.

[0003] Small open reading frames (sORFs) refer to open reading frames in the genome that are 300 bases or less in length. These ORFs have long been labeled as "transcriptional noise" or considered to lack coding capacity due to their small number of bases and difficulty in identification with traditional annotation tools. However, with the recent development of computer technology, ribosome analysis, and experimental techniques, increasing evidence has shown that sORFs can be transcribed and encoded into short peptides of 100 amino acids or less in length, which have been revealed to be involved in an increasingly wide range of basic biological processes. SEPs are characterized by their small size and molecular weight, which facilitates drug development; they are specifically expressed in tissues and have low cytotoxicity, and may have great potential in screening new drugs; they have a short translation time and can quickly respond to changes in the intracellular and extracellular environment; and they have strong target specificity. Because their molecular structure is similar to endogenous human peptides, SEPs can precisely act on inflammation-related targets (such as cytokine receptors or signaling pathway proteins), reducing interference with non-target tissues. Studies have shown that SEPs are widely involved in biological processes such as anti-cancer, antibacterial, immune regulation and embryonic development. However, the exploration of their functions in the anti-inflammatory field, especially the systematic exploration of SEPs and their anti-inflammatory mechanisms from bacterial infection models, is still relatively scarce. Therefore, the discovery of lead molecules for anti-inflammatory drugs has great practical significance.

[0004] Based on this, we established a bacterial infection mouse model, collected plasma, and obtained samples using an organic solvent extraction method. The samples were analyzed by LC-MS / MS, and differential analysis and screening were performed among the identified SEPs. At the same time, a series of anti-inflammatory activity detection experiments were carried out to detect inflammation regulation, and the SEPs with the strongest anti-inflammatory effect were selected. This SEP is expected to be used to develop a promising drug for the treatment of excessive inflammatory response. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for selectively screening SEPs with anti-inflammatory activity by obtaining samples through constructing a bacterial infection model, combining MTBE organic solvent extraction and LC-MS / MS analysis to identify SEPs, as well as the application of SEPs with anti-inflammatory activity in the treatment of excessive inflammatory reactions.

[0006] The above-mentioned invention objectives are achieved through the following technical solutions: extracting samples from a mouse model infected with Acinetobacter baumannii, enriching SEPs via MTBE organic solvent extraction, identifying them via LC-MS / MS analysis, and preparing them through chemical synthesis for use in anti-inflammatory research. The resulting SEPs have a simple structure and low molecular weight, allowing for efficient chemical synthesis. More importantly, they exhibit strong anti-inflammatory activity and high biosafety, making them suitable for treating excessive inflammatory responses.

[0007] The SEPs provided by the present invention are obtained by using a bacterial infection mouse model constructed using Acinetobacter baumannii. LL-37, which has both antibacterial and anti-inflammatory activity, is used as an indicator to detect changes in its content before and after infection. This is used to determine the sampling time and sampling site of the sample, which is a plasma sample 24 hours after infection. 201 SEPs were identified by MTBE organic solvent extraction combined with LC-MS / MS analysis, and 8 candidate SEPs were screened for chemical synthesis and anti-inflammatory activity testing based on their basic characteristics, difference multiples, related properties, and chemical synthesis feasibility. After NO content detection, SEP-5 with the best anti-inflammatory effect was screened out, and its anti-inflammatory activity was detected by RT-qPCR in the RAW264.7 cell inflammation model after treatment.

[0008] The beneficial effects of the present invention are: successfully identifying under-annotated SEPs from the plasma of bacterially infected mouse models, which have small molecular weight and short sequence, and have the advantage of greatly reducing production costs; having good anti-inflammatory therapeutic effects, and having the potential to be a lead molecule for the development of anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Statistical graph of LL-37 levels in mouse plasma at different times after infection.

[0010] Figure 2: Statistical graph of LL-37 expression in mouse plasma and various tissues.

[0011] Figure 3 : Purification chromatograms of candidate SEPs after chemical synthesis and mass spectrometry detection results of target peaks.

[0012] Figure 4 : NO detection results in the supernatant of RAW264.7 cell culture medium after treatment with candidate SEPs.

[0013] Figure 5 : RT-qPCR quantification of gene mRNA expression in RAW264.7 cells after SEPs-5 treatment. A, interleukin-1β; B, nitric oxide synthase; C, interleukin-6; D, tumor necrosis factor-α; E, cyclooxygenase-2; F, interleukin-10. DETAILED DESCRIPTION

[0014] The present invention provides 8 embodiments in total, and the specific information of the embodiments is shown in the following table.

[0015]

[0016] The eight SEPs were efficiently prepared through peptide solid-phase chemical synthesis. The measured molecular weights were consistent with the theoretical molecular weights, demonstrating their efficient preparation via chemical synthesis. Their effects on NO levels and mRNA expression levels of related inflammatory genes after treatment were systematically tested.

[0017] The sampling time of the bacterial infection mouse model was determined as follows Figure 1 As shown in the figure, to determine the sampling time after infection, 8 time gradients were set, namely 15 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours and 36 hours after infection, and plasma was taken for detection. Starting from 2 hours, the LL-37 content in the Ab infection group was statistically significantly different from that in the control group (P<0.0001; P<0.001), and the content difference reached its maximum at 24 hours, and the content decreased significantly at 36 hours, recovering to a level similar to that in the control group. Since LL-37 is widely distributed in the body, we referred to the literature and tested the LL-37 content in the plasma, heart, liver, spleen, lung and kidney homogenates of mice 24 hours after model induction. The test results are shown in the figure. Figure 2As shown, the LL-37 content in the plasma of the control group and the Ab-induced group showed a very significant difference (P<0.0001), with a difference of about 2.5 times, while there was no significant statistical difference in LL-37 in the homogenates of other tissues. In summary, the mouse plasma samples were collected 24 hours after infection for subsequent experiments. 201 SEPs were identified by MTBE organic solvent extraction combined with LC-MS / MS analysis, and 8 candidate SEPs were screened for chemical synthesis based on their basic characteristics, difference times, related properties and feasibility of chemical synthesis. The synthesis results are shown in the figure below. Figure 3 shown.

[0018] The NO level can directly reflect the activation state and inflammation degree of RAW264.7 cells, and is an important indicator for evaluating the efficacy of anti-inflammatory drugs. We detected NO in the supernatant of RAW264.7 cell culture medium after treatment with candidate SEPs. The results are as follows: Figure 4 As shown, the NO content in the LPS-induced group was significantly upregulated compared with the Control group (P<0.0001), indicating that LPS successfully stimulated the polarization of macrophages. In the drug-treated group, the NO content of SEPs-2, SEPs-4, SEPs-6 and SEPs-7 was downregulated at a final concentration of 10nM, but the trend was not significant (P<0.01); the NO content of SEPs-5 was significantly downregulated at a final concentration of 10nM (P<0.0001), and only SEPs-5 had a more significant downward trend at a final concentration of 1nM (P<0.001). This suggests that SEPs-5 may have good anti-inflammatory activity at low concentrations, so we used SEPs-5 at a final concentration of 10nM to perform subsequent detection of inflammation-related gene mRNA expression. The results are shown in Figure 5 As shown, the mRNA expression levels of proinflammatory factors such as IL-1β, INOS, IL-6, TNF-α, and COX-2 were significantly elevated under inflammatory pathological conditions (LPS-stimulated polarization), indicating that the cellular inflammation model was successfully induced. Furthermore, the mRNA expression levels of IL-1β, INOS, IL-6, TNF-α, and COX-2 in the drug-treated group were significantly downregulated after SEPs-5 treatment. As an anti-inflammatory cytokine, increased expression of IL-10 generally reflects compensatory suppression of the inflammatory response. Increased IL-10 expression can inhibit the release of proinflammatory factors by macrophages, thereby reducing inflammatory damage. In summary, we know that SEPs-5 can exhibit significant anti-inflammatory activity at the mRNA level.

[0019] 1. Construction of bacterial infection mouse model ( Figure 1 and Figure 2 ).

[0020] The bacterial species used in the present invention is Acinetobacter baumannii, which is frozen in 50% glycerol and stored at -80°C. After natural thawing, 1 mL of the frozen bacterial solution is added to 5-6 mL of BHI medium and cultured in a shaking incubator at 37°C until the logarithmic phase; the bacterial solution and BHI medium are then passaged at a ratio of 1:9 and cultured until the third logarithmic phase, and then diluted to 1×10 9 CFU / mL, keep on ice until ready; administer via intraperitoneal injection. Gently press the injection site with sterile gauze to prevent spillage of the bacterial solution. Mark the appropriate area of the mouse's body (such as the tail) with a marker, and record the injection time and dose. Provide the mouse with a comfortable environment and closely observe its behavior, diet, and mental state. At specific time points, collect samples for testing.

[0021] 2. Database search with LC-MS / MS analysis and raw data.

[0022] The present invention uses a Thermo Fisher Orbitrap Eclipse Tribrid mass spectrometer equipped with a FAIMS Pro interface for mass spectrometry analysis.

[0023] (1) The enzymatic peptide fragments were loaded onto a 2 cm PEPMAP Trap column (Thermo Fisher Scientific) for desalting.

[0024] (2) Separation was performed on a 25 cm PepMap C18 column (Thermo Fisher Scientific) using a linear gradient from 3% to 38% buffer (80% ACN, 0.1% formic acid) for 102 min and equilibrated in 100% buffer for 10 min.

[0025] (3) By setting up high-field asymmetric waveform ion mobility spectrometry (FAIMS), the CV values were switched between −35 V, −45 V, and −65 V in sequence, and the cycle time was set to 1 s.

[0026] (4) Primary spectra were collected using an electrostatic orbitrap (Orbitrap, Resolution: 60k; AGC target: Standard; MaxIT: Auto; RF lens: 50%; mass range: 350 to 1500). The dynamic exclusion time was set to 40 s. Secondary spectra were collected using a linear ion trap (isolation window: 1.6 m / z; scan rate: rapid; AGQ target: standard; MaxIT: Auto; HCDCE: 35%; data type: centroid) to obtain raw data.

[0027] Maxquant software (version 2.5, San Jose, CA) was used for mass spectrometry data library search and label-free relative quantitative analysis of the proteome. The SmProt mouse database (downloaded on November 24, 2024, containing 9472 sequences) was used for library search, and the parameters were set as follows.

[0028] (1) Methionine oxidation and protein N-terminal acetylation were set as variable modifications, and cysteine alkylation was set as a fixed modification.

[0029] (2) The mass tolerances for precursor ions and fragment ions were set to 10 ppm and 0.6 Da, respectively. The minimum and maximum peptide lengths were 6 and 144 amino acids, respectively.

[0030] (3) The number of missed cleavage sites allowed for the peptide segment was 2, the FDR was set to 0.01, and the other parameters used the default values.

[0031] 3. Solid-phase peptide chemical synthesis of SEPs ( Figure 3 ).

[0032] This invention utilizes the Fmoc solid-phase peptide synthesis method to synthesize linear peptides. Specifically, Rink resin is used as a support, and peptides are synthesized sequentially from the C-terminus to the N-terminus, using Fmoc (9-fluorenylmethoxycarbonyl) as the amino protecting group for the amino acids. After coupling, the newly synthesized peptide is cleaved from the Rink resin using a cleavage buffer, then precipitated with glacial ether and identified by mass spectrometry. Based on a 0.1 mmol peptide synthesis scale, the specific steps are as follows: (1) Resin expansion. Weigh 0.1 mmol of Rink resin and place it in a synthesis tube. Add 3 mL of DMF and expand it for 1 hour. Then filter out the DMF in the tube.

[0033] (2) Deprotection of the resin. After the resin was expanded, 3 mL of 20% piperidine was added to the resin and deprotected on a rotator for 7 minutes. The piperidine was then removed and 3 mL of 20% piperidine was added to the resin for a second deprotection on a rotator for 8 minutes. After deprotection, the piperidine was removed and the resin was washed with DMF 8 times.

[0034] (3) Activation of amino acids. Activate the amino acids while performing the first deprotection. Take one portion each of HATU and HOBT, add 0.75 mL of N-methylmorpholine to each, mix the two, and then add them to the pre-weighed amino acids. Mix on a rotary mixer. The activation time is 15-20 min.

[0035] (4) Coupling of amino acids. Add the activated amino acids to the resin in step (2), shake to mix the resin and amino acid solution thoroughly, and perform the coupling reaction on a rotator for 1 hour.

[0036] (5) Peptide chain extension. Repeat steps (2)-(4) until the last amino acid coupling reaction is completed.

[0037] (6) Peptide chain cleavage. After the last amino acid coupling reaction is completed, deprotect the peptide with 20% piperidine, then wash with DMF 8 times and anhydrous methanol 4-5 times. After washing, drain and add 6 mL of lysis buffer. React in a rotary mixer for 2 h. Collect the lysis buffer in a 50 mL centrifuge tube and add ice ether to the lysis buffer for precipitation twice. The lower precipitate is the crude peptide. The crude peptide after precipitation is placed in a fume hood for 2-3 minutes to evaporate the ether in the tube. Then, seal and store at 4°C.

[0038] 4. NO content determination ( Figure 4 ).

[0039] The present invention uses an NO detection kit to detect the NO content in the supernatant of the cell culture medium.

[0040] (1) Cell inoculation: Add 100 μL of uniform cell suspension to each well of a 96-well plate except the outermost circle. Add 100 μL of PBS to the outermost circle to avoid the influence of edge effects on the cell state. Culture for 24 hours until the cell density reaches about 80%.

[0041] (2) Preparation of drugs: Use serum-free DMEM medium and add LPS to a final concentration of 2μg / mL to stimulate macrophage polarization. Two final concentration gradients of 10nM and 1nM were set for each drug group. The peptide drug solution was added to serum-free medium containing LPS. When adding drugs, the old medium in the wells was discarded and 100μL of the corresponding drug was added to each well. Six replicates were performed for each gradient. (3) Set up a blank group (cultured only with serum-free DMEM medium) and a control group (no drug, cultured with serum-free DMEM medium containing LPS at a final concentration of 2 μg / mL) and treat the cells for 24 h.

[0042] (4) Collect the culture medium supernatant for detection. The specific operation should be carried out according to the instructions of the NO detection kit.

[0043] 5. Real-time fluorescence quantitative PCR detection ( Figure 5 ).

[0044] (1) RNA extraction: RNA was extracted using a chloroform-free extraction method using an RNA extraction kit purchased from Accurate Biology. For specific steps, refer to the instructions.

[0045] (2) One-step RT-qPCR: A one-step kit purchased from Vazyme Biotech was used to perform quantitative PCR using RNA as a template. The PCR primer templates are shown in the following table:

Claims

1. A novel SEPs or a pharmaceutically acceptable salt thereof, characterized in that: The SEPs comprise eight polypeptides, which can be prepared by solid-phase peptide chemical synthesis. Their names, sequences, and molecular weights are as follows: SEPs-1; ISKKTSDPR; 1330.35 Da. SEPs-2; KNLFMMEQSRTHFVEQ; 2024.94 Da. SEPs-3; MVEGKKQPLRTHNLKPRDLNVLTPTGF; 3090.67 Da. SEPs-4; VPHHGRGQRPGPPLCSRVRPAPGAAGSLGHQHAEQ; 3616.43 Da. SEPs-5; MRSRMHQSKPAS; 1415.37 Da. SEPs-6; RVESGRKSTELLERKT; 1888.31 Da. SEPs-7; ITENAGGKSMHGPSRK; 1669.70 Da. SEPs-8; MLSGLGKVLRNSSPTM; 1691.61 Da.

2. The novel SEPs or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The SEPs also include a pharmaceutically acceptable carrier.

3. The novel SEPs or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The SEPs also include a pharmaceutically acceptable diluent.

4. The novel SEPs or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The SEPs also include pharmaceutically acceptable adjuvants.

5. The novel SEPs or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The SEPs also include a pharmaceutically acceptable vehicle.

6. Use of a novel SEPs or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The preparation can be prepared for preventing or treating inflammatory infectious diseases, including but not limited to acute and chronic inflammatory diseases caused by bacteria, immunity, drugs, etc.

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