CN111117990B - Polypeptide for inhibiting MMP9 activity and application thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide specifically combined with human mantle cell lymphoma cells and application thereof. The polypeptide is the amino acid sequence of any one of the following (1): TFKEPVPDLC (M3); (2) Polypeptide derivatives which have deletion, insertion or substitution of one or more amino acids in the polypeptide molecules described in (1) and have the same biological functions as the polypeptide molecules described in (1). The polypeptide of the invention can inhibit the activity of MMP9 protein, and can be specifically combined with mantle cell lymphoma cells to inhibit the proliferation, invasion and metastasis of the mantle cell lymphoma cells. The polypeptide has obvious effect and provides reliable scientific basis for clinical early diagnosis and the research and development of targeted drugs.

Description

Polypeptide for inhibiting MMP9 activity and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to a polypeptide for inhibiting activity of matrix metalloproteinase 9 (MMP 9), and particularly relates to a polypeptide which has specific binding to MMP9 and has the function of inhibiting the activity of MMP9 and application thereof.

Background

Cancer is currently the leading cause of death worldwide and therefore the search for new formulations with potential anti-cancer properties is of great interest. Matrix Metalloproteinases (MMPs) are closely involved in processes such as apoptosis, cell migration, angiogenesis, etc., and play a crucial role in the remodeling of extracellular matrix (ECM) and the progression of cancer. Meanwhile, the expression and activity of MMPs in tissues and blood of different tumor patients are increased, and the MMPs can be used as a marker for predicting invasiveness and indicate the risk of distant metastasis. Therefore, matrix metalloproteinase inhibitors (MMPIs) may be targets of anticancer drugs, and the development of MMPIs creates more possibilities for treating cancer patients.

Since the 90 s of the 20 th century, a great deal of capital was invested in the development of the novel antitumor drug MMPIs by various pharmaceutical companies in the world. Currently, over a dozen MMPIs have entered clinical trials and there are a number of compounds under preclinical investigation. These chemically synthesized MMPIs can be broadly classified into peptide MMPIs, non-peptide MMPIs, tetracyclines MMPIs, bisphosphonates MMPIs, and the like. Broad-spectrum inhibitors of MMPs, such as batimastat, show very superior anti-tumor effects in preclinical animal models. However, these broad-spectrum inhibitors of MMPs are often associated with intolerable side effects in clinical trials, limiting their use. Relatively specific MMPs inhibitors are developed in the later period, and experiments prove that the MMPs inhibitor has anti-tumor and anti-angiogenesis effects. However, the results of clinical trials with these inhibitors are disappointing. The role of MMPs in the tumor development stage is determined, and the specificity of MMPIs is ensured to be the basis for better application of the MMPIs. MMP9 is specifically and highly expressed in tumor tissues, is related to each stage of tumor growth and metastasis, is related to poor prognosis, and is a better drug design target. Due to the similarity between different MMPs, achieving specificity of MMP9 inhibitors to avoid off-target effects on other MMPs is a powerful challenge to the development of MMP9 inhibitors. To date, no effective specific MMP9 inhibitor has been clinically applied. There is a strong need for a safe and effective MMP9 inhibitor.

The peptide drug has the advantages of low adverse reaction, low immunogenicity and easy synthesis and transformation, and the peptide inhibitor targeting MMP9 is designed based on the analysis of the crystal structure of MMP9 in the research. And (3) screening stable inhibitory peptides by using a molecular docking module in MOE software.

Disclosure of Invention

The invention aims to provide a novel polypeptide for inhibiting MMP9 activity and application thereof, wherein the polypeptide can be specifically combined with MMP9 and inhibit the activity thereof, can be specifically combined with tumor cells in mantle cell lymphoma cells with high expression of MMP9, and can inhibit the proliferation and invasion and metastasis capacities of the tumor cells, so that the polypeptide has important effects on the aspects of early diagnosis of the mantle cell lymphoma, research and development of targeted drugs and the like.

MMP9 generally exists in a zymogen form, and its propeptide part is inserted into the active site gap to block the binding of zinc ion of the catalytic active center to the substrate, and when activated, the propeptide is detached to expose the active site, activating MMP9 function. The invention uses the characteristic of propeptide, selects the amino acid sequence TPRCGVPDL containing the key amino acid 97-100 PRCG in propeptide sequence as the template peptide for inhibiting the activation of MMP9. Four key site amino acids in the template peptide are replaced according to the physicochemical properties of the amino acids, and amino acids with different sequences are designed to be candidate peptides. And (3) introducing the candidate peptides and the MMP9 structure into MOE software, docking the MMP9 with each candidate peptide by using a molecular docking module, and analyzing and screening the targeted inhibitory peptides according to the S score and the interaction. The polypeptide can further inhibit the cell proliferation and invasion and metastasis of mantle cell lymphoma by inhibiting the activity of MMP9, and is expected to become a novel medicine for treating the mantle cell lymphoma.

In order to achieve the above object, the present invention adopts the following technical solutions.

A polypeptide that inhibits MMP9 activity, the polypeptide being any of: the amino acid sequence of the polypeptide is as follows: TFKEPVPDLC, and (2) polypeptide derivatives which have deletion, insertion or substitution of one or several amino acids in the polypeptide molecule of (1) and have the same biological function as the polypeptide molecule of (1).

Further, the polypeptide has targeting inhibition on MMP9, and can be specifically combined with tumor cells and inhibit the activity of the tumor cells.

Further, the tumor cell is a mantle cell lymphoma cell.

An application of polypeptide for inhibiting MMP9 activity in preparing tumor diagnosis kit.

Further, the kit comprises the polypeptide or polypeptide conjugate.

Use of a polypeptide that inhibits MMP9 activity in the manufacture of a medicament for the treatment of mantle cell lymphoma, the medicament comprising said polypeptide and a pharmaceutically active ingredient, or comprising said polypeptide and a delivery vehicle.

Further, the medicament is in any pharmaceutically therapeutically acceptable dosage form.

Further, the preferred dosage form of the medicament is an injection preparation.

Further, the medicament is in any pharmaceutically therapeutically acceptable dose.

Compared with the prior art, the invention has the following beneficial effects.

(1) The invention uses MOE software molecule docking technology, has the advantages of simple operation, high flux panning, high efficiency, capability of simulating the combination of polypeptide and target protein, screening of optimal combination conformation, low cost and the like. The search for new compounds with specific biological activities requires enormous effort and costs. This effort stems from the large number of compounds that need to be synthesized and subsequently bioassayed. Therefore, pharmaceutical companies show great interest in theoretical approaches that enable rational design of drugs. In recent years, bioinformatics has undergone tremendous evolution with the development of professional software and the increase in computer power. MOE software molecular docking module searches for favorable junction between ligand and macromolecular target

Synthetic structures have been used in the design and virtual screening of new drugs.

(2) The invention can specifically combine the polypeptide screened by applying MOE molecular docking technology with the activated MMP9 protein and inhibit the activity thereof by researching pro-MMP9 activation mode and propeptide function, can inhibit the activity of tumor cells, has obvious effect and provides reliable scientific basis for clinical diagnosis, treatment and new drug research and development.

Drawings

FIG. 1 is a band diagram of the pro-MMP 9D crystal structure, in which the catalytic domain is shown in pink, the three FnII domains are shown in red, yellow and blue, respectively, the calcium of the active center is shown as a red sphere and the zinc is shown as an orange sphere, and the amino acid residues of the hydrophobic regions S1, S2, S1 ', S2' are shown as red-white globules.

FIG. 2 is an amino acid substitution scheme for designing a candidate peptide, wherein A is a non-polar aromatic amino acid (F/Y/W) substituted for the first amino acid P; b is a nonpolar aromatic amino acid (F/Y/W) substituted for the first amino acid G.

FIG. 3 is a receptor-ligand amino acid residue interaction, M0 polypeptide.

FIG. 4 is a receptor-ligand amino acid residue interaction, M1 polypeptide.

Figure 5 is a receptor-ligand amino acid residue interaction, M2 polypeptide.

FIG. 6 is a receptor-ligand amino acid residue interaction, M3 polypeptide.

FIG. 7 is a 3D structure of the interaction of a polypeptide with the MMP9 active site, where A-D correspond to M0-M3, respectively, and the designed inhibitory peptides are shown as green backbone peptide fragments.

FIG. 8 is the M0 polypeptide structure.

FIG. 9 is the M1 polypeptide structure.

FIG. 10 is the M2 polypeptide structure.

FIG. 11 is the M3 polypeptide structural formula.

Fig. 12 is a prediction of M0 polypeptide properties, M0 sequence: TPRCGVPDLC, chemical Formula: C43H73N13O14S2, extinction coefficient: 0M-1 cm-1, GRAVY:0.07, average molecular weight: 1060.24g/mol, theoretical isoelectric point: the pH was 6.13. GRAVY = arithmetic mean of hydrophobicity, negative numbers representing hydrophilicity, smaller being more hydrophilic.

Fig. 13 is a prediction of M1 polypeptide properties, M1 sequence: TFKCGVPDLC, chemical Formula: C47H75N11O14S2, extinction coefficient: 0M-1 cm-1, GRAVY:0.57, average molecular weight: 1082.29g/mol, theoretical isoelectric point: the pH was 6.13. GRAVY = arithmetic mean of hydrophobicity, negative numbers representing hydrophilicity, smaller being more hydrophilic.

Figure 14 is a prediction of M2 polypeptide properties, M2 sequence: TFKEGVPDLC, chemical Formula: C49H77N11O16S, extinction coefficient: 0M-1 cm-1, GRAVY: -0.03, average molecular weight: 1108.26g/mol, theoretical isoelectric point: the pH was 4.07.GRAVY = arithmetic mean of hydrophobicity, negative numbers representing hydrophilicity, smaller being more hydrophilic.

FIG. 15 is a M3 polypeptide property prediction, TFKEPVPDLC, chemical Formula: C52H81N11O16S, extinction coefficient: 0M-1 cm-1, GRAVY: -0.15, average molecular weight: 1148.32g/mol, theoretical isoelectric point: the pH was 4.07.GRAVY = arithmetic mean of hydrophobicity, negative numbers representing hydrophilicity, smaller being more hydrophilic.

FIG. 16 shows the binding specificity of M0 peptide to mantle cell lymphoma detected by flow cytometry, and the cells were classified into Jeko-1 group, 5. Mu.M peptide, 50. Mu.M peptide, 100. Mu.M peptide, 150. Mu.M peptide and MMP9 antibody group, and the binding rate was gradually increased with increasing peptide concentration, but was lower than that of MMP9 antibody group. Concentration unit: mu M; c: jeko-1 group; ab: MMP9 antibody panel.

FIG. 17 shows flow cytometry for binding specificity of M1 peptide to mantle cell lymphoma cells divided into Jeko-1, 5. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M and MMP9 antibody groups, with increasing peptide concentration, increasing binding rates but lower than those of the MMP9 antibody group. Concentration unit: mu M; c: jeko-1 group; ab: MMP9 antibody panel.

FIG. 18 shows the flow cytometry analysis of the binding specificity of M2 peptide to mantle cell lymphoma, which divided the cells into Jeko-1 group, 5. Mu.M peptide, 50. Mu.M peptide, 100. Mu.M peptide, 150. Mu.M peptide, and MMP9 antibody group, and the binding rate was gradually increased with increasing peptide concentration, but all were lower than that of MMP9 antibody group. Concentration unit: mu M; c: jeko-1 group; ab: MMP9 antibody panel.

FIG. 19 shows flow cytometry for detecting the binding specificity of M3 peptide to mantle cell lymphoma, where the cells were divided into Jeko-1 group, 5. Mu.M peptide, 50. Mu.M peptide, 100. Mu.M peptide, 150. Mu.M peptide, and MMP9 antibody group, and the binding rate was gradually increased with increasing peptide concentration but was lower than that of MMP9 antibody group, and the M3 binding rate was decreased when nonfluorescent MMP9 antibody and 150. Mu. M M3 were added. Concentration unit: mu M; c: jeko-1 group; ab: MMP9 antibody panel.

FIG. 20 shows that the template peptide M0 inhibits MMP9 activity by gelatin zymography, and M0 can significantly inhibit MMP9 activity without affecting MMP9 content.

FIG. 21 shows that peptide M3 inhibits MMP9 activity by gelatinase assay, and M3 can significantly inhibit MMP9 activity without affecting MMP9 content.

FIG. 22 shows the MMP9 content in Jeko-1 cell culture medium after different concentrations of M3 were added by ELISA.

FIG. 23 shows that after 100. Mu. M M3 was added to Jeko-1 cells and cultured for 48h, immunofluorescence detects fluorescent peptide M3 (FITC) expression, and cell membranes were counterstained with DIL at magnification × 200.

FIG. 24 shows CCK-8 detects changes in cell proliferation after addition of 50. Mu.M, 100. Mu. M M3 and MMP9 antibody to Jeko-1 cells,. P <0.05,. P <0.01, compared to Jeko-1 cells.

FIG. 25 is an examination of the cell cycle of Jeko-1 cell groups separately by flow cytometry.

FIG. 26 shows the assay for loss cytometry at 50. Mu. M M3, respectively. The cell cycle of the group.

FIG. 27 shows that the 10. Mu.M 3 cell cycles were measured separately by flow cytometry.

FIG. 28 is an individual detection of MMP9 antibody group cell cycle by shed cytometry.

Figure 29 is Jeko-1 cells, 50 μ M M,/10 μ M3 and MMP9 antibody panel cell cycle quantification data, p <0.05, p <0.01, compared to Jeko-1 cells; # p <0.05, # p <0.01, compared to the MMP9 antibody group.

FIG. 30 is a Transwell assay to detect changes in cell invasiveness of Jeko-1 cells after addition of 50 μ M, 100 μ M M, and MMP9 antibody,. P <0.01, compared to Jeko-1 cells; # p <0.05, compared to the MMP9 antibody panel.

FIG. 31 is a graph showing the cell migration ability of Jeko-1 cells after addition of 50. Mu.M, 100. Mu. M M3 and MMP9 antibody <0.05,. P <0.01, compared to Jeko-1 cells, as measured by the Transwell assay; # p <0.05, # p <0.01, compared to the MMP9 antibody group. Data are presented as mean ± standard pyramidal error, with three replicates per treatment group.

Detailed Description

The present invention is further described in the following examples and the accompanying drawings, the following examples are only preferred embodiments of the present invention, and the present invention is not limited thereto, and various modifications and changes can be made to the present invention by those skilled in the art, and any modifications, equivalents, improvements and the like within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Examples are given.

1. Test materials.

1.1 cells.

Jeko-1 cells were purchased from Shanghai cell Bank, chinese academy of sciences.

1.2 And (4) experimental reagents.

MMP Zymography assay kit Primay Gene technology, inc.

MMP9 antibody, abcam, UK.

Human MMP9 ELISA kit, abcam, UK.

Propidium Iodide (PI) Sigma, USA.

RNase A Sigma, USA.

Paraformaldehyde, beijing Sorleibao technologies, inc.

DIL Invitrogen USA.

RPMI-1640 medium, gibco, USA.

Trypsin: gibco, USA.

Fetal Bovine Serum (FBS): hyclone, USA.

Transwell cell: corning, USA.

Matrigel gel: corning corporation, USA.

CCK-8 kit: bilun sky, inc.

1.3 And (4) experimental working solution.

Preparing a gelatin zymogram experimental reagent.

(1) Eluent (1 ×): measuring TritonX-100 ml, adding deionized water to the volume of 1000 ml.

(2) Incubation solution (1 ×): weighing 6.06 g of Tris alkali, 0.74 g of CaCl2.2H2O (or 0.555 g of CaCl 2), 0.2 g of Brij-35 and 11.7 g of NaCl. Water was added to 800 ml. The pH was adjusted to 7.6 with concentrated HCl and the volume was 1000 ml.

(3) 2 XLoading buffer (without beta mercaptoethanol).

0.5 M Tris-HCl，pH 6.8 ： 2.5 ml。

Glycerin: 2.0 ml.

10%（w/v）SDS ： 4.0 ml。

0.1% bromophenol blue: 0.5 ml.

dH2O： 1 ml。

Total： 10 ml。

(4) 5 × loading buffer (without β mercaptoethanol): the following reagents were weighed and placed in 15ml plastic centrifuge tubes.

1M Tris-HCl（pH6.8）： 2.5 ml。

SDS： 1 g。

Bromophenol blue: 0.05 g.

Glycerol: 5 ml.

Adding dH2O to a constant volume of 10 ml, oscillating and mixing evenly, subpackaging in small parts (1 ml/tube), and storing at the temperature of 20 ℃.

(5) 0.5% (w/v) Coomassie Brilliant blue R-250 ml: weighing 2g Coomassie brilliant blue R-250, placing in a 1L beaker, measuring 100 ml isopropanol, adding into the beaker, stirring for dissolving, adding 40 ml glacial acetic acid, stirring uniformly, adding 260 ml deionized water, stirring for dissolving, filtering with filter paper to remove particulate matters, and storing at room temperature.

(6) Coomassie brilliant blue destaining solution (400 ml): methanol: acetic acid: water =200:40:160=5:1: 4.

(7) Gelatin stock solution (10 mg/ml): gelatin 0.1 g is weighed and dissolved in deionized water 10 ml. After compounding, the mixture was stored at 4 ℃. Thawing in water bath at 55 deg.C if the gel is coagulated.

(8) A10% SDS-PAGE gel (containing 1.0 mg/ml gelatin).

dH2O： 3ml。

30% acrylamide solution: 3.3 ml.

1.5 M Tris-HCl（pH 8.8）： 2.5 ml。

10% SDS ： 0.1 ml。

10% ammonium persulfate: 0.1 ml.

TEMED ： 0.004 ml。

10mg/ml gelatin: 1 ml.

Total ： 10 ml。

And (5) configuring the concentrated glue.

H2O： 4.1ml。

30% acrylamide solution: 1 ml.

1.0mol/L Tris-HCl： 0.75 ml。

10% SDS： 0.06 ml。

10% ammonium persulfate: 0.06 ml.

TEMED ： 0.006 ml。

2. Experimental methods.

2.1 Receptor optimization was performed in MOE (molecular operating environment) software.

The peptides were molecularly interfaced with activated MMP9 using Molecular Operating Environment (MOE) software. The 3D crystal structure of MMP9 was obtained from the Protein Database (PDB) (PDB ID:1L 6J). The structure was optimized by software addition of H atoms. Meanwhile, in order to optimize the structure, H ₂ The O molecules are removed from the structure and subsequently three-dimensionally protonated, bringing it to the level of ionization. In addition, specific parameters are required to minimize the energy of the receptor prior to molecular docking.

2.2 Selecting a template peptide and designing a candidate peptide.

MMP9 is normally present in a zymogen form (proMMP 9), and the proMMP9 structure, including the propeptide, catalytic domain, and triple fibronectin type II (FnII) F domain, is shown in figure 1. The 97-100 amino acid residues of the propeptide are inserted into the cleft of the active site of the catalytic domain, so that the combination of the catalytic center Zn < 2+ > and a substrate is blocked. proMMP9 can cleave the propeptide portion into active MMP9 by autoproteolytic degradation, stimulated in vivo or in vitro by organomercury compounds or other proteases. Cleavage of the propeptide opens the active site, facilitating the interaction of the catalytic domain with a substrate or inhibitor. Through the research on the propeptide, the amino acid sequence TPRCGVPDL is screened from the propeptide and is used as a template peptide segment, wherein the template peptide segment comprises key amino acid residues (PRCG) at positions 97-100. Optimizing key amino acid residues according to the physicochemical properties of the amino acid. There are two options for adding non-polar aromatic amino acids (F/Y/W) to key amino acids: either the first position amino acid P or the fourth position G. The second position residue of the key amino acid interacts similarly with the positively charged polar amino acid residue K, while the third position amino acid residue belongs to a polar amino acid, which can be selected for substitution (R/N/D/Q/E/H/C/S/T) (FIG. 2). Thirty or more candidate peptides were designed for subsequent docking with an activated MMP9 molecule. The designed peptide fragments were converted to the respective 3D structures using ChemSketch software.

2.3 Peptide optimization was performed in MOE.

All candidate peptides were optimized using MOE software. First, similar to the receptor optimization process, H atoms need to be added to the peptide fragments. Setting gradient:0.05, force Field: parameters such as MMFF94X, central Constraint and Current Geometry achieve energy minimization of the peptide. In addition, these peptides were subjected to conformational search using the LowModeMD method and the search results were saved in the mdb database for further docking analysis.

2.4 The molecular docking module in MOE performed peptide-to-receptor docking.

These peptides were molecularly interfaced with activated MMP9 using the algorithm of the MOE software. The parameters are set to Re-targeting function: london dG, displacement: triangle mather, retain: 5. refinement: force Field and Re-rating 2: london dG. The molecular docking procedure of MOE provides the correct conformation for the ligand through the rotation of chemical bonds and flexible molecular structure. The S-score is the basis for selecting the optimal peptide fragment and the optimal conformation. The optimal conformation further studies hydrogen bonds and pi-pi conjugation conditions through amino acid interactions.

2.5 The flow cytometry technology detects the binding specificity of the peptide and the tumor cells.

1X 106 Jeko-1 cells were collected, washed 2 times with PBS, and then added with fluorescent-labeled peptides M0 (50. Mu.M, 100. Mu.M, 150. Mu.M), M1 (50. Mu.M, 100. Mu.M, 150. Mu.M), M2 (50. Mu.M, 100. Mu.M, 150. Mu.M), M3 (50. Mu.M, 100. Mu.M, 150. Mu.M) and MMP9 antibody, respectively, and incubated at 37 ℃ for 1 hour. And (4) centrifuging the mixture at a low speed of 1000rpm for 8min by PBS, washing the mixture for 3 times, and then detecting the mixture on a machine.

2.6 Gelatin zymogram experiments.

The Jeko-1 cells in log phase were cultured in serum-free medium for 24h. The next day, the supernatant was collected, transferred to a centrifuge tube and centrifuged at 2000 rpm for 10min, and stored at-70 ℃ for further use. The protein concentration in the cell culture supernatants of each group was adjusted according to the cell count. Mix with 5 XLoading buffer, 13ul sample +4ul loading buffer. Separating gel and concentrating gel are prepared, and SDS-PAGE electrophoresis is carried out at 4 ℃ for 100V for about 1.5h under the conditions of 16 ul/Kong Shangyang. After electrophoresis, the gel is placed in an eluent to be shaken and eluted for 2 times, then the gel is rinsed for 2 times by using a rinsing solution, and then the gel is placed in an incubation solution to be incubated for 42 hours at 37 ℃. After incubation, the cells were stained with a staining solution for 3 hours, and decolorized with a decolorizing solution A, B, C (methanol concentration 30%, 20%, 10%, acetic acid concentration 10%, 5%) for 0.5, 1, and 2 hours, respectively, to show that MMP-9 (92 KD) is a clear band on a blue background.

2.7 MMP9 ELISA assay.

The cell supernatant sample is detected after being diluted by at least 100 times, and the diluted sample can be obtained by mixing 10 mul of sample and 90 mul of diluent by 100 times. The microplate was removed from the sealed bag equilibrated to room temperature, and 100 μ l of detection solution was added to each well. And respectively adding the standard substances with different concentrations and the experimental samples into corresponding holes, wherein each hole is 100 mu l. The reaction wells were sealed with a sealing plate of gummed paper, placed on a shaker (diameter 3 mm) and incubated at 500 rpm/min for 2h at room temperature. And (5) sucking the liquid in the plate and washing the plate. And adding 400 mul of washing liquid into each hole, and then absorbing the washing liquid in the plate. The operation was repeated 4 times. And (5) after the last washing of the plate is finished, sucking all liquid in the plate to be dry. 200 μ l of enzyme-labeled detection antibody was added to each microwell. Sealing the reaction hole with sealing plate gummed paper, placing on a shaking instrument (the diameter is 3 mm), incubating at room temperature for 1h at 500 rpm/min, adding 200 mul of chromogenic substrate into each micropore after washing the plate, and incubating at room temperature for 30min. 50 mul of stop solution 1 was added to each well, and the color of the solution in the wells changed from blue to yellow. Within 30min after the addition of the stop solution 1, the absorbance value of 450nm was measured using a microplate reader, and 540 nm or 570 nm was set as a calibration wavelength. And (3) calculating the result: the corrected absorbance (OD 450-OD540/OD 570) for each of the standards and samples was averaged and subtracted by the OD at the zero point of the standard curve. And (3) drawing a 4-parameter (4-PL) linear standard curve by using software carried by a microplate reader, wherein the abscissa of the curve is the concentration value of the human MMP-9 at the point of the standard curve, and the ordinate of the curve is the OD average value of the point of the standard curve. From the OD value of the sample, the concentration of human MMP-9 in the sample can be obtained from a standard curve.

2.8 Cell immunofluorescence assay.

Jeko-1 cells were centrifuged at 1000rpm at 4 ℃ for 5min, aspirated from the medium and resuspended in PBS. PBS was resuspended 3 times, then PBS was removed by centrifugation, cells were resuspended in 1ml of 4% paraformaldehyde, and fixed for 30min. And (4) centrifuging. The fixative was aspirated and the cells were resuspended 3 times in PBS. Add 1ml 100 μ M M and stand at 4 ℃ for 1h. PBS was washed 3 times. DIL 2. Mu.l was added for 30min and washed 2 times with PBS. The cells were centrifuged and resuspended in a small amount of PBS, the cell suspension was dropped onto a glass slide, a cover slip was added, and the fluorescence intensity was observed under a confocal laser microscope (Nikon, japan).

2.9 And (4) detecting the activity of CCK-8 cells.

Jeko-1 cells in logarithmic growth phase were seeded in 6-well plates, 1X 105 cells per well were inoculated, the cells were placed in a 5 CO2 incubator overnight at 37 ℃, and after 0.67. Mu.g of si-h-MMP9 was transfected by EntransTM-R for 24h, the cells were seeded in 96-well plates at a density of 4X 103 cells/well, 3 duplicate wells were set for each group of cells, and the cells were collected at 24h, 48h, and 72h, respectively. Each well was treated with 10. Mu.l of CCK-8 (Dojindo Molecular Technologies Inc., japan) for 1 hour. Absorbance at 450nm was measured using a multimode reader microplate reader (LD 942, beijing, china). The horizontal axis represents time, and the vertical axis represents absorbance.

2.10 Transwell experiment.

Transwell cells containing 8 μm microwells were placed in 24-well plates, and the upper chamber was plated with 1. 24h after cell transfection, 200. Mu.l of the cell suspension was added to the upper chamber, and the number of cells was 1X 104/well, and 800. Mu.l of a 30% FBS-containing culture medium was added to the lower chamber. The 24-well plate was incubated at 37 ℃ and 5% CO2 under saturated humidity conditions for 24 hours. The lower chamber cell suspension was collected and 10. Mu.l was added to a total volume of 100. Mu.l PBS (one drop of Trypan blue dye). From the diluted cell suspension, 10. Mu.l of the suspension was taken out and added to a counting plate, and counted under a microscope.

2.11 Flow cytometry detects cell cycle and apoptosis.

Cells were counted and collected by conventional methods (about 2X 106) and washed once with PBS (1000 rpm,5 min).

Pre-cooled 75% ethanol fixation was applied and incubated overnight at 4 ℃. 75% ethanol was discarded and washed once with PBS (1000 rpm,5 min). Resuspend the cells in 800ul 1 XPBS +1% BSA solution, add 100ul PI staining solution (3.8X 10-2 sodimucitrate, pH 7.0), add 100ul RNase (RNase A,10 mg/ml), incubate in the dark at 37 ℃ for 30min, and test on the machine. The HCV value of the signal received by each amplifier was <2% and was analyzed using cell Modifit software. Cell proliferation index PI = (S + G2/M)/(S + G2/M + G0/G1), and PI index represents cell proliferation level. The sub-G0 cell ratio represents the percentage of apoptosis.

3. And (5) experimental results.

3.1 And designing a template peptide fragment and a candidate peptide fragment.

MMP9 is commonly found in the zymogen form (proMMP 9), and the proMMP9 structure in the PDB database includes a propeptide, a catalytic domain, and a fibronectin type II (FnII) F domain, as shown in figure 1. According to the proMMP9 activation process, an amino acid sequence TPRCGVPDL is screened from the propeptide as a template peptide fragment (M0), which comprises 97-100 key amino acid residues (PRCG). Key amino acid residues are optimized according to the physicochemical properties of amino acids, a substitution scheme is shown in figure 2, and more than thirty candidate peptides are designed for subsequent docking screening.

3.2 The docking results for candidate peptide M3 were superior to template peptide M0.

In the MOE molecular docking program, each peptide fragment is set to store ten conformations, and the conformation with the minimum S value is selected as the optimal conformation. The optimal conformation of peptide stretch M3 was considered to be more stable in binding and secondly M1 and M2 according to the S score, see table 1. The optimal conformations of the three peptide fragments are further subjected to interaction analysis to find hydrogen bonds and pi-pi conjugated interactions. The results show that M1, M2 and M3 can be respectively matched with Zn of the active center ²⁺ Binding, affecting enzyme activity. In addition to having an optimal S value, M3 also interacts with the three amino acid residues (Pro 193, his405, and Leu 409) of the activation center of activated MMP9. Therefore, M3 can be a candidate drug for inhibiting activated MMP9. In addition, the S-score for M1 ranked second, and it potentially interacted with the active center amino acid His 411. Other peptidesThe segments (M0 and M2) have no potential interaction with the active site, but have a hydrophobic effect on the active residues of the catalytic domain. The interaction of amino acid residues with the active site is shown in table 1. The interaction between the receptor and the ligand is shown in FIGS. 3-6. The binding pattern of the ligand to the receptor protein is shown in FIG. 7. The solid phase synthesis method synthesizes four polypeptides of M0, M1, M2 and M3, and the prediction of structural formula and polypeptide property is shown in FIGS. 8-11 and 12-15.

3.3 The MMP9 targeted inhibitory peptide M3 can be compatible with mantle cell lymphoma cells and inhibit MMP9 activity.

3.3.1 Flow cytometry identified the binding specificity of the four fluorescent synthetic peptides to Jeko-1.

In order to identify the specificity of the binding of the M0, M1, M2 and M3 peptides to the Jeko-1 cell line and to find the preferential binding of each fluorescent peptide to Jeko-1, the binding of the fluorescent modified peptides to Jeko-1 cells was detected by flow cytometry in this section. By setting different incubation times, the optimal incubation time is selected to be 1h.

The results in FIGS. 16-19 show that the four fluorescently-modified polypeptides M0, M1, M2 and M3 all bind specifically to Jeko-1 cells, and the binding rate increases with increasing peptide concentration, but is lower than that of MMP9 antibody. When 150 μ M M3 was added after the addition of MMP9 non-fluorescent antibody, the M3 binding rate was significantly reduced, as shown in fig. 19.

3.3.2 The targeting inhibitory peptide M3 can specifically inhibit MMP9 activity.

As shown in fig. 20-21, MMP9 activity gradually decreased with increasing concentrations of peptides M0 and M3 (5 μ M, 50 μ M, 100 μ M, and 150 μ M), and M3 inhibited MMP9 activity more strongly than M0. However, both of these polypeptides inhibit less than MMP9 inhibitors. Whereas cellular MMP9 activity did not change significantly upon addition of peptides M1 and M2. The MMP9 expression level of each group of Jeko-1 cells after adding different concentrations of M3 is not changed obviously as shown in FIG. 22, which shows that the polypeptide M3 only inhibits the activity of MMP9 without reducing the expression level. After 100 mu M M and Jeko-1 cells were co-cultured for 48h, M3 was tested for affinity to cells using cellular immunofluorescence, confirming that M3 could indeed be associated with Jeko-1 cells, as shown in FIG. 23.

MMP2 also belongs to the MMPs family, and is structurally similar to MMP9. The results of the experiment suggest that MMP2 levels secreted into serum-free medium were not affected, as shown in FIGS. 20-21. The results show that M3 is specific for MMP9.

3.4 MMP9 targeted inhibitory peptide M3 can inhibit the proliferation, apoptosis and invasion and migration of mantle cell lymphoma cells.

To further verify the function of peptide M3 in the MCL cell line, jeko-1 cells were divided into MMP9 antibody group, 50 μ M M group, and 100 μ M M group for cell proliferation function assay. With the increase of M3 concentration in Jeko-1 cells, cell proliferation was significantly inhibited in a dose-dependent manner, with the extent of inhibition being most significant at 48h, as shown in FIG. 24.

Flow cytometry was used to detect apoptosis and cell cycle and assess the effect of M3 on MCL cell function. For detecting apoptosis, jeko-1 cells are respectively added with 50, 100 mu M M or MMP9 antibody to be cultured for 48 h. Flow cytometry analysis showed 12.42% and 20.31% of apoptotic cells in the 50 and 100 μ M M3 Jeko-1 cell groups, and 25.87% in the MMP9 antibody group. In the cell cycle analysis, the Jeko-1 cell proliferation index decreased from 0.51 to 0.48 or 0.46 as the M3 concentration increased from 0 to 50 or 100. Mu.M, as shown in FIGS. 25-29.

In the invasion experiment, jeko-1 cells were cultured in 1h by adding MMP9 antibody, 50 and 100. Mu. M M3, respectively, and then transferred to gelatin-coated cells previously placed in a 24-well plate for further 24 hours. The M3 groups (50 and 100 μ M) significantly inhibited the Jeko-1 cell invasion capacity (54.5% and 63.6%), but the inhibition effect was not as significant as the MMP9 antibody. In the migration experiment, the migration capacity of Jeko-1 cells in 50 and 100. Mu. M M3 groups was reduced by 27.3% and 36.4%. Again, this inhibitory effect was not as pronounced as the MMP9 antibody as shown in FIGS. 30-31.

SEQUENCE LISTING

<110> university of Chinese medical science

<120> polypeptide inhibiting MMP9 activity and application thereof

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Artificial sequence

<400> 1

Thr Phe Lys Glu Pro Val Pro Asp Leu Cys

1 5 10

Claims (8)

1. A polypeptide that inhibits MMP9 activity, the polypeptide having the amino acid sequence: TFKEPVPDLC.

2. Use of the polypeptide inhibiting MMP9 activity of claim 1 in the preparation of a kit for tumor diagnosis.

3. Use according to claim 2, wherein said polypeptide or polypeptide conjugate is contained in the kit.

4. Use of a polypeptide that inhibits MMP9 activity as defined in claim 1 in the manufacture of a medicament for the treatment of mantle cell lymphoma.

5. The use of claim 4, wherein the medicament comprises the polypeptide of claim 1 and a pharmaceutically active ingredient, or comprises the polypeptide of claim 1 and a delivery vehicle.

6. The use according to claim 4, wherein the medicament is in any pharmaceutically and therapeutically acceptable dosage form.

7. The use according to claim 4, wherein the medicament is in the form of an injectable formulation.

8. The use of claim 4, wherein the medicament is in any pharmacotherapeutically acceptable dose.

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