Use of peptides as inhibitors of the biological activity of histone acetyltransferase MOF

Abstract

The application belongs to the technical field of biological medicine, and provides application of a peptide in a histone acetyltransferase MOF biological activity inhibitor, and an amino acid sequence of the peptide is PYQRRGYGKF, as shown in SEQ ID No. 1. The application finds a biological activity domain of the histone acetyltransferase MOF, identifies an amino acid sequence capable of inhibiting the biological activity of the MOF, and proves that the sequence small molecule peptide can play a role in inhibiting the biological function and acetylation catalytic activity of the MOF, and can be applied as the MOF inhibitor to alleviate or treat diseases caused by the increase of the biological activity of the MOF.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a peptide in a histone acetyltransferase MOF biological activity inhibitor. BACKGROUND

[0002] Proteins are important components of various tissues, organs and cells of the human body, and are also the main body of cells to exert biological functions. Most protein molecules have active centers, and replacement of key amino acid residues in these sites will significantly change their biological activity and function, while replacement or deletion of non-key amino acid residues will not significantly change their activity and biological function. Therefore, discovery and identification of the biological activity domain or amino acid sequence of a protein are of great significance for targeted regulation of the biological function of the protein. In fact, functional proteins in the body are regulated by polypeptides thereon. In recent years, many biologically active polypeptides have been discovered and used for treating diseases, such as antibacterial effect of active peptides, blood pressure / lipid lowering, treatment of myocardial infarction, and the like. Some active peptides are also used for improving immunity, antibacterial ability, and improving food flavor, etc.

[0003] MOF (also known as MYST1, KAT8) is a member of the histone acetyltransferase MYST family, which is composed of 458 amino acids, and the GenBank number is NP_115564. MOF was first discovered in Drosophila as an important component of the dosage compensation system of the male X chromosome. Human MOF is the ortholog of Drosophila dMOF protein and is widely present and expressed in different tissues. According to the current research results, deletion of MOF in human cells can cause a wide range of reduction of H4K16 acetylation (H4K16ac), abnormal DNA damage repair response, genomic instability, chromosomal aberration, cell cycle disorder and nuclear morphology change, etc. In addition, the levels of MOF and H4K16ac in most clinical tumor tissues are lower than those in normal tissues, but are higher in lung cancer and glioblastoma. Recent studies have also found that the level of histone acetylation is increased in cells with myocardial hypoxia / ischemia injury, myotube atrophy and pulmonary fibrosis, indicating that the protein expression amount and enzyme activity of MOF are widely involved in various biological functions in cells. Therefore, it is particularly important to change the expression amount and biological function of MOF in cells by biological small molecule peptides, and then to improve diseases caused by the expression disorder of MOF. At present, the application research of biologically active small peptides in different fields is gradually increasing, and most of the biologically active small peptides are extracted from plants or total proteins, and lack of action on specific targets. Therefore, designing and identifying a small peptide and acting on a specific protein will be more helpful for targeted therapy. SUMMARY

[0004] The purpose of this invention is to provide the application of peptides as inhibitors of histone acetyltransferase MOF bioactivity, aiming to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The application of peptides as inhibitors of histone acetyltransferase MOF bioactivity, wherein the amino acid sequence of the peptide is: PYQRRGYGKF, as shown in SEQ ID No. 1.

[0007] Compared with the prior art, the beneficial effects of the present invention are:

[0008] This invention has discovered the bioactive domain of histone acetyltransferase MOF, identified an amino acid sequence (PYQRRGYGKF) that can inhibit MOF bioactivity, and confirmed that the small peptide of this sequence can play a role in inhibiting MOF biological function and acetylation catalytic activity, and can be used as a MOF inhibitor to alleviate or treat diseases caused by elevated MOF bioactivity. Attached Figure Description

[0009] Figure 1 The predicted structure of the MOF protein is shown using Surface (top left), Dots (bottom left), and Sticks (top right), respectively. In the figure, the left wall of the amino acid composition pocket (MOF-pocket) entrance is marked in yellow, the right wall of the amino acid composition pocket entrance is marked in green, and the bottom of the amino acid composition pocket is marked in red.

[0010] Figure 2 This diagram illustrates the combination of Rg3 and MOF-pocket. In the diagram, yellow represents Arg-325 (R325) (Å=2.1); red represents Gly-327 (G327) (Å=2.8); blue represents Lys-432 (K432) (Å=1.8, 2.0); and orange represents Lys-433 (K433) (Å=2.2).

[0011] Figure 3 In the figure, A represents the MOF protein level of cells treated with Rg3 for 1 hour; B is the protein quantification diagram of Figure A; C represents the MOF protein level of cells treated with Rg3 for 48 hours; and D is the protein quantification diagram of Figure C.

[0012] Figure 4In the diagram, A is a schematic diagram of the preparation of Rg3-PEGA resin and Rh2-PEGA resin; B is a diagram of the detection of MOF and Annexin A2 protein levels in Input, pulldown and flow throw using pulldown combined with Western blotting; C is a diagram of the detection of MOF protein levels after Rg3-pulldown under gradient increase of free Rg3 using pulldown combined with Western blotting.

[0013] Figure 5 In the diagram, A represents the presence of four hydrogen bond binding sites between wild-type MOF and Rg3; B represents the hydrogen bond binding result between Rg3 and MOF due to mutations in R325A, R326A, and R325A / R326A; C represents the hydrogen bond binding result between Rg3 and MOF due to the G327E mutation; D represents the hydrogen bond binding result between Rg3 and MOF due to the T319A mutation; and E represents the hydrogen bond binding result between Rg3 and MOF due to the L359A mutation.

[0014] Figure 6 In Figure A, MOF protein levels and histone H4K16 acetylation levels were detected; B shows the quantification results of MOF protein and H4K16ac levels in Figure A.

[0015] Figure 7 In the diagram, A represents the position of the pocket in the MOF structure and the designed pocket-independent sequence (brown); B represents the designed and synthesized small molecule peptides; C represents the UV spectrophotometers of Rg3 after binding with different concentrations of small molecule peptides; D represents the cell viability results detected by the CCK-8 assay kit; and E represents the effect of the synthesized small molecule peptides on Rg3's inhibition of gastric cancer colony formation. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0018] An embodiment of the present invention provides the application of a small molecule peptide as a bioactive inhibitor of histone acetyltransferase MOF, wherein the amino acid sequence of the small molecule peptide is: PYQRRGYGKF, as shown in SEQ ID No. 1.

[0019] Example 1: Using the "AlloFinder" software platform, the structurally variable region consisting of 17 amino acids in the histone acetyltransferase MOF protein structure was analyzed and identified. This structurally variable region resembles a pocket (MOF-pocket).

[0020] The design and analysis process is as follows:

[0021] This invention utilizes the AlloFinder interactive platform (used to study potential endogenous or exogenous protein allosteric regulators) to input the MOF amino acid sequence (177-447) into the software system and predict possible allosteric domains or structurally variable regions in the MOF structure based on the system's default parameters. The software prediction yields 25 possible candidate amino acid sites, including W(191), F(270), L(271), I(317), L(318), T(319), L(320), Y(323), Q(324), R(325), R(326), G(327), Y(328), G(329), K(330), L(332), I(333), S354, L(356), G(357), L(359), S(360), Y(361), S(363), and Q(430). The structure of the MOF was then predicted using the Alphafold2 molecular docking interaction platform and visualized using Pymol. The structural features that the aforementioned amino acid sites might form were analyzed, and finally, the minimum number of amino acid sites required to compose the pocket was determined. Figure 1 As shown, this includes Q(324), R(325), R(326), G(327), Y(328), G(329) on the left side of the entrance, K(330), L(356), G(357), L(359), S(360), Y(361), S(363) on the right side of the entrance, and amino acid sites I(317), L(318), T(319), L(320) at the bottom.

[0022] Example 2: Molecular docking analysis confirmed the binding of rare ginsenoside Rg3 to MOF-pocket;

[0023] The design and analysis process is as follows:

[0024] The MOF structure predicted using the Alphafold2 platform in Example 1 was subjected to molecular docking analysis with rare ginsenoside 20(S)-Ginsenoside Rg3 using Autodock Vina software. This analysis confirmed that Rg3 has four hydrogen bond binding sites with the MOF-pocket. Figure 2As shown, the MOF-pockets include arginine R325 (Å=2.1); glycine G327 (Å=2.8); lysine K432 (Å=1.8, 2.0) and lysine K433 (Å=2.2). This indicates that the MOF-pockets obtained from the analysis may be important structural domains for MOFs to perform their biological functions.

[0025] Example 3: In BGC823 cells, the cellular thermal shift assay (CETSA) combined with Western blotting was used to confirm that Rg3 binds to MOF and stabilizes the protein. CETSA has been widely used in drug screening to detect the interaction between small molecule drugs and proteins.

[0026] The experimental design and procedure are as follows:

[0027] (1) Place 1.5×10⁻⁶ mol / L of the mixture into a 6cm petri dish. 6 BGC823 gastric cancer cells were treated with Rg3 for 1 hour after 24 hours, and the cells were collected. After washing with PBS (phosphate-buffered saline), a complete protease inhibitor was added to PBS (to prevent protein degradation in subsequent operations). This PBS (300 μl) was added to 6 cm culture dishes, and the cells were collected. The harvested cell mixture was divided into 7 aliquots and heated separately at 37, 42, 45, 48, 51, 54, and 57 °C for 3 min, then cooled to room temperature for 3 min. Three freeze-thaw cycles were then performed using liquid nitrogen and a 25 °C water bath. The cells were centrifuged at 12500 rpm for 15 min at 4 °C to separate the protein supernatant from cell debris, and the protein supernatant was collected quantitatively. The supernatant was transferred to new EP tubes and finally analyzed by denaturation, SDS-PAGE protein separation, and Western blotting. Figure 3 (A). GAPDH is the internal reference protein. The quantitative graphs of MOF protein levels in the treated and untreated Rg3 groups are shown below. Figure 3 As shown in B.

[0028] (2) The same experimental method as in step (1) was used, except that the treatment time for Rg3 was 48 hours. For example... Figure 3 The C-cell diagram shows the MOF protein level (GAPDH is the internal reference protein), such as... Figure 3 The middle D plot shows a quantitative map of MOF protein levels.

[0029] Depend on Figure 3 It can be seen that Rg3 treatment of cells for 1 hour ( Figure 3 (A) or treated cells for 48 hours ( Figure 3 Both C and α can bind to MOF and stabilize MOF protein, and the MOF protein level is significantly increased compared with the DMSO (without Rg3) group. Figure 3(B and D). This confirmed that ginsenoside Rg3 binds to MOF and stabilizes the MOF protein, verifying the results of molecular docking.

[0030] Example 4: Using the Rg3-beads pulldown experiment and the competition experiment of Free Rg3 in BGC823 gastric cancer cells, it was confirmed that Rg3 has specificity in binding to MOF protein.

[0031] The experimental design and procedure are as follows:

[0032] (1) Synthesis of (20S)-Rh2-PEGA resin (Rh2-beads) and (20S)-Rg3-PEGA resin (Rg3-beads): Dry PEGA resin (containing approximately 0.4 mmol / g of free amino groups, purchased from Novabiochem) was placed in a small ground-glass stoppered flask, and 10 mL of pyridine was added to swell the resin. The mixture was stirred at room temperature for 1 h. An equimolar amount of 4-nitrophenol ethyl bromide was added, and the mixture was stirred at room temperature for another 3 h. The resulting resin was obtained by centrifugation and then washed with approximately 10 mL of dichloromethane, methanol, and DMF, respectively. 15 mL of DMF, an equimolar amount of K2CO3, and an equimolar amount of ginsenoside (20S)-Rh2 or (20S)G-Rg3 were added to the activated PEGA resin, and the mixture was stirred at room temperature for 3 days. The resulting resin was obtained by centrifugation and then washed with approximately 10 mL of dichloromethane and methanol, respectively. The synthesized resin is stored in methanol to prevent bacterial growth. The resin is washed with the appropriate buffer solution before use.

[0033] (2) BGC823 gastric cancer cells cultured in a 10cm culture dish (1×10⁻⁶) 7 Whole-cell proteins were extracted from the cells and divided into a negative control group, an Rg3 pulldown group, and an Rh2 positive control group. Then, the Rg3-beads and Rh2-beads prepared in step (1) were bound in a cold room for 4 hours. The levels of MOF and Annexin A2 proteins in the Input, Pulldown, and Flowthrow samples were detected by pulldown-based immunoblotting assay. Figure 4 (B) Annexin A2 is known to bind to Rh2 and served as a positive control for successful resin preparation. The results showed that Rg3 can bind to endogenous MOF proteins in cells, but not to Annexin A2 protein; conversely, Rh2 only binds to Annexin A2 and not to MOF, indicating that the binding of Rg3 to MOF is specific.

[0034] (3) Similarly, BGC823 gastric cancer cells (1×10⁻⁶) cultured in 10 cm culture dishes 7Whole-cell proteins were extracted from the cells and randomly divided into five groups: negative control (Lane 1), Rg3 pulldown group (Lane 2), low-dose Rg3-pulldown + Free Rg3 (50 μg / mL) group (Lane 3), medium-dose Rg3-pulldown + Free Rg3 (100 μg / mL) group (Lane 4), and high-dose Rg3-pulldown + Free Rg3 (200 μg / mL) group (Lane 5). After 4 hours of reaction, MOF protein levels in the Input and Rg3-pulldown samples were detected using a pulldown-based immunoblotting assay. GAPDH was used as an internal control protein.

[0035] The results showed that the number of MOFs bound to Rg3-PEGA resin gradually decreased after the gradient increase of Free Rg3, further demonstrating that the binding of Rg3 and MOF is specific (Figure C).

[0036] Example 5: Analysis using Autodock Vina and Pymol visualization software clarified that mutations in amino acids in the MOF-pocket alter the MOF structure, leading to Rg3 no longer binding to the MOF-pocket.

[0037] The design and analysis process is as follows:

[0038] To further verify the binding of Rg3 to the MOF-pocket, point mutations were performed on several amino acid sites that make up the pocket, including R325A, R326A, G327E, T319A, and L359A. The structure was then re-predicted using Alphafold2, followed by molecular docking analysis using Autodock Vina software. The changes in the Rg3-MOF binding position were then visualized using Pymol. There are four hydrogen bond binding sites between MOFwt and Rg3 (…). Figure 5 (A), compared with MOF wt, point mutations were performed on the above amino acid sites ( Figure 5In Figure B (right side), the brown-yellow Dots amino acids, and in Figure CE (bottom), the brown-yellow Dots amino acids represent mutated amino acids. The binding position of Rg3 with MOF has changed significantly. The R325A mutation causes Rg3 to bind to hydrogen bonds at K304 (Å=2.4), K306 (Å=2.6), S308 (Å=2.3), P309 (Å=2.7), T345 (Å=2.6), and V346 (Å=2.7) in MOF; the R326A mutation causes Rg3 to bind to hydrogen bonds at E338 (Å=2.3, 2.8), Q439 (Å=2.3), and W440 (Å=2.1) in MOF; the R325A / R326A mutation causes Rg3 to bind to hydrogen bonds at Q439 (Å=1.9, 2.1) and W440 (Å=2.2) in MOF. Figure 5 (B); The G327E mutation can lead to hydrogen bond binding between Rg3 and MOF at E305 (Å=2.7), K306 (Å=2.2), N312 (Å=2.2), and V346 (Å=2.2). Figure 5 (C); The T319A mutation can lead to hydrogen bond binding between Rg3 and MOF at R377 (Å=2.5), G378 (Å=2.1), L380 (Å=3.2), I415 (Å=3.0), and V417 (Å=3.2). Figure 5 (D); The L359A mutation can cause Rg3 to bind to the K432 (Å=2.5) hydrogen bond of MOF ( Figure 5 (E). It was clarified that mutations in amino acids in the MOF-pocket alter the MOF structure, thereby preventing Rg3 from binding to the MOF-pocket.

[0039] Example 6: Mutating the amino acid sites in the MOF-pocket can inhibit the activity of MOF catalyzing histone acetylation at the H4K16 site, indicating that the identified MOF-pocket is crucial for the biological function and catalytic activity of MOF.

[0040] The experimental design and procedure are as follows:

[0041] (1) In order to further clarify the importance of MOF-pocket in exerting its biological function, R325A and R325A / R326A double mutant plasmids were constructed.

[0042] (2) HEK293T cells (an immortalized human kidney embryonic epithelial cell line transfected with adenovirus E1A gene and simultaneously expressing SV40 large T antigen) were seeded in 2 mL of DMEM culture medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) in 6-well plates, with 7.5 × 10⁶ cells per well. 5Cells were collected, and after 24 hours, the cell coverage was approximately 40%. 1 μg of pcDNA3.1 (Vector group in the figure), pcDNA-Flag-MOFwt (Flag-MOFwt group in the figure), pcDNA-Flag-MOF R325A (MOFR325A in the figure), or Flag-MOF R325A / R326A (MOF R325A / R326A group in the figure) mutant plasmids were transfected. After 48 hours, samples were collected, and the activity of MOF in catalyzing histone H4K16 acetylation was assessed using Western blotting. Figure 6 (A, IB: H4K16ac). GAPDH is an internal reference protein.

[0043] (3) Figure 6 Quantification results of MOF protein and H4K16ac levels in A showed that Figure 6 The results showed that mutations at the important amino acid sites R325 and R326 in the MOF-pocket reduced the MOF's biological activity in catalyzing histone H4K16 acetylation.

[0044] Example 7: The synthesized small molecule peptide has the activity of inhibiting the biological function of MOF.

[0045] The experimental design and procedure are as follows:

[0046] (1) Based on the results of the above examples, in MOF-pocket sequences and sequences unrelated to MOF-pocket ( Figure 7 In section A), a small peptide modified with FITC and N-terminus green fluorescence was designed and synthesized: PYQRRGYGKF (322-331). Simultaneously, small peptides with mutant R325A (Mt1: PYQARGYGKF, as shown in SEQ ID No. 2), mutant G325E (Mt2: PYQRREYGKF, as shown in SEQ ID No. 3), and mutant R325A / G327E (Mt3: PYQAREYGKF, as shown in SEQ ID No. 4), as well as other peptides with irrelevant sequences, were synthesized. Figure 7 (B) The synthesized small molecule peptides were dissolved in purified water and mixed with Rg3 at different concentration gradients. The absorbance peaks of the small molecule peptides at different concentrations of Rg3 alone and Rg3+ were then detected using a Thermo NanoDrop 2000 ultra-micro spectrophotometer to compare the changes. Figure 7 (C). Detection revealed that the absorption peak of the mixture of small molecule peptide and Rg3 shifted to the left from 240 nm when Rg3 was used alone, indicating that the small molecule peptide and Rg3 are bound together. Therefore, it is speculated that the small molecule peptide has the activity of competitively inhibiting the biological function of MOF.

[0047] (2) CCK-8 assay: 2000 BGC823 gastric cancer cells were added to each of 96 wells and incubated in a CO2 incubator for 24 h. The experiment included a blank group (0 μM), small peptide wt (Peptide-wt), small peptide Mt1 (Peptide-Mt1), small peptide Mt2 (Peptide-Mt2), small peptide Mt3 (Peptide-Mt3), and an unrelated small peptide group. The small peptide concentrations were set at 0.25, 2.5, 5, 10, 20, 40, 80, and 160 μM, with three replicates per group. The cells were incubated in a CO2 incubator for 48 h. 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance at 450 nm was then measured using a multi-mode microplate reader. The values ​​were recorded and processed using Excel and Graphpad Prism for analysis. Figure 7 (D). The results showed that the small molecule peptide (peptide-wt) inhibited the viability of BGC823 cells in the range of 40-160 μM, indicating that it has biological activity. However, other small molecule peptides (including peptide-Mt1, peptide-Mt2, peptide-Mt3, and other peptides) did not affect the viability of BGC823 cells in the same concentration range. In other words, other small molecule peptides may not exert biological activity in cells.

[0048] (3) Cell colony formation assay: BGC823 gastric cancer cells in logarithmic growth phase were trypsinized and resuspended in 1640 medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) to form a cell suspension, and the cells were counted. 2000 cells / well were seeded into each experimental group in a 6-well plate and incubated in a CO2 incubator for 24 h. The experiment included a DMSO control group, an Rg3 group (100 μg / ml), an Rg3 (100 μg / ml) + Peptide-wt (10 μM) group, and an Rg3 (100 μg / ml) + Peptide-mt (10 μM) group. After drug administration, the cells were incubated in a CO2 incubator. Cells were cultured until the cell count in most individual clones exceeded 50 cells / well after 14 days. The culture medium was changed every 3 days, and the cell status was observed. After cloning, the cells were photographed under a microscope, then washed once with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation for 30-60 min, followed by another wash with PBS. 1 mL of crystal violet staining solution was added to each well for staining for 10-20 min. The cells were washed several times with PBS, air-dried, and photographed with a digital camera (photographing the entire six-well plate and each well individually). The results are shown below. Figure 7As shown in Figure E. The results showed that Rg3 significantly inhibited the proliferation of gastric cancer cells. However, after Rg3 was co-treated with small molecule peptides, the original anti-gastric cancer effect of Rg3 was inhibited, indicating that the small molecule peptides competitively bind to Rg3, thereby inhibiting the anti-gastric cancer effect of Rg3 through binding to MOF.

[0049] The preparation instructions for the drugs and solutions used in Examples 1-7 are as follows:

[0050] 1. Preparation of synthetic small peptides: Wild-type (wt) and point-mutant peptides were synthesized at Jier Biochemical (Shanghai) Co., Ltd. Each tube of lyophilized peptide powder was centrifuged at 3000 rpm for 5 min to obtain a sample from the bottom of the tube. Sterile ultrapure water was added to the clean bench in the cell culture room to prepare a 1 mM stock solution, as shown in Table 1.

[0051] Table 1. Preparation of synthetic small peptides

[0052]

[0053] 2. Preparation of cell culture medium: Add 50 mL of fetal bovine serum and 5 mL of penicillin / streptomycin dual antibiotic mixture (penicillin, 10 U / mL; streptomycin, 10 μg / mL) to 500 mL of DMEM (purchased from MeilunBio) cell culture medium or 1640 medium. Both fetal bovine serum and the penicillin / streptomycin dual antibiotic mixture were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0054] 3. Preparation of Phosphate Buffered Saline (PBS): Dissolve 8g sodium chloride, 0.2g potassium chloride, 3.581g disodium hydrogen phosphate dodecahydrate, and 0.24g potassium dihydrogen phosphate in ultrapure water and bring the volume to 1L. All the above reagents were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0055] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The application of peptides in the preparation of drugs for treating gastric cancer, characterized in that, The amino acid sequence of the peptide is: PYQRRGYGKF, as shown in SEQ ID No. 1; and the peptide is an inhibitor of histone acetyltransferase MOF bioactivity.

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