CN114591400B - Group of targeted FoxM1-DBD polypeptides and application thereof - Google Patents

Abstract

The invention discloses acquisition of a group of targeted FoxM1-DBD polypeptides and application thereof in tumor resistance and fibrosis resistance. The polypeptide sequence is obtained by biological panning of a phage heptapeptide library, 4 types of high affinity and specificity are determined by reverse screening, and polypeptide modification is carried out, so that the modified polypeptide can effectively inhibit and kill cancer cells, promote cancer cell apoptosis and inhibit cancer cell migration; meanwhile, the compound can inhibit the proliferation of fibrocyte and has potential anti-fibrosis effect. The polypeptide of the invention has research value for developing anti-tumor and anti-fibrosis drugs.

Description

A group of targeted FoxM1-DBD polypeptides and their applications

technical field

The invention relates to the field of biotechnology and pharmacy, in particular to a group of targeted FoxM1-DBD polypeptides and their application in anti-tumor and fibrosis.

Background technique

Cancer-targeted therapy blocks the growth of cancer cells by specifically targeting molecules required for cell growth and tumorigenesis. Compared with other traditional treatment options, cancer-targeted therapy has a more specific effect on cancer cells. Good efficacy and low side effects. FoxM1 is a transcription factor of the forkhead box protein family and is involved in a variety of biological processes, including cell proliferation, differentiation, cell cycle transition, migration, apoptosis, DNA damage repair, etc. FoxM1 is expressed in highly proliferating cells and disappears in quiescent and terminally differentiated cells. FoxM1 is closely related to the occurrence and development of cancer. It is overexpressed in most human cancers including liver cancer, lung cancer, breast cancer, etc. FoxM1 overexpression is closely related to a variety of cancer characteristics, including immortal proliferation, invasion and metastasis, angiogenesis, resistance, etc. Numerous studies have demonstrated that the inhibition of FoxM1 in cancer cells can lead to reduced cell proliferation and migration, invasion, angiogenesis, EMT, and drug resistance, indicating that it is a promising target for cancer-targeted therapy.

Fibrotic diseases such as idiopathic pulmonary fibrosis (PF) or scleroderma (systemic sclerosis) are chronic fibroproliferative diseases for which there is currently no effective treatment. The study found that FoxM1 is also closely related to fibroblasts and fibrosis. FoxM1 is required for pulmonary fibrosis and epithelial-mesenchymal transition (EMT). Excessive fibroblast proliferation is a key event in promoting pulmonary fibrosis, and inhibition of FoxM1 expression can ameliorate the progression of fibroblast proliferation during pulmonary fibrosis. In addition, FoxM1 promotes renal fibrosis by activating the Wnt/β-catenin signaling pathway, and the FoxM1 inhibitor salinamycin A attenuates renal fibrosis in UUO mice.

At present, although some achievements have been made in anti-cancer molecules targeting FoxM1 at home and abroad, most of them are limited to non-peptide organic synthetic drugs, which are greatly limited by their shortcomings such as difficulty in obtaining, poor biocompatibility, and metabolic toxicity. application in clinical treatment. In contrast, peptide drugs have good biocompatibility, easy structure modification, and simple synthesis, making them a hot spot in biomedical research and development. Peptide drugs targeting FoxM1 are rarely reported. In the early stage, our research group obtained a polypeptide 9R-P201 targeting FoxM1 DNA binding domain (DBD) protein by screening phage-displayed dodecapeptide library, which has a selective inhibitory and killing effect on tumor cells. Its IC50 value for liver cancer cells is 13.1 μM, see patent ZL201510783946.4 for details. And it is disclosed in patent application 201910086295.1 that its combined use with 5-Fu can enhance the sensitivity of HepG2 cells to 5-Fu. Patent application 202010150842.0 discloses an optimized peptide based on the construction of a biased peptide library based on the P201 sequence, which has a stronger inhibitory and killing effect on tumor cells. Unconstrained linear random peptide libraries can assume millions of different conformations, but only a few of them are likely to bind to receptors, however, cyclized peptide libraries may reduce conformational freedom, reduce peptide entropy, and hopefully separate ligands with higher affinity. In addition, heptapeptide has the advantages of smaller molecular weight, shorter sequence and lower synthetic modification cost than the dodecapeptide we screened before. Therefore, we screened the phage cyclic heptapeptide library to find new peptides with higher affinity to the target FoxM1-DBD protein and better inhibitory and killing effect on tumor cells, which has important research and social significance for the development of anti-tumor drugs . In addition, the treatment of fibrotic diseases with peptides targeting FoxM1 is rarely reported, so it is of pioneering significance to study the effect of peptides targeting FoxM1-DBD protein on fibrosis.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a group of targeting FoxM1-DBD polypeptides and their application in anti-tumor and fibrosis.

The targeted FoxM1-DBD polypeptide with anti-tumor and fibrosis applications provided by the present invention is screened from a phage display cyclic heptapeptide library.

The polypeptide is a cyclic peptide formed of disulfide bonds between two cysteines, and the sequences are shown in SEQ ID NO: 1-NO: 21. Cyclic peptides with high affinity and specificity for the target FoxM1-DBD were obtained through reverse screening, named CP13, CP18, CP29, and FCP20, and the cyclic peptide sequences are shown in SEQ ID NO: 1-NO: 4, respectively.

The cyclic peptide sequence with high affinity and specificity obtained by screening was modified and designed, the cysteines at both ends of the sequence were deleted, and the N-terminus was connected with 9 D-type arginine penetrating peptides through a dimerized glycine serine linking peptide Linear peptides were formed, named 9R-CP13L, 9R-CP18L, 9R-CP29L, 9R-FCP20L, respectively, and the sequences were shown in SEQ ID NO: 22-NO: 25, respectively. The modified four polypeptides can inhibit tumor cells, and the 9R-CP29L polypeptide has the strongest inhibitory effect.

The method for obtaining the targeted FoxM1-DBD polypeptide, the specific steps are as follows:

S1, phage biopanning; specifically includes the following sub-steps:

S11. Add the recombinantly expressed FoxM1-DBD protein to a high-affinity microplate and place it on an ice box to coat overnight, and wash once with TBST solution; add BSA blocking solution at 4°C to block for at least 1 hour, and quickly wash the plate with TBST 6 times.

S12, this step is performed according to the following two methods:

Method 1. Add phage to the well plate coated with BSA, shake at room temperature for 1 hour, and then suck it into the well plate coated with FoxM1-DBD protein;

Method 2. Directly add the phage to the well plate coated with FoxM1-DBD protein;

Step S13, wash the plate 6-8 times with TBST, eluate the bound phage, suck the eluate into a microtube, add 1M Tris-HCl to neutralize the eluate; take part of the phage for titer determination, and the remaining phage for amplification And titer determination was performed for the next round of panning, for a total of four rounds.

S2, phage monoclonal sequencing and reverse screening;

S21. Sequencing: randomly pick a single plaque from the test titer plate for a small amount of amplification, draw part of the bacterial solution, design primers according to the M13KE vector for sequencing, and deduce the sequence of the displayed polypeptide according to the amino acid sequence of the phage.

S22. Reverse screening: Amplify the sequenced monoclonal phage, and perform titer determination, add 1×10 ⁹ per well to the microplate, and then elute the bound phage and perform titer determination , according to the P/N value, that is, the titer of the phage eluted from the hole coated with FoxM1-DBD protein/the titer of the phage eluted from the well blocked with BSA only, to judge the affinity and specificity of the phage monoclonal and the target. Affinity and specificity.

S3. Polypeptide transformation: The high affinity and specific cyclic heptapeptide obtained by screening is connected to form a ring by a disulfide bond. Since FoxM1 is an intracellular target, the disulfide bond is unstable and easily broken by reduction under the reducing environment in tumor cells. The Cys at both ends of the peptide sequence was preliminarily designed to be deleted, and 9 D-type arginines were added to the N-terminus of the peptide sequence to achieve the purpose of penetrating the membrane, and the target-binding peptide was connected by dimeric glycine serine to obtain a newly designed 9R-containing peptide. Linear polypeptide of 20 amino acids.

Preferably, the step S21 is specifically: randomly picking a single plaque from the plates on which the titers of the third and fourth rounds of eluate are measured, performing a small amount of amplification, drawing part of the bacterial liquid, and designing according to the M13KE vector The primers were sequenced (F: CTGTCTTTCGCTGCTGAGG, R: AACCCCGCTAATCCTAATCC), and the sequence of the displayed polypeptide was deduced from the amino acid sequence of the phage. In the step S22, four cyclic heptapeptides with high affinity and specificity, CP13 (CHGYPWSLC), CP18 (CMHSNTLYC), CP29 (CAWWNTEWC), and FCP20 (CDSYFWRPC), are finally screened out. In the step S3, the cyclic heptapeptides CP13, CP18, CP29 and FCP20 obtained by screening are used for modification and design. The four modified polypeptide sequences are 9R-CP13L (RRRRRRRRGSGSHGYPWSL), 9R-CP18L (RRRRRRRRGSGSMHSNTLY), 9R-CP29L (RRRRRRRRRGSGSAWWNTEW), and 9R-FCP20L (RRRRRRRRRGSGSDSYFWRP).

S4. Peptide synthesis. (Synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.)

The synthetic steps of the peptide are as follows:

(1) Resin swelling: put 2-chlorotrityl chloride resin (2-ChlorotritylChloride Resin with a degree of substitution of 1.1 mmol/g) into a reaction tube, add DMF (15 mL/g), and shake for 60 min.

(2) Connect the first amino acid: filter out the solvent through sand core suction, add 3 times molar excess Fmoc-Trp(Boc)-OH, the first amino acid at the C-terminus of Fmoc-Arg(pbf)-OH, then add 10 Molar excess of DIEA was added, and finally DMF (10 mg/g) was added to dissolve, and the solution was shaken for 30 min; the methanol was capped for 30 min.

(3) Deprotection: remove DMF, add 20% piperidine DMF solution (15 mL/g) for 5 min, remove and add 20% piperidine DMF solution (15 mL/g) for 15 min.

(4) Detection: remove the piperidine solution, take more than ten resins, wash with ethanol three times, add a drop of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5min, and turn dark blue as a positive reaction.

(5) Washing: DMF (10 mL/g) twice, methanol (10 mL/g) twice, DMF (10 mL/g) twice.

(6) Condensation: add 3 times molar excess of Fmoc to protect amino acid, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, finally add DMF (10mg/g) to dissolve, shake for 45min.

(7) Detection: Take a dozen resins, wash three times with ethanol, add one drop each of ninhydrin, pyridine and phenol solution, heat at 105℃-110℃ for 5min, colorless is a negative reaction.

(8) Washing: DMF (10 mL/g) once, methanol (10 mL/g) twice, DMF (10 mL/g) twice.

(9) Repeat steps (3) to (8) to connect amino acids in the sequence from right to left.

(10) Wash the resin according to the following method and drain it:

DMF (10 mL/g) twice, DCM (10 mL/g) three times, methanol (10 mL/g) four times, and drained for 10 min.

(11) Cutting: prepare cutting solution (10/g) TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1% cutting time: 180min.

(12) Drying and washing: The lysate was blown dry with nitrogen as much as possible, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed six times with ether, and then evaporated to dryness at room temperature.

(13) Purification preparation:

First, take a little crude product and dissolve H ₂ O/ACN. Then, take a small amount of sample and analyze it on the HPLC analyzer to determine the target peak corresponds to the peak time. Use C18 reversed-phase chromatography preparation system: avelength: 220 nm; Flow Rate: 15 mL/min; Inj.Vol: 20 mL Column Temp: 25°C Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution. Use a 1.5mL centrifuge tube to take a small amount of the target peak solution for mass spectrometry confirmation and purity detection.

(14) freeze-drying the qualified target peak solution to obtain a finished product.

A small amount of finished polypeptides were taken respectively for molecular weight identification by MS and purity identification by HPLC analysis. Store the white powdery peptides in a sealed package and store at -20 degrees.

Compared with the prior art, the advantages of the present invention are:

The present invention provides a group of polypeptide molecules with anti-tumor activity targeting FoxM1, which can effectively inhibit and kill cancer cells, promote cancer cell apoptosis and inhibit cancer cell migration. Meanwhile, the polypeptide can inhibit the proliferation of fibroblasts and has the potential to treat fibrotic diseases. In particular, the polypeptide 9R-CP29L has the best effect, which can effectively inhibit highly metastatic liver cancer cells, triple-negative breast cancer cells, and fibroblasts. It has laid a good working foundation for the development and design of new drug molecules with anti-tumor and fibrotic diseases.

Other advantages, objects, and features of the present invention will appear in part from the description that follows, and in part will be appreciated by those skilled in the art from the study and practice of the invention.

Description of drawings

Figure 1. Changes in phage elution recovery rate in four rounds of biopanning.

Figure 2. Histogram of phage monoclonal reverse screening results.

Figure 3. Bar graph of the inhibitory effect of four synthetic polypeptides on the viability of HCCLM3 and MDA-MB-231 cells.

Figure 4. The effect of polypeptide 9R-CP29L on the viability of HCCLM3 cells.

Figure 5. The effect of polypeptide 9R-CP29L on the viability of MDA-MB-231 cells.

Figure 6. Flow chart of the apoptosis of HCCLM3 cells by polypeptide 9R-CP29L.

Figure 7. Histogram of apoptosis of HCCLM3 cells by polypeptide 9R-CP29L.

Figure 8. Transwell diagram of polypeptide 9R-CP29L on the migration of HCCLM3 cells.

Figure 9. Histogram of the migration of HCCLM3 cells by polypeptide 9R-CP29L.

Figure 10. Inhibition diagram of polypeptide 9R-CP29L on L929 cell viability.

Figure 11. The effect of polypeptide 9R-CP29L on L929 cell clone formation.

Figure 12. Histogram of L929 cell clones formed by polypeptide 9R-CP29L.

Figure 13. The expression of FoxM1 protein in L929 cells after polypeptide 9R-CP29L treatment.

Figure 14. Molecular docking pattern diagram of polypeptide CP29L and FoxM1-DBD protein.

Detailed ways

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

A method for obtaining a set of FoxM1 targeting peptides, the steps are as follows:

S1, phage biopanning;

15 μg of recombinantly expressed FoxM1-DBD protein was added to a high-affinity microplate and placed on an ice box to coat overnight, and washed once with TBST solution (TBS+Tween20, four rounds of Tween concentration were 0.1%, 0.1%, 0.3%, respectively. , 0.3%), add 300 μL BSA blocking solution at 4°C for at least 1 h, wash the plate 6 times with TBST, first add 1 × 10 ¹¹ phage to the BSA-coated well plate, shake for 1 h at room temperature, and then aspirate the FoxM1-coated well plate. -DBD protein in the well plate. This is the subtraction method. A non-subtractive method was also used here, ie, the phage was directly added to the wells coated with FoxM1-DBD. Wash the plate 6-8 times with TBST, add 200 μL of Glycine-HCl (pH 2.2) and 1 mg/mL BSA to each well to elute the bound phage, shake gently for about 10 min, suck the eluate into a microtube, add 30 μL of The eluate was neutralized with 1M Tris-HCl (pH 9.1). 10 μL of phage were taken for titer determination, and the remaining phage were amplified and titered for the next round of panning, for a total of four rounds. The results are shown in Figure 1: through the four-round binding-elution-amplification cycle of the phage display library, the recovery rate of phage was gradually increased, and it was considered that the phage bound to the target was effectively enriched.

S2, phage monoclonal sequencing and reverse screening;

S21. Sequencing: randomly pick a single plaque from the plates on which the titers of the third and fourth rounds of eluate are measured, perform a small amount of amplification, draw part of the ER2738 bacterial solution, and design primers according to the M13KE vector for sequencing (F: CTGTCTTTCGCTGCTGAGG, R: AACCCCGCTAATCCTAATCC), the sequence of the displayed polypeptide was deduced from the amino acid sequence of the phage, and the polypeptide sequence results are shown in the following table.

serial number filter number sequence Filter occurrence frequency SEQ ID: NO5 CP7 CMSYGGAPC 1 SEQ ID: NO6 CP9 CHPSWNTFC 1 SEQ ID: NO1 CP13 CHGYPWSLC 1 SEQ ID: NO7 CP14 CTLWGTYEC 2 SEQ ID: NO8 CP15 CSGHLPTLC 1 SEQ ID: NO2 CP18 CMHSNTLYC 1 SEQ ID: NO3 CP29 CAWWNTEWC 10 SEQ ID: NO9 CP40 CNTWPWQFC 1 SEQ ID: NO10 CP43 CSQWWFGAC 2 SEQ ID: NO11 FCP7 CNVEMFWRC 2 SEQ ID: NO12 FCP9 CPVEFKPLC 1 SEQ ID: NO13 FCP10 CSHLLAVKC 1 SEQ ID: NO14 FCP11 CDEHLHRTC 1 SEQ ID: NO15 FCP15 CLNMPISHC 1 SEQ ID: NO16 FCP18 CEPMLGPRC 1 SEQ ID: NO4 FCP20 CDSYFWRPC 1 SEQ ID: NO17 FCP29 CNSDSFKLC 1 SEQ ID: NO18 FCP4N CFGVYTNVC 1 SEQ ID: NO19 FCP8N CLSGPLSKC 1 SEQ ID: NO20 FCP12N CLPTGLIAC 1 SEQ ID: NO21 FCP18N CMTPIDWRC 1

S22. Reverse screening: Amplify the sequenced monoclonal phage, and measure the titer. Add 1×10 ⁹ per well to the microplate (1 μg of the coating target), and wash the plate 8 times with TBST. Then the bound phage was eluted and the titer was measured, and the phage monoclonal was judged by the P/N value, that is, the titer of the phage eluted from the well coated with FoxM1-DBD protein/the titer of the phage eluted from the well blocked with BSA only. Affinity and specificity with the target. The results are shown in Figure 2: the monoclonal P/N value of the phage displaying the CP13, CP18, CP29, and FCP20 peptides was the highest, and the frequency of CP29 was the highest during the screening process, which was 10 times.

S3. Polypeptide transformation: the screened cyclic heptapeptides CP13, CP18, CP29, and FCP20 (named CP13 (sequence: CHGYPWSLC), CP18 (sequence: CMHSNTLYC), CP29 (sequence: CAWWNTEWC), FCP20 (sequence: CDSYFWRPC)) are linked by disulfide bonds to form a loop. Since FoxM1 is an intracellular target, the disulfide bond is unstable and easy to be broken by reduction in the reducing environment of tumor cells. Therefore, the Cys at both ends of the peptide sequence (named CP13L (sequence: HGYPWSL) and CP18L (sequence: MHSNTLY) were initially designed to be deleted. ), CP29L (sequence: AWWNTEW), FCP20L (sequence: DSYFWRP)), in order to enable the polypeptide to enter the cell through the cell membrane and interact with the target, 9 D-type arginines (9R) were added to the N-terminus of the peptide sequence. ) to achieve the purpose of transmembrane, through dimerization of glycine serine and the target binding peptide. Accordingly, newly designed linear polypeptides of 20 amino acids including 9R (respectively named 9R-CP13L (sequence: RRRRRRRRGSGSHGYPWSL), 9R-CP18L (sequence: RRRRRRRRGSGSMHSNTLY), 9R-CP29L (sequence: RRRRRRRRGSGSAWWNTEW), 9R - FCP20L (sequence: RRRRRRRRGSGSDSYFWRP).

S4, peptide synthesis, the steps are as follows:

(1) Resin swelling: put 2-chlorotrityl chloride resin (2-ChlorotritylChloride Resin with a degree of substitution of 1.1 mmol/g) into a reaction tube, add DMF (15 mL/g), and shake for 60 min.

(2) Connect the first amino acid: filter out the solvent through sand core suction, add 3 times molar excess Fmoc-Trp(Boc)-OH, the first amino acid at the C-terminus of Fmoc-Arg(pbf)-OH, then add 10 Molar excess of DIEA was added, and finally DMF (10 mg/g) was added to dissolve, and the solution was shaken for 30 min; the methanol was capped for 30 min.

(3) Deprotection: remove DMF, add 20% piperidine DMF solution (15 mL/g) for 5 min, remove and add 20% piperidine DMF solution (15 mL/g) for 15 min.

(4) Detection: remove the piperidine solution, take more than ten resins, wash with ethanol three times, add a drop of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5min, and turn dark blue as a positive reaction.

(5) Washing: DMF (10 mL/g) twice, methanol (10 mL/g) twice, DMF (10 mL/g) twice.

(6) Condensation: add 3 times molar excess of Fmoc to protect amino acid, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, finally add DMF (10mg/g) to dissolve, shake for 45min.

(7) Detection: Take a dozen resins, wash three times with ethanol, add one drop each of ninhydrin, pyridine and phenol solution, heat at 105℃-110℃ for 5min, colorless is a negative reaction.

(8) Washing: DMF (10 mL/g) once, methanol (10 mL/g) twice, DMF (10 mL/g) twice.

(9) Repeat steps (3) to (8) to connect amino acids in the sequence from right to left.

(10) Wash the resin according to the following method and drain it:

DMF (10 mL/g) twice, DCM (10 mL/g) three times, methanol (10 mL/g) four times, and drained for 10 min.

(11) Cutting: prepare cutting solution (10/g) TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1% cutting time: 180min.

(12) Drying and washing: The lysate was blown dry with nitrogen as much as possible, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed six times with ether, and then evaporated to dryness at room temperature.

(13) Purification preparation:

First, take a little crude product and dissolve H ₂ O/ACN. Then, take a small amount of sample and analyze it on the HPLC analyzer to determine the target peak corresponds to the peak time. Use C18 reversed-phase chromatography preparation system: avelength: 220 nm; Flow Rate: 15 mL/min; Inj.Vol: 20 mL Column Temp: 25°C Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution. Use a 1.5mL centrifuge tube to take a small amount of the target peak solution for mass spectrometry confirmation and purity detection.

(14) freeze-drying the qualified target peak solution to obtain a finished product.

A small amount of finished polypeptides were taken respectively for molecular weight identification by MS and purity identification by HPLC analysis. Store the white powdery peptides in a sealed package and store at -20 degrees.

The performance of the polypeptides obtained by the above method is studied as follows:

1. Inhibitory effect of synthetic peptides on tumor cell viability

The effects of four synthetic peptides on the viability of human highly metastatic liver cancer cells HCCLM3 cells and human triple-negative breast cancer cells MDA-MB-231 cells were detected by CCK8 assay. The HCCLM3 and MDA-MB-231 cells in good condition were inoculated into 96-well plates, 5×10 ³ cells per well, and the peptides were added after overnight culture. Dilute into concentration gradient (30, 50, unit μM), 4 duplicate wells for each concentration, add pre-mixed CCK8 mixture after 24h and 48h (CCK8 solution: medium volume ratio is 1:10), incubator Incubate for about 2h, detect the absorbance values at 450nm and 630nm (reference wavelength) with a microplate reader, take OD450-OD630 as the reading value after correction, and calculate the cell viability inhibition rate according to the formula value-background value)/(control group reading-background value)]×100. The results are shown in Figure 3, the four polypeptides have different degrees of inhibitory effect on the two tumor cells, and the 9R-CP29L peptide has the strongest inhibitory effect on the two tumor cells.

2. Inhibitory effect of polypeptide 9R-CP29L on tumor cell viability

The effect of polypeptide 9R-CP29L on the viability of HCCLM3 cells and MDA-MB-231 cells was detected by CCK8 method. The method is the same as described in 1. The peptide concentration is diluted into a concentration gradient (5, 10, 20, 30, 40, 50, in μM), with 4 replicate wells for each concentration. background value)/(control group reading value-background value)×100. The results are shown in Figure 4 and Figure 5: with the concentration gradually increasing, the cell viability decreased significantly. At 20 μM concentration, the cell viability of HCCLM3 decreased by 74.46%, 91.83%, and 86.62% at 12h, 24h, and 48h, respectively; MDA-MB The cell viability of -231 cells decreased by 38.26%, 77.00%, and 92.87% at the concentration of 20 μM; it can be found that 9R-CP29L inhibits the viability of both tumor cells in a dose-dependent manner, while 9R-CP29L exerts an inhibitory effect on HCCLM3 cells. The time was significantly lower than that of MDA-MB-231 cells. The IC50 values of 9R-CP29L on HCCLM3 cells at 12h, 24h and 48h were 12.87μM, 7.20μM, and 8.57μM, respectively, and the IC50 values on MDA-MB-231 cells in three time periods were 20.10μM, 11.63μM, and 7.27μM, respectively.

3. The pro-apoptotic effect of polypeptide 9R-CP29L on tumor cells

The effect of polypeptide 9R-CP29L treatment on apoptosis of HCCLM3 cells was detected by AnnexinV-FITC/PI double staining method. Plate 1×10 ⁵ HCCLM3 cells in each well of a 12-well plate, add 10 and 20 μM peptides overnight for treatment for 24 h, collect the cell culture medium after the action time, wash the cells once with PBS and collect them, digest the cells with EDTA-free trypsin to After turning into a circle, the digestion was terminated, mixed with the previously collected cells and centrifuged for collection, washed the cell pellet twice with PBS, added 100 μL of Binding buffer to resuspend the cells, added 5 μL of AnnexinV-FITC and blown gently, and then added 5 μL of PI to mix well; incubate at room temperature for 10 min in the dark, Add 400 μL of Binding Buffer, mix gently, and perform flow-through detection, with 2 replicates per group. The results are shown in Figure 6 and Figure 7: the average early and late apoptosis rates were 4.90% and 6.70% after 10 μM treatment for 24 h, and the average early and late apoptosis rates were 17.44% and 25.09 after 20 μM treatment for 24 h. % (Figure 6 shows only one of the results, Figure 7 is the average of the two). Compared with untreated cells, the rate of apoptosis was significantly increased in the peptide-treated group in a dose-dependent manner.

4. Inhibitory effect of polypeptide 9R-CP29L on tumor cell migration

Transwell method was used to detect the effect of polypeptide 9R-CP29L treatment on the migration of HCCLM3 cells. Plate 1×10 ⁵ cells per well in a 12-well plate, treat overnight with 10 and 15 μM peptides for 24 h, wash twice with PBS to remove dead cells after reaching the action time, trypsinize cells, and collect by centrifugation after termination, PBS Wash the cell pellet once, collect the cells by centrifugation, resuspend in DMEM medium and count, add 1.5×10 ⁴ cells of 200 μL cell suspension per well to the chamber, and move the chamber to 24 wells with 600 μL 20% fetal bovine serum medium After 24 hours, the cells in the inner chamber were gently wiped off with a cotton swab, fixed with 4% paraformaldehyde for 25 minutes, stained with 0.1% crystal violet for 25 minutes, and photographed under a microscope. repeat. Counting was performed using Image J software. The results are shown in Figure 8 and Figure 9: the number of cells passing through the chamber in the polypeptide-treated group was significantly reduced, and with the increase of the polypeptide concentration, the number of cells passing through the chamber showed a decreasing trend.

5. Inhibitory effect of polypeptide 9R-CP29L on fibroblast viability

The effect of polypeptide 9R-CP29L treatment on the viability of mouse fibroblast L929 was detected by CCK8 method. The CCK8 experiment was performed according to the method described in 1, with a polypeptide concentration gradient (10, 20, 30, 40, 50, 60, 70, in μM), with 5 replicate wells for each concentration. The results are shown in FIG. 10 , the inhibitory effect of 9R-CP29L peptide on L929 cells was dose- and time-dependent. The IC50 values at 24h and 48h were 20.64 μM and 16.43 μM, respectively.

6. Inhibitory effect of polypeptide 9R-CP29L on fibroblast clone formation

The clone formation assay was used to detect the effect of polypeptide 9R-CP29L on the clone formation of L929 cells. 700 cells were seeded in each well of a six-well plate, and 30 μM and 50 μM drugs were added overnight after treatment for two weeks. The medium was changed every three days. After reaching the time, PBS was washed once, and 4% paraformaldehyde was fixed for 30 min. Stain with 0.1% crystal violet for 30 min, wash with PBS for 3-4 times until the background is clear, and take pictures with a camera. The experiment was repeated three times. The results are shown in Figure 11 and Figure 12 , compared with the control group, the number of colonies formed in the drug-added group was significantly reduced, and the higher the concentration, the more obvious the inhibitory effect.

7. Inhibitory effect of polypeptide 9R-CP29L on FoxM1 protein expression in fibroblasts

The effect of polypeptide 9R-CP29L on FoxM1 protein expression in L929 cells was detected by Western Blot assay. 1.5×10 ⁵ L929 cells were inoculated in each well of a 12-well plate. After overnight culture, blank medium and 30 μM polypeptide were added for treatment for 24 h and 48 h. After reaching the action time, the cells were lysed with RIPA, centrifuged at 13,000 rpm for 10 min, and the supernatant was aspirated. A small amount of protein supernatant was tested for concentration with BCA kit, and the rest was added to 5× protein loading buffer, boiled for denaturation for 10 minutes, then run off the gel, wet transferred to membrane, blocked for 3 hours, incubated with FoxM1 primary antibody overnight at 4°C, and washed with TBST for 3 hours. -4 times, incubate with secondary antibody for 1 h, wash 3-4 times with TBST, and add ECL chromogenic solution for exposure. The results are shown in Figure 13, the FoxM1 protein expression level decreased after the polypeptide treatment.

8. Docking of the polypeptide CP29L with the target FoxM1-DBD molecule

Download the FoxM1-DBD protein structure file (3G73) from the PDB database, use the online protein-peptide docking website HPEPDOCK for molecular docking, delete the nucleic acid in FoxM1-DBD and upload the file in pdb format. Points were docked, and the Discovery Studio Visualizer Client was used to analyze the interaction between the predicted protein and the optimal binding conformation of the peptide, and the results were visualized by PyMOL software. The results are shown in Figure 14. The polypeptide CP29L can form hydrogen bonds with Asn283, His287, Asn288, and His292, form Pi-Cation interaction and electrostatic attraction with Arg236, and form Pi-Pi T-shaped interaction with His287. The major contributors to the DNA-binding specificity of FoxM1 are three invariably conserved residues: Asn-283, Arg-286 and His-287. Therefore, it is speculated that CP29L can bind to these sites and interfere with the binding of FoxM1-DBD to DNA, thereby affecting the expression of downstream genes and exerting corresponding biological activities.

To sum up, the implementation cases show that the linear polypeptides 9R-CP13L, 9R-CP18L, 9R-CP29L, 9R-FCP20L screened and designed and modified by the present invention are effective in highly metastatic liver cancer cells HCCLM3 and triple negative breast cancer cells MDA-MB- 231 all have different degrees of inhibitory effect, among which 9R-CP29L showed the strongest inhibitory effect on HCCLM3 and MDA-MB-231, and the IC50 value of 9R-CP29L peptide on cancer cells was almost the same as that reported from phage random peptide library against The lowest concentration of anti-tumor polypeptide molecules screened by FoxM1 target has high research value; in addition, 9R-CP29L can promote HCCLM3 cell apoptosis and inhibit cell migration, and can also inhibit the proliferation of fibroblast L929 and FoxM1 in cells The expression of the protein has laid a good foundation for the development and design of new anti-tumor and fibrotic disease drug molecules.

The above are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Technical personnel, within the scope of the technical solution of the present invention, can make some changes or modifications to equivalent embodiments of equivalent changes by using the technical content disclosed above, but any content that does not depart from the technical solution of the present invention, according to the present invention Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solutions of the present invention.

sequence listing

<110> Southwest Jiaotong University

<120> A group of targeted FoxM1-DBD polypeptides and their applications

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial series

<400> 1

Cys His Gly Tyr Pro Trp Ser Leu Cys

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial series

<400> 2

Cys Met His Ser Asn Thr Leu Tyr Cys

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial series

<400> 3

Cys Ala Trp Trp Asn Thr Glu Trp Cys

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial series

<400> 4

Cys Asp Ser Tyr Phe Trp Arg Pro Cys

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial series

<400> 5

Cys Met Ser Tyr Gly Gly Ala Pro Cys

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial series

<400> 6

Cys His Pro Ser Trp Asn Thr Phe Cys

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial series

<400> 7

Cys Thr Leu Trp Gly Thr Tyr Glu Cys

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial series

<400> 8

Cys Ser Gly His Leu Pro Thr Leu Cys

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial series

<400> 9

Cys Asn Thr Trp Pro Trp Gln Phe Cys

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial series

<400> 10

Cys Ser Gln Trp Trp Phe Gly Ala Cys

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial series

<400> 11

Cys Asn Val Glu Met Phe Trp Arg Cys

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial series

<400> 12

Cys Pro Val Glu Phe Lys Pro Leu Cys

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial series

<400> 13

Cys Ser His Leu Leu Ala Val Lys Cys

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial series

<400> 14

Cys Asp Glu His Leu His Arg Thr Cys

1 5

<210> 15

<211> 9

<212> PRT

<213> Artificial series

<400> 15

Cys Leu Asn Met Pro Ile Ser His Cys

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial series

<400> 16

Cys Glu Pro Met Leu Gly Pro Arg Cys

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial series

<400> 17

Cys Asn Ser Asp Ser Phe Lys Leu Cys

1 5

<210> 18

<211> 9

<212> PRT

<213> Artificial series

<400> 18

Cys Phe Gly Val Tyr Thr Asn Val Cys

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial series

<400> 19

Cys Leu Ser Gly Pro Leu Ser Lys Cys

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial series

<400> 20

Cys Leu Pro Thr Gly Leu Ile Ala Cys

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial series

<400> 21

Cys Met Thr Pro Ile Asp Trp Arg Cys

1 5

<210> 22

<211> 20

<212> PRT

<213> Artificial series

<400> 22

Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ser Gly Ser His Gly Tyr

1 5 10 15

Pro Trp Ser Leu

20

<210> 23

<211> 20

<212> PRT

<213> Artificial series

<400> 23

Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ser Gly Ser Met His Ser

1 5 10 15

Asn Thr Leu Tyr

20

<210> 24

<211> 20

<212> PRT

<213> Artificial series

<400> 24

Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ser Gly Ser Ala Trp Trp

1 5 10 15

Asn Thr Glu Trp

20

<210> 25

<211> 20

<212> PRT

<213> Artificial series

<400> 25

Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ser Gly Ser Asp Ser Tyr

1 5 10 15

Phe Trp Arg Pro

20

Claims (2)

1. The FoxM1-DBD targeting polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO:1-NO:4 or SEQ ID NO:22-NO:25, respectively.

2. The use of the targeted FoxM1-DBD polypeptide of claim 1, wherein the polypeptide molecule can be used as a lead molecule for the preparation of drugs against liver cancer, triple negative breast cancer and fibrotic diseases.

Images