Anti-inflammatory therapeutic application of a MNSFβ / RC3H1 PPI interfering peptide
By designing interference polypeptides that interfere with MNSFβ-RC3H1 interaction, the problems of poor stability and large side effects of existing TNFα inhibitors have been solved, and the effect of significantly inhibiting TNFα expression has been achieved, providing new targets and drug molecules for anti-TNFα therapy.
Patent Information
- Application Number
- CN202310475140.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-28
- Publication Date
- 2025-03-18
- Estimated Expiration
- 2043-04-28
AI Technical Summary
Existing drugs that directly inhibit TNFα such as monoclonal antibodies and fusion proteins have poor stability, high production costs and possible serious side effects, and still have potential risks to pregnancy. It is urgent to develop new small-molecule TNF-α inhibitors.
By studying the effect of MNSFβ and/or RC3H1 expression on TNFα expression in macrophages, an interfering polypeptide capable of interfering with MNSFβ-RC3H1 interaction was designed. The specific sequence is shown in SEQ ID NO: 2 to inhibit the expression of TNFα.
This interfering polypeptide significantly inhibits the expression of TNFα in macrophages and has no macrophage toxicity, providing new targets and drug molecules for anti-TNFα therapy.
Smart Images
Figure SMS_1 
Figure HDA0004205226110000011 
Figure HDA0004205226110000012
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to an anti-inflammatory therapeutic application of a MNSFβ / RC3H1 PPI interfering polypeptide. Background Art
[0002] Tumor necrosis factor α (TNFα) is a pleiotropic proinflammatory cytokine, also known as cachectin and TNFSF1A, which is mainly secreted by active macrophages and monocytes. TNFα exerts its biological functions by binding to type 1 and type 2 TNFα receptors (TNF-R1 or TNF-R2). The biological functions of TNFα are diverse and its mechanism of action is relatively complex. On the one hand, it regulates the body to fight against certain types of infections, and on the other hand, it can cause pathological complications. Studies have found that elevated TNF-α levels are associated with tumors, diabetes, and especially autoimmune diseases such as rheumatoid arthritis (RA), psoriatic arthritis, multiple sclerosis, and Crohn's disease. Therefore, direct inhibition of TNF-α has become the main method for treating these diseases. In addition, high levels of TNFα at the maternal-fetal interface are believed to be associated with pathological pregnancies such as spontaneous abortion, premature birth, and preeclampsia (PE). Therefore, TNFα has become an important target for drugs for the treatment of obstetric complications, and the intervention effect of TNFα blockers in some cases related to adverse obstetric outcomes has been confirmed.
[0003] Monoclonal antibodies and fusion protein drugs can directly bind to TNFα and prevent its interaction with tumor necrosis factor receptor (TNFR). They are the main drugs on the market that are used to antagonize the function of TNFα. Although these biological macromolecule preparations show good efficacy and high specificity in the treatment of autoimmune diseases. However, they have poor stability, high production costs, and may have serious side effects. There is still a potential risk to pregnancy. Therefore, further research and development of new small molecule TNF-α inhibitors is urgently needed. Through in-depth research on the regulation mechanism of TNFα expression in macrophages and the discovery of potential drug targets, it is particularly important to design and develop new drugs targeting TNFα. Summary of the invention
[0004] The present invention studies the effect of down-regulating the expression of MNSFβ and / or RC3H1 in macrophages on the expression level of TNFα and finds that MNSFβ is involved in the expression regulation of TNFα, down-regulating MNSFβ can inhibit the expression of TNFα, and MNSFβ depends on RC3H1 to regulate the expression of TNFα. In addition, the present invention verifies the binding of MNSFβ to the HEPN-N (81-326aa) domain of RC3H1 through in vitro protein binding experiments. On this basis, the present invention designs and provides an interfering polypeptide capable of interfering with the MNSFβ-RC3H1 interaction (PPI), the sequence of the interfering polypeptide is shown in SEQ ID NO: 2, and experiments prove that the interfering polypeptide of the present invention can significantly inhibit the binding of MNSFβ to RC3H1. In addition, fluorescence quantitative PCR and CCK-8 methods verify that the interfering polypeptide of the present invention can significantly inhibit the expression of TNFα in macrophages without macrophage toxicity.
[0005] Therefore, the first object of the present invention is to provide an application of the signaling molecule complex MNSFβ-RC3H1 for use as a target for screening or designing drugs for anti-TNFα treatment.
[0006] According to the present invention, MNSFβ of the signaling molecule complex MNSFβ-RC3H1 binds to the HEPN-N (81-326aa) domain of RC3H1.
[0007] Furthermore, the amino acid sequence of the HEPN-N (81-326aa) domain is shown in SEQ ID NO: 1.
[0008] The second object of the present invention is to provide a MNSFβ / RC3H1 PPI interfering polypeptide for anti-TNFα treatment, wherein the amino acid sequence of the interfering polypeptide is shown in SEQ ID NO:2.
[0009] The third object of the present invention is to provide the use of the MNSFβ / RC3H1 PPI interfering polypeptide for preparing drugs for anti-TNFα treatment.
[0010] The beneficial effects of the present invention are as follows: the present invention discovered a new signaling molecule complex MNSFβ-RC3H1 that regulates the expression of inflammatory factors such as TNFα, providing a new target for the development of anti-TNFα drugs; in addition, the present invention provides an interfering polypeptide that can interfere with the MNSFβ-RC3H1 interaction (PPI), which can be used as a new drug molecule for anti-TNFα treatment and applied in clinical drug development. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1This is a graph showing the result of Example 1, showing the expression level of TNFα after down-regulating the expression of MNSFβ and / or RC3H1.
[0012] Figure 2 This is a graph showing the results of western blotting detection of different incubation groups in Example 2.
[0013] Figure 3 This is a graph showing the results of western blotting detection of different incubation groups in Example 3.
[0014] Figure 4 This is a graph showing the binding strength of MNSFβ and RC3H1 in different incubation groups in Example 3.
[0015] Figure 5 This is a graph showing the results of detecting the TNFα expression levels in different treatment groups using the qPCR method in Example 4.
[0016] Figure 6 This is a graph showing the results of the WB experiment in Example 4 to detect the expression levels of TNFα in different treatment groups.
[0017] Figure 7 This is a graph showing the results of detecting the cytotoxicity of different treatment groups using the CCK-8 method in Example 5. DETAILED DESCRIPTION
[0018] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions, and the materials used, such as vectors, strains, etc., can all be obtained from commercial sources unless otherwise specified.
[0019] Example 1: Study on the effect of down-regulating the expression of MNSFβ and / or RC3H1 in macrophages on the expression level of TNFα
[0020] Human monocytic cell line THP1 (purchased from American Type Culture Collection (ATCC)) was cultured at 5×10 5 Cells / well were seeded in a 12-well plate, and 200ng / ml phorbol myristate ester (PMA) was added to culture for 24 hours. Negative control (NC), MNSFβ-specific siRNA (siMNSFβ) and RC3H1-specific siRNA were transfected using lipofectamine2000. After further culture for 24 hours, LPS (100ng / ml) was used to stimulate for 1 hour. Each well of cells was washed twice with 500ul PBS, and 500ul Trizol was added to extract total RNA from the cells. Reverse transcription was performed using the reverse transcription kit of TAKARA, and the expression level of the inflammatory factor TNFα was detected by fluorescent quantitative PCR. The results are shown in Figure 2. Figure 1 shown.
[0021] Figure 1 In the figure, NC indicates macrophages transfected with negative control siRNA; siMNSFβ indicates macrophages transfected with MNSFβ-specific siRNA; siRC3H1 indicates macrophages transfected with RC3H1-specific siRNA; siMNSFβ+siRC3H1 indicates macrophages transfected with both MNSFβ- and RC3H1-specific siRNAs; *: P<0.05, P<0.05 indicates statistically significant difference; ns: P>0.05, P>0.05 indicates no statistically significant difference.
[0022] Figure 1 The results showed that in macrophages induced by human mononuclear cells THP1, the expression level of TNFα was significantly reduced after downregulating MNSFβ compared with the control group; while the expression level of TNFα was significantly increased after downregulating RC3H1 or downregulating both MNSFβ and RC3H1, and there was no significant difference in the expression levels of TNFα between the two. This indicates that downregulating MNSFβ can inhibit the expression of TNFα, while downregulating RC3H1 can promote the expression of TNFα, and MNSFβ depends on RC3H1 to regulate the expression of TNFα.
[0023] Experimental conclusion: A new signaling pathway that regulates TNFα expression in macrophages, MNSFβ is involved in the expression regulation of TNFα, downregulation of MNSFβ can inhibit the expression of TNFα, and MNSFβ depends on RC3H1 to regulate the expression of TNFα.
[0024] Example 2: Identification of the binding region between MNSFβ protein and RC3H1 protein
[0025] (1) MNSFβ was constructed into the prokaryotic expression vector pGEX-6p-1 vector and transformed into the BL21 strain. After induction of expression, beads bound to the fusion protein GST-MNSFβ (GST-MNSFβ group) and beads bound to the GST protein (GST group) were purified.
[0026] (2) The 81-326aa fragment and the 174-410aa fragment of the RC3H1 protein were constructed into the prokaryotic expression vector pet28a plasmid, transformed into the BL21 strain, and induced to express the fragment protein of RC3H1 fused with His tag (His-tag-RC3H1-81-326aa and His-tag-RC3H1-174-410aa).
[0027] (3) GST-MNSFβ beads and GST beads were incubated with bacterial suspension expressing His-tag-RC3H1-81-326aa and His-tag-RC3H1-174-410aa, respectively, at 4°C overnight. The beads were collected, washed four times, added with 1× loading buffer, and boiled at 95°C for 10 min.
[0028] (4) Pull-down experiments were performed using beads fused with GST-MNSFβ protein and the 81-326aa and 174-410aa fragments of RC3H1. The samples were used for subsequent western blotting detection. The results are shown in Figure 4. Figure 2 shown.
[0029] Figure 2 In the figure, His-tag-RC3H1-81-326aa: fusion protein of RC3H1 protein 81-326aa fragment containing 6×His tag (without HEPN-C); His-tag-RC3H1-174-410aa: fusion protein of RC3H1 protein 174-410aa fragment containing 6×His tag (without HEPN-N); GST: GST-beads; GST-MNSFβ: GST-MNSFβ-beads; +: indicates that the fusion protein was added to the co-incubation system for pull down experiment.
[0030] Figure 2 The results showed that a band was produced in the group co-incubated with GST-MNSFβ and His-tag-RC3H1-81-326aa, while no band was produced in the group co-incubated with GST-MNSFβ and His-tag-RC3H1-174-410aaa, indicating that MNSFβ binds to the 81-326aa fragment of RC3H1.
[0031] Experimental conclusion: MNSFβ binds to the HEPN-N(81-326aa) domain of RC3H1.
[0032] Example 3: Study on the inhibition of MNSFβ-RC3H1 interaction by MNSFβ / RC3H1 interfering peptide (M-peptide)
[0033] The amino acid sequence of the HEPN-N (81-326aa) domain of the RC3H1 protein is shown in SEQ ID NO: 1. In this embodiment, according to the MNSFβ and RC3H1 binding sites, an interfering polypeptide capable of interfering with the MNSFβ-RC3H1 interaction (PPI) is designed, namely, the MNSFβ / RC3H1 interfering polypeptide (M-peptide), which is fused with the TAT sequence and commissioned to be synthesized by Qiangyao Biotechnology Co., Ltd., with a purity of >95%. The sequence of the interfering polypeptide (M-peptide) is shown in SEQ ID NO: 2, and the sequence of the control random polypeptide (S-peptide) is shown in SEQ ID NO: 3.
[0034] In this example, a pull-down experiment was performed. Based on the original detection of the purified protein GST-MNSFβ binding to RC3H11, reaction groups were set up to add equal amounts of interfering peptide M-peptide and control peptide S-peptide to explore the interference of peptides on protein interactions. The specific experimental steps are as follows.
[0035] Using the GST-MNSFβ beads (GST-MNSFβ group) and the GST protein-bound beads (GST group) obtained in Example 2, 1×10 7 THP1-Mφ cells were lysed and divided into four groups:
[0036] 1) Negative control group: lysate was co-incubated with GST beads;
[0037] 2) Blank group: lysate was co-incubated with GST-MNSFβ beads;
[0038] 3) M-peptide group: lysate + 5ug M-peptide and GST-MNSFβ beads were co-incubated;
[0039] 4) S-peptide group: Lysate + 5ug S-peptide were co-incubated with GST-MNSFβ beads.
[0040] After incubation at 4°C overnight, the beads were washed four times, collected, added with 1× loading buffer, and boiled at 95°C for 10 min. Pull-down experiments were performed using GST beads (GST, negative control) and GST-MNSFβ beads (GST-MNSFβ). The samples were used for subsequent western blotting detection. The results are shown in Figure 3The results were analyzed by grayscale analysis using Image Studio software, with GAPDH from the same experiment as the internal reference, and each group was repeated three times. The experimental data were analyzed using GraphPad Prism8 software, and statistical graphs were made using GraphPad Prism 8 software to show the relative binding strength of MNSFβ and RC3H1 in different incubation groups. The results are shown in Figure 4 shown. Figure 4 In the table, *: P < 0.05, ***: P < 0.001, P < 0.05 indicates statistically significant difference.
[0041] Figure 3 The results showed that the color of the band in the M-peptide group was lighter than that in the blank group and the S-peptide group, indicating that M-peptide inhibited the binding of MNSFβ to RC3H1; Figure 4 The results showed that M-peptide could significantly inhibit the binding of MNSFβ to RC3H1 in macrophage lysate.
[0042] Experimental conclusion: M-peptide significantly inhibits the binding of MNSFβ to RC3H1.
[0043] Example 4: M-peptide inhibits the expression of inflammatory cytokine TNFα in macrophages
[0044] Human monocytic cell line THP1 was cultured at 5×10 5 Cells / well were seeded in a 12-well plate and cultured for 24 hours after adding 200ng / ml phorbol ester (PMA). Then, the cells were stimulated with DMSO (blank group), LPS (100ng / ml), LPS+1uM M-peptide, LPS+2uM M-peptide, LPS+4uM M-peptide, LPS+1uM S-peptide, LPS+2uM S-peptide, and LPS+4uM S-peptide for 2 hours. Each well of cells was washed twice with 500ul PBS, and 500ul Trizol was added to extract total RNA from the cells. Reverse transcription was performed using the reverse transcription kit of TAKARA, and the expression level of the inflammatory factor TNFα was detected by fluorescent quantitative PCR. The results are shown in Figure 2. Figure 5 As shown, GAPDH is an internal reference protein.
[0045] As above, human monocytic cell line THP1 was seeded in 6-well plates at 1.5×106 cells / well. After stimulation under the same grouping conditions, each well of cells was washed twice with 500ul PBS, and 200ul RIPA lysis buffer was added to extract the total protein in the cells. The expression of TNFα in the cells was detected by western blotting. The results are shown in Figure 6 shown. Figure 6 In the table, *: P < 0.05, ***: P < 0.001, P < 0.05 indicates statistically significant difference.
[0046] Figure 5 and Figure 6 The results showed that compared with the LPS group, the TNFα expression level in the M-peptide group with an addition amount of 2uM and 4uM was significantly reduced, and the band color was lighter, while there was no significant difference in the negative control S-peptide group. This shows that M-peptide can significantly inhibit the expression of TNFα in a dose-dependent manner, while the negative control peptide S-peptide has no effect.
[0047] Experimental conclusion: M-peptide significantly inhibits the expression of TNFα in macrophages.
[0048] Example 5: Detection of cytotoxicity of M-peptide by CCK-8 method
[0049] Human monocytic cell line THP1 was seeded in a 96-well plate at 1×104 cells / well, and 200 ng / ml phorbol ester (PMA) was added and cultured for 24 hours. Then, DMSO (blank group), M-peptide (10ug / ml, 20ug / ml, 40ug / ml) and S-peptide (10ug / ml, 20ug / ml, 40ug / ml) were used for treatment, and three replicate wells were added to each well. After further culture for 48 hours, 10ul CCK8 solution was added to each well. After culture for 30 minutes, the OD450 absorbance was detected by a microplate reader, and the cell viability of each group at four final drug concentrations (0, 10ug / ml, 20ug / ml, 40ug / ml) was calculated (cell viability = (OD450 value of treatment group / OD450 value of blank group) %). The results are shown in FIG. Figure 7 shown.
[0050] Figure 7 The results showed that there was no significant difference in cell viability between the M-peptide group and the S-peptide group at different concentrations, and there was no significant difference in cell viability between the two groups. This indicates that within the experimental concentration range of 0 to 40ug / ml, M-peptide and S-peptide will not affect macrophage viability, that is, there is no cytotoxicity within this range.
[0051] Experimental conclusion: M-peptide does not affect the proliferation activity of macrophages, that is, M-peptide has no cytotoxicity.
[0052] In summary, the present invention has discovered a new signaling molecule complex (MNSFβ-RC3H1) that regulates the expression of TNFα in macrophages, and the interfering polypeptide (M-peptide) of the present invention can significantly inhibit the expression of TNFα in macrophages without macrophage toxicity by interfering with the MNSFβ-RC3H1 interaction. Therefore, the signaling molecule complex (MNSFβ-RC3H1) discovered by the present invention provides a new target for the development of clinical anti-TNFα drugs; in addition, the interfering polypeptide (M-peptide) of the present invention can be used as a new drug molecule for anti-TNFα treatment in clinical drug development. The interfering polypeptide can be further prepared into a pharmaceutical composition of the interfering polypeptide using methods known in the art, which is obvious to those skilled in the art. It can be seen from this that the interfering polypeptide of the present invention has good development and application value in the development of clinical anti-TNFα drugs.
[0053] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.
[0054] Sequence Listing
[0055] Sequence Listing Information:
[0056] DTD version: V1_3
[0057] File name:231043sequence list.xml
[0058] Software Name:WIPO Sequence
[0059] Software version: 2.2.0
[0060] Date of creation: 2023-04-17
[0061] Basic Information:
[0062] Current application / applicant file name: 231043
[0063] Applicant's name: Shanghai Institute of Biomedical Technology
[0064] Applicant's name / language:zh
[0065] Applicant's name or title / Latin name:SIBPT
[0066] Invention Title: Anti-inflammatory therapeutic application of a MNSFβ / RC3H1 PPI interfering peptide (zh)
[0067] Total number of sequences: 3
[0068] sequence:
[0069] Serial number (ID): 1
[0070] Length:270
[0071] Molecular type:AA
[0072] Feature Position / Qualifier:
[0073] -source,1..270
[0074] >mol_type,protein
[0075] >organism,Peptide
[0076] Residue:
[0077]
[0078] Serial Number(ID):2
[0079] Length:45
[0080] Molecular type:AA
[0081] Feature Position / Qualifier:
[0082] -source,1..45
[0083] >mol_type,protein
[0084] >organism,Peptide
[0085] Residue:
[0086] PLSSARGVGL NSTTQSVLSR PMQRKLVTLV HCQLYGRKKR RQRRR 45Serial number (ID): 3
[0087] Length:45
[0088] Molecular type:AA
[0089] Feature Position / Qualifier:
[0090] -source,1..45
[0091] >mol_type,protein
[0092] >organism,Peptide
[0093] Residue:
[0094] TEMLDQTENR QLGSNMKSNT QLLELKEVQE QESLKLRKKR RQRRR 45END。
Claims
1. A MNSFβ / RC3H1 PPI interfering polypeptide for anti-TNFα treatment, characterized in that: The amino acid sequence of the interfering polypeptide is shown in SEQ ID NO:
2.
2. The use of the MNSFβ / RC3H1 PPI interfering polypeptide according to claim 1, characterized in that: Used for preparing medicines for anti-inflammatory treatment.