Application of natural small molecule compound taking DCTPP1 as target in preparation of medicine for treating lung cancer

Abstract

The invention discloses a drug application of a natural small molecule compound Colletopetide A taking DCTPP1 as a target, and the natural small molecule compound Colletopetide A realizes proliferation inhibition and apoptosis promotion of lung cancer cells by targeted inhibition of activity of DCTPP1 protein and reduction of expression level of DCTPP1 gene, and provides a new strategy for treatment of lung cancer. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the use of natural small molecule compounds with DCTPP1 as a target in preparing drugs for treating lung cancer, and in particular to the use of cyclic peptide compounds with DCTPP1 inhibitory activity in preparing drugs for treating lung cancer. Background Art

[0002] Lung cancer is one of the most common malignant tumors, most of which are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is an important member of the epidermal growth factor, hepatocyte growth factor (HGF) or tyrosine kinase receptor (ErbB) family and is considered to be a key oncogene in NSCLC and other epithelial cancers. Abnormal EGFR gene activity can activate downstream signaling pathways, inhibit cell apoptosis, enhance cell metabolism and proliferation, and thus promote the occurrence and development of cancer.

[0003] With the continuous progress of targeted chemotherapy, small molecule inhibitors targeting EGFR kinase have been shown to significantly prolong the survival of NSCLC patients. However, due to bottlenecks such as adverse reactions, multifactorial resistance and off-target toxicity of chemotherapeutic drugs, the treatment of lung cancer still faces major challenges. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide a new drug application of a natural inhibitor targeting DCTPP1 in the treatment of lung cancer.

[0005] Technical solution: The cyclic peptide compound Colletopeptide A of the present invention is used in the preparation of drugs for treating lung cancer, wherein the structure of the cyclic peptide compound Colletopeptide A is as follows:

[0006]

[0007] The present invention studies the metabolites of the endophytic fungus Colletotrichum sp. of the aster plant, and successfully obtains the natural cyclic peptide compound Colletopeptide A by using silica gel column chromatography, RP-18 and Sephadex LH-20 gel column chromatography. Compared with traditional chemical synthetic drugs, natural products can show significant advantages in the field of new anti-tumor drug discovery due to their structural complexity, structural diversity, low toxicity, and low resistance.

[0008] Preferably, the drug is a drug for treating non-small cell lung cancer.

[0009] Preferably, the drug is a drug that inhibits tumor cell proliferation and promotes tumor cell apoptosis.

[0010] More preferably, the drug is a drug that inhibits tumor cell proliferation and promotes tumor cell apoptosis in a dose-dependent manner.

[0011] Preferably, the drug is a drug for treating lung cancer with high expression of DCTPP1.

[0012] The present invention uses bioinformatics methods and clinical sample tissue chip analysis to find that DCTPP1 is generally highly expressed in lung cancer patients, and its high expression is significantly correlated with the poor prognosis of patients, which not only reveals the clinical significance of DCTPP1 protein in lung cancer, but also provides a potential drug target for lung cancer treatment. Specifically, downregulating the expression level of DCTPP1 can significantly inhibit the proliferation of lung cancer cells and promote cell apoptosis, while upregulating the expression level of DCTPP1 can promote cell growth.

[0013] Preferably, the drug is a drug that inhibits the expression of DCTPP1.

[0014] More preferably, the drug is a drug that targets and inhibits the expression of DCTPP1.

[0015] Preferably, the drug is a drug that inhibits the activity of DCTPP1 protein.

[0016] More preferably, the drug is a drug that targets and binds to DCTPP1 protein.

[0017] Preferably, the preparation method of the cyclic peptide compound Colletopeptide A comprises the following steps:

[0018] (1) fermenting the endophytic fungus Colletotrichum sp. from Aster plant with rice solid culture medium and extracting with ethyl acetate to obtain an extract;

[0019] (2) Separating and purifying the extract obtained in step (1) to obtain the cyclic peptide compound Colletopeptide A.

[0020] Specifically, Colletotrichum sp. is cultured until the mycelium completely covers the culture dish, and then the mycelium is chopped and transferred under a sterile environment for static fermentation.

[0021] The fermented product was extracted with ethyl acetate several times, and the extract was concentrated under reduced pressure to obtain the total extract. It was then chromatographed on a silica gel column and gradient eluted with petroleum ether / acetone in different ratios (1:0 to 1:1) to obtain 5 fractions (Fr.1 to Fr.5). Fr.3 was chromatographed on an RP-18 silica gel column and gradient eluted with methanol / water (10% to 90%) to obtain 7 fractions (Fr.3-1 to Fr.3-7). Fr.2-4 was chromatographed on a Sephadex LH-20 column and isocratically eluted with chloroform / methanol (1:1) to obtain 6 fractions (Fr.2-4-1 to Fr.2-4-6). Fr.2-4-2 was purified by YMC-Pack ODS-AC 18 The compound Colletopeptide A was separated by column semi-preparative high performance liquid chromatography.

[0022] The invention discloses an application of a pharmaceutical composition comprising a cyclic peptide compound, Colletopeptide A, as an active ingredient in the preparation of a drug for treating lung cancer.

[0023] Specifically, the pharmaceutical composition is composed of a cyclic peptide compound Colletopeptide A and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0024] The pharmaceutical composition can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0025] The pharmaceutical composition can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0026] Preferably, the drug is a drug for treating non-small cell lung cancer.

[0027] Preferably, the drug is a drug that inhibits tumor cell proliferation and promotes tumor cell apoptosis.

[0028] Preferably, the drug is a drug that inhibits the expression of DCTPP1.

[0029] More preferably, the drug is a drug that targets and inhibits the expression of DCTPP1.

[0030] Preferably, the drug is a drug that inhibits the activity of DCTPP1 protein.

[0031] More preferably, the drug is a drug that targets and binds to DCTPP1 protein.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0033] The present invention finds that the cyclic peptide compound Colletopeptide A inhibits the activity of DCTPP1 protein by targeting, reduces the expression level of DCTPP1 gene, achieves proliferation inhibition and apoptosis promotion of lung cancer cells, and provides a new strategy for the treatment of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 To investigate the expression of DCTPP1 gene in lung cancer and its relationship with poor prognosis;

[0035] Figure 2 The results of the difference in expression levels of DCTPP1 gene in lung cancer tissues and adjacent paracancerous tissues (n=5);

[0036] Figure 3 To verify the efficiency of DCTPP1 gene overexpression or knockdown;

[0037] Figure 4 The relationship between DCTPP1 gene expression and lung cancer cell proliferation;

[0038] Figure 5 The result is that Colletopeptide A inhibits the proliferation of lung cancer cells;

[0039] Figure 6 This is the effect of Colletopeptide A on the expression and activity of DCTPP1 protein and the binding results. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with embodiments.

[0041] Example 1: Expression of DCTPP1 in lung cancer and its relationship with poor prognosis

[0042] 1. Experimental methods

[0043] This experiment analyzed the expression level of DCTPP1 gene in lung cancer and its relationship with patient prognosis and survival by accessing TCGA, GTEx and UALCAN databases (http: / / ualcan.path.uab.edu / analysis.html). In addition, we detected the expression level of DCTPP1 protein by including different lung cancer tumor tissues and adjacent tissue chips. In order to evaluate the effect of DCTPP1 on the overall survival of lung cancer patients, the univariate Cox regression model and Kaplan-Meier survival curve analysis were used to reveal the value of this gene as a potential prognostic marker.

[0044] 2. Experimental results

[0045] like Figure 1 and Figure 2 As shown in the study, the expression of DCTPP1 in lung cancer tissue was significantly higher than that in normal lung tissue; lung cancer patients with high expression of DCTPP1 gene had a poor prognosis and a significantly shorter overall survival period than patients with low expression of DCTPP1 gene (P=0.02). The above results suggest that DCTPP1 is generally highly expressed in lung cancer patients, and high expression of DCTPP1 is closely related to poor prognosis of patients, making it a potential target for lung cancer treatment.

[0046] Example 2: Effects of knockdown and overexpression of DCTPP1 on the proliferation of LLC lung cancer cells

[0047] 1. Experimental methods

[0048] (1) siRNA cell transfection experiment: The DCTPP1 siRNA used in this experiment was chemically synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., and the siRNA stock solution (20 μM) was subsequently prepared according to the siRNA synthesis report and instruction manual. The primer sequences were F: AGUUCCAUCAGCCUCGGAATT, R: UUCCGAGGCUGAUGGAACUTT.

[0049] LLC cells were seeded into a 6-well plate at an appropriate concentration, allowed to adhere to the wall in an incubator for 24 hours, and replaced with 1.6 mL of fresh antibiotic-free culture medium containing 10% FBS. Take 2 sterile centrifuge tubes, fill each with 200 μL of serum-free culture medium, add 4 μL siRNA to one tube and 4 μL Lipofectamine RNAiMAX to one tube, mix them gently, let them stand at room temperature for 5 minutes, then mix the two tubes of solution evenly and let them stand for 15 minutes. Pour the siRNA transfection complex solution drop by drop into the well plate, shake it gently, and return it to the 37°C carbon dioxide incubator for culture. Collect the cells after 48 hours. Use immunoblotting to verify the transfection efficiency, such as Figure 3 As shown, DCTPP1 was significantly knocked down.

[0050] (2) DCTPP1 overexpression experiment: LLC cells were inoculated into 6-well plates at an appropriate concentration and allowed to adhere to the plate in an incubator for 24 hours. Take a pair of sterile EP tubes and fill them with 200 μL of serum-free culture medium. Add 2 μg of the extracted overexpression plasmid to one tube and 4 μL of Lipofectamine 2000 to the other tube. Mix them gently and let them stand for 5 minutes. Then mix the two tubes gently to form a complex and let it stand for 10 minutes. Add the liquid dropwise to the culture medium and shake gently. Return the culture medium to the incubator and change the medium after 8 hours. Collect the cells after 24 hours. Use immunoblotting to verify the overexpression efficiency. Figure 3 As shown, DCTPP1 expression was significantly enhanced.

[0051] (3) SRB detection of cell proliferation: LLC lung cancer cells were subjected to the aforementioned DCTPP1 knockdown and overexpression operations, digested with trypsin and centrifuged, and then seeded into 96-well plates at an appropriate density. After 48 h, the SRB colorimetric assay was performed, and the absorbance value was detected at 540 nm to calculate the cell survival rate.

[0052] 2. Experimental results

[0053] like Figure 4 As shown in the figure, compared with the control group, the apoptosis level of lung cancer cells LLC was significantly increased and the cell proliferation activity was decreased after knocking down DCTPP1; on the contrary, overexpression of DCTPP1 promoted cell proliferation.

[0054] Example 3: Preparation of cyclic peptide Colletopeptide A

[0055] Colletotrichum sp. was cultured in PDA medium at 28°C until the mycelium completely covered the culture dish. The mycelium was then chopped and transferred to 100 1L conical flasks filled with rice medium (each flask contained 180g rice and 220mL tap water, and sterilized by high pressure at 121°C for 30min) under a sterile environment. Static fermentation was carried out for 28 days under the same temperature conditions.

[0056] The rice fermentation product of strain Colletotrichum sp. was extracted with ethyl acetate four times (35L×3 times), and the extract was concentrated under reduced pressure to obtain the total extract. It was then chromatographed on a silica gel column and gradient eluted with petroleum ether / acetone in different ratios (1:0-1:1) to obtain 5 fractions (Fr.1-Fr.5). Fr.3 was chromatographed on an RP-18 silica gel column and gradient eluted with methanol / water (10%-90%) to obtain 7 fractions (Fr.3-1-Fr.3-7). Fr.2-4 was chromatographed on a Sephadex LH-20 column and isocratically eluted with chloroform / methanol (1:1) to obtain 6 fractions (Fr.2-4-1-Fr.2-4-6). Fr.2-4-2 was purified by YMC-Pack ODS-AC 18 The compound Colletopeptide A was separated by column semi-preparative high performance liquid chromatography.

[0057] Example 4: Evaluation of the effect of cyclic peptide Colletopeptide A on the proliferation of lung cancer cells LLC using sulforhodamine B (SRB) colorimetry

[0058] 1. Experimental Principle

[0059] Sulforhodamine B dye can specifically bind to the basic amino acid residues of intracellular proteins under slightly acidic conditions to form a complex and produce an absorption peak at a wavelength of 540nm. The measured absorbance value is linearly positively correlated with the number of cells. Therefore, this method can be used to determine the survival rate of living cells to reflect the toxicity of drugs to cells.

[0060] 2. Experimental methods

[0061] LLC cells with appropriate density and good growth status were digested and resuspended, and inoculated into 96-well plates. When the cells in the wells were attached to the wall and the density was about 50%, the prepared Colletopeptide A was added to each well for 24h and 48h. The final concentrations of the drugs were 20μM, 10μM, 5μM, 2.5μM, and 1.25μM, respectively, and three parallel wells were set for the blank group and each drug group. After the intervention end point, 25μL of 50% acetic acid was added to each well and fixed at room temperature for 1h, and then the liquid in the well was discarded and slowly rinsed under water and inverted at room temperature to dry. 100μL of 0.4% SRB dye solution was added to each well and stained at room temperature for 30min. After the dye solution was discarded, it was washed 3 times with 1% acetic acid solution. After the oven was dried, 100μL of 10mM Tris was added and shaken for more than 5min, and the absorbance value was detected at a wavelength of 540nm to calculate the IC of the drug. 50 value.

[0062] 3. Experimental results

[0063] like Figure 5 As shown, the cyclic peptide Colletopeptide A compound intervention for 24h and 48h can inhibit the proliferation of lung cancer cells LLC in a dose-dependent manner.

[0064] Example 5: Experiment on the binding ability of cyclic peptide Colletopeptide A compound to DCTPP1 protein

[0065] 1. Experimental methods

[0066] Microscale thermophoresis (MST): This method is used to detect interactions between biomolecules. Using the characteristic that biomolecules can undergo directional movement in a microscopic temperature gradient, proteins are labeled with fluorescent dyes to detect and quantify their directional movement in the microscopic temperature gradient field formed by infrared laser irradiation. When drug molecules, i.e. ligands, bind to proteins, they affect the fluorescent groups and produce changes in fluorescence intensity. Therefore, the binding constant K can be calculated based on the rate of change of the detection signal after different concentrations of ligands bind to fluorescent proteins. D The value of fitting.

[0067] The experimental method is to fluorescently label the DCTPP1 protein with a His tag with the RED-tris-NTA dye in the His-Tag-LabelingKitRED-tris-NTA kit (Cat. No.: LO18) at room temperature for 30 minutes in the dark according to the instructions. The sample was then centrifuged at 4°C, 15000rpm for 10 minutes and the supernatant was taken. Prepare 16 PCR tubes and mark them as 1 to 16. Add 20μL of ColletopeptideA drug to tube 1, and add 10μL of PBS-T to the other 15 tubes. Take 10μL of ColletopeptideA from tube 1 and add it to tube 2 and mix evenly. Then perform a 2-fold gradient dilution in each tube in this way, and discard 10μL of tube 16. Add 10μL of the labeled protein to each of the 16 PCR tubes and mix thoroughly. Use a capillary to absorb the sample and then test it on the machine. The instrument was set to 25°C, 100% LED / excitation power and medium MST power on the MO.Control 2 software and run according to the set program. The experimental data obtained were then analyzed using MO.AfnityAnalysis Software.

[0068] 2. Experimental results

[0069] like Figure 6 As shown in Figure 2, MST experiments showed that Colletopeptide A can stably bind to PRDX1 protein in vitro, and its binding affinity constant K D The value is 4.32μM.

Claims

1. Use of a cyclic peptide compound Colletopeptide A in the preparation of a drug for treating lung cancer, 2. The use according to claim 1, characterized in that: The medicine is a medicine for treating non-small cell lung cancer.

3. The use according to claim 1, characterized in that: The medicine is a medicine that inhibits tumor cell proliferation and promotes tumor cell apoptosis.

4. The use according to claim 3, characterized in that: The drug is a dose-dependent drug that inhibits tumor cell proliferation and promotes tumor cell apoptosis.

5. The use according to claim 1, characterized in that: The medicine is a medicine for treating lung cancer with high expression of DCTPP1.

6. The use according to claim 1, characterized in that: The drug is a drug that inhibits the expression of DCTPP1.

7. The use according to claim 6, characterized in that: The drug is a drug that targets and inhibits the expression of DCTPP1.

8. The use according to claim 1, characterized in that: The drug is a drug that inhibits the activity of DCTPP1 protein.

9. The use according to claim 8, characterized in that: The drug is a drug that targets and binds to DCTPP1 protein.

10. The use according to claim 1, characterized in that: The preparation method of the cyclic peptide compound Colletopeptide A comprises the following steps: (1) fermenting the endophytic fungus Colletotrichum sp. from Aster plant with rice solid culture medium and extracting with ethyl acetate to obtain an extract; (2) Separating and purifying the extract obtained in step (1) to obtain the cyclic peptide compound Colletopeptide A.

Images