CN115317622A - Preparation and application of anti-tumor polypeptide coupled drug - Google Patents
Preparation and application of anti-tumor polypeptide coupled drug Download PDFInfo
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- CN115317622A CN115317622A CN202210966541.4A CN202210966541A CN115317622A CN 115317622 A CN115317622 A CN 115317622A CN 202210966541 A CN202210966541 A CN 202210966541A CN 115317622 A CN115317622 A CN 115317622A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Engineering & Computer Science (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- General Chemical & Material Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an anti-tumor polypeptide conjugate drug and application thereof. The molecular sequence of the polypeptide coupled drug is as follows: MTX-Gly-Phe-Leu-Gly-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH 2 Wherein the Gly-Phe-Leu-Gly sequence linked to MTX is cleaved by cathepsin-B or the like. The antitumor polypeptide provided by the invention has membrane penetrability, and can be used as a carrier of a small-molecule antitumor drug to prepare a polypeptide coupled drug, so that the antitumor drug can be promoted to penetrate a cell membrane of a tumor cell, the release of the small-molecule drug is completed, and the tumor treatment is carried out.
Description
Technical Field
The invention relates to the field of protein polypeptide drugs, in particular to the field of anti-tumor drugs involved in the treatment process of liver cancer, ovarian cancer and lymph cancer, and specifically relates to a coupling of polypeptide and MTX drugs and application thereof.
Background
Cancer has developed as a major health problem that afflicts the world and is one of the leading causes of human death worldwide. According to the statistics of the world health organization, nearly 1000 million people died from cancer in 2018. More seriously, the new cancer cases in the world grow at a rate of approximately twenty million per year, and only in 2020, the new cancer cases reach 1930 million, and the death cases approach 1000 million.
There are many treatments for cancer, such as chemotherapy, radiotherapy, immunotherapy and surgical resection. Of these treatments, surgical resection and radiotherapy are often used for the treatment of non-metastatic cancer, and anticancer drugs (chemotherapy, hormones) are often used for the treatment of metastatic cancer.
Small molecules have significant advantages in metabolic stability, membrane permeability, etc., but also have higher off-target effects and therefore higher toxicity risks.
Compared with other small molecule drugs, peptide drugs have some unique advantages: peptide molecules have lower potential toxicity than small molecules, relatively fewer off-target side reactions, and the like.
Peptide-drug conjugates (PDC) are a novel molecular drug delivery system, generally consisting of a polypeptide, a covalent linker, and a payload. Generally, after the drug is combined with the polypeptide through a covalent linker, the polypeptide and the drug are endowed with bidirectional functions, so that the effect of promoting the killing or targeting of the drug can be achieved, and the PDC drug is a promising cancer treatment mode.
Melittin is a bioactive polypeptide extracted from the venom of European honeybee. Melittin, a cell-penetrating alpha-helical peptide containing 26 amino acid residues. Melittin is capable of producing multi-target antitumor effects including cell membrane disruption, antiproliferative, anti-invasive and anti-metastatic effects.
Methotrexate (MTX) is a drug commonly used for the treatment of leukemia, but its drug resistance etc. limits its further use. In addition, low cellular uptake of MTX is a challenge in the course of MTX cancer therapy.
Covalent linkers are of various types, including aliphatic chains, polyethylene glycol, amino acids, and the like. Among them, amino acids are one of the most commonly used covalent linkers in chemical synthesis. The GFLG tetrapeptide sequence has been widely applied to a spacer which can be cleaved in the presence of cathepsin-B, and the GFLG tetrapeptide spacer is most common in molecular delivery systems and can realize site-directed cleavage of cathepsin-B in tumor environment, thereby playing a role of a covalent linker in the molecular delivery system.
Aiming at the defect of low cellular uptake rate of MTX, polypeptide conjugate drugs of MTX are designed. MTX is connected with Melittin through GFLG to form a drug molecule delivery system taking Melittin polypeptide as a carrier and MTX as a payload. Wherein, the chemical synthesis of the MTX and the glycine in the polypeptide is realized through an amido bond, so that the MTX-GFLG-Melitin molecular delivery system is successfully constructed.
Solid Phase Polypeptide Synthesis (SPPS) is currently the predominant method of use for the synthesis of natural and non-natural polypeptides. Condensation systems commonly used in solid phase polypeptide synthesis such as HCTU/DIPEA, HATU/HOAt/DIPEA and DIC/HOBt. The temperature conditions used in these condensation systems are generally 30 ℃ and, relatively speaking, the lower the temperature and the longer the condensation reaction time of the amino acid.
Data have shown that rapid synthesis of polypeptides can be achieved using the DIC/Oxyma condensation system at 50 ℃. The following advantages are achieved by using DIC/Oxyma condensation system to perform condensation reaction of amino acid under 50 deg.C: the synthesis speed of the polypeptide is relatively high, the condensation time of the amino acid can be reduced by about 1/2, (2) the condensation of the amino acid has strong robustness, (3) the racemization rate of the amino acid in the condensation process is low, and the racemization rate of the amino acid in the condensation process is not higher than 3%. The method adopts a high-temperature method to synthesize the polypeptide part in the PDC medicament
Disclosure of Invention
In order to solve the defects of methotrexate in antitumor application, especially the drug resistance and low cell uptake rate of tumor cells caused by frequent medication, thereby reducing the antitumor effect. The invention mainly aims to provide an anti-tumor polypeptide conjugate drug and application thereof.
In a first aspect, the invention provides a polypeptide conjugate drug, which comprises the anti-tumor polypeptide, a cytotoxic drug and a linker. The molecular sequence is MTX-GFLG-GIGAVLKVLTTGLPALISSWIKRRQQ-NH 2
Further, the cytotoxic drug is a tumor chemotherapy small molecule drug.
Preferably, the tumor chemotherapeutic is methotrexate.
Further, the linker has four amino acid sequences of Gly-Phe-Leu-Gly, GFLG. GFLG can be cleaved by highly expressed enzymes in the tumor microenvironment and tumor cells, such as cathepsin-B.
Further, the polypeptide conjugate drug has a structure shown in formula A, wherein Melitin represents the antitumor polypeptide of claim 1 and the peptide shown in formula A, and the amino acid sequence of the polypeptide conjugate drug is H-GIGAVLKVLTTGLPALILKIKRRQQ-NH 2 。
The invention provides a preparation method of the polypeptide coupled drug, which comprises the following steps:
(1) Synthesis of the Melitin polypeptide chain was accomplished by DIC/Oxyma condensation system at 50 ℃. Fmoc-Gln-OH is coupled to Rink-Amide AM resin by adopting Fmoc solid phase synthesis method, and then Fmoc-Gln-OH, fmoc-Arg-OH, fmoc-Lys-OH, fmoc-Ile-OH, fmoc-Trp-OH, fmoc-Ser-OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Ala-OH, fmoc-Pro-OH, fmoc-Leu-OH, fmoc-Gly-OH, fmoc-Thr-OH, fmoc-Leu-OH, fmoc-Val-OH, fmoc-Lys-OH, fmoc-Leu-OH, fmoc-Val-OH, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Iloc-Leu-OH, fmoc-Val-Gly-OH and Meoc-Gly-OH are sequentially coupled to obtain polypeptide chain containing resin.
(2) After step (1) is completed, steps 2-a and 2-b are performed.
Step 2-a: according to the solid phase synthesis method, four amino acids in the linker are sequentially coupled with Melitin polypeptide chain according to the sequence of Fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Phe-OH and Fmoc-Gly-OH.
Step 2-b: according to the solid-phase synthesis method, MTX is connected to a glycine-containing resin through an amido bond to obtain a polypeptide-coupled drug resin, a TFA lysate is used for cleaving the polypeptide from the resin, and the polypeptide-coupled drug is obtained through subsequent separation and purification.
The second aspect of the invention provides an application of the polypeptide conjugated drug in antitumor drugs.
Further, the antitumor application is mainly to evaluate the killing effect of tumor cells including HepG2, hepa1-6, ES-2, U937, daudi, COC1 and A20 cells.
The invention has the beneficial effects that:
1. a method for rapid synthesis of Melitin polypeptide chains is provided. The synthesis of Melittin polypeptide chains by means of the DIC/Oxyma condensation system at 50 ℃ has the following advantages: the condensation of amino acid can be completed quickly, a large amount of reaction time is saved, the racemization rate of the amino acid in the synthesis process is low, and a condensation system has strong robustness and can resist high temperature of 50 ℃.
2. The anti-tumor polypeptide provided by the invention has penetrability, and the polypeptide is used as a carrier of a small-molecule anti-tumor medicament to prepare a polypeptide coupled medicament, so that the anti-tumor polypeptide can penetrate a cell membrane of a cancer cell, thereby playing a role in killing the tumor cell on one hand, promoting the uptake of the small-molecule medicament at a tumor part on the other hand, and increasing the treatment effect of the anti-tumor medicament MTX.
3. The Melittin and methotrexate are connected through the linker, the enzyme digestion group of the Gly-Phe-Leu-Gly tetrapeptide can be degraded by cathepsin-B and the like at a tumor part, and the polypeptide coupled drug belongs to an enzymolysis linker and has the characteristics of strong specificity, high selectivity and the like compared with the traditional linker.
Drawings
FIG. 1 is a schematic diagram of the synthetic process of MTX-GFLG-Melitin polypeptide conjugate drug;
FIG. 2 is a mass spectrum (ESI-MS) analysis spectrum (MW: 2845.8) of pure Melittin;
FIG. 3 is a mass spectrum (ESI-MS) analysis spectrum (MW: 3657.2) of a pure polypeptide conjugate drug A;
FIG. 4 analytical reversed phase high performance liquid chromatography (RP-HPLC) analysis spectra of crude Melittin and purified Melittin peptides;
FIG. 5 analytical reversed phase high performance liquid chromatography (RP-HPLC) analysis spectra of crude peptide and purified peptide of polypeptide conjugate drug A;
FIG. 6MTT method for detecting MTX-GFLG-Melitin anti-adherent tumor cell (HepG 2, hepa1-6 and ES-2) activity;
FIG. 7CCK8 method for detecting MTX-GFLG-Melitin anti-suspension tumor cell (U937, daudi, COC1 and A20) activity;
FIG. 8 is a graph of MTX anti-adherent tumor cell (HepG 2, hepa1-6 and ES-2) activity;
FIG. 9MTX anti-suspension tumor cell (U937, daudi, COC1 and A20) activity profile;
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method without specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition suggested by an instrument manufacturer.
Example 1: synthesis of the native sequence of the polypeptide and linker (FIG. 1).
(1) Weighing Rink-Amide AM resin
Taking the Rink-Amide AM resin out of the refrigerator, standing at room temperature and recovering the temperature to room temperature, and then weighing the Rink-Amide AM resin on a weighing balance. The weighing process needs to be rapid, and the moisture absorption of resin in the weighing process is avoided. The resin weighed 600mg and the degree of substitution of the resin was 0.27mmol/g.
(2) Activation of the resin
The weighed resin was put into a polypeptide synthesis tube, 4mL of DMF/DCM (v: v = 9) mixed reagent was added, and resin activation was performed at 30 ℃ for 1 hour or more to sufficiently activate the resin.
(3) Standard Wash of resins
The fully activated resin was subjected to standard washing. The standard washing method is as follows: the resin of the synthesis tube was washed with DMF first and twice. The resin was then washed twice with DCM. After that, the resin was washed again with DMF and then with DCM. Finally, the resin was washed three times with DMF.
(4) Removal of Fmoc protecting group on Rink-Amide AM resin
Removal of the Fmoc protecting group on the resin was performed using Fmoc deprotection reagent (piperidine/DMF = 1. Fmoc removal reaction was performed at 30 ℃ twice, 5min for the first time and 10min for the second time.
(5) Kaiser reagent preparation and detection of Fmoc protecting group removal
The Kaiser reagent is a reagent for detecting amino groups, and mainly consists of 2 reagents. The preparation method of the two reagents is as follows: reagent 1:1g of ninhydrin was dissolved in 20mL of absolute ethanol. Reagent 2:16g of phenol was dissolved in 4mL of absolute ethanol.
After the first Fmoc removal reaction, we performed a Kaiser test. The Kaiser test procedure was: a small amount of Rink-Amide AM resin was taken from a sample application capillary in a 200. Mu.L centrifuge tube. Then, 1 drop of ninhydrin solution and 1 drop of phenol solution were added to the centrifuge tube in this order. After the solution was mixed well, it was put into a beaker and heated. Heating for 1-2 min, taking out the centrifuge tube after heating, and observing the color of the resin in the centrifuge tube. If the color of the resin changed, it was confirmed that the Fmoc protecting group on the resin had been removed.
(6) Activation of the first Fmoc-amino acid
3eq of Fmoc-Gln-OH and 6eq of Oxyma racemization inhibitor were weighed into a 5mL centrifuge tube, then neat DMF was added to dissolve the solid powder, and ultrasonic cleaning was used to facilitate dissolution of the solid powder. 6eq of DIC compound were added to the dissolved solution and shaken up. Then, activating for 3min-5min under the condition of normal temperature. The activated solution is added into the resin with the Fmoc protecting group removed, and the synthesis tube is put into a constant temperature shaking table (50 ℃,120 rpm) for reaction. The reaction is carried out twice, the first time is 15min-20min, and the second time is 20min-30min. Two reactions ensure the condensation reaction yield.
(7) Condensation of amino acids by Kaiser reagent
After the first amino acid condensation reaction is completed, we need Kaiser reagent test to detect the first condensation reaction of amino acids. Kaiser test procedure was as described in (5). Finally, the color of the resin is observed, and if the color of the resin becomes colorless, the amino acid condensation reaction is successful.
(8) Extension of polypeptide sequences
The polypeptide chain elongation was carried out by repeating the following synthetic procedure according to the above synthetic procedure: removing Fmoc protecting groups by Kaiser detection, condensing amino acids by Kaiser detection and removing Fmoc protecting groups. Fmoc-Gln-OH, fmoc-Arg-OH, fmoc-Lys-OH, fmoc-Ile-OH, fmoc-Trp-OH, fmoc-Ser-OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Ala-OH, fmoc-Pro-OH, fmoc-Leu-OH, fmoc-Gly-OH, fmoc-Thr-OH, fmoc-Leu-OH, fmoc-Val-OH, fmoc-Lys-OH, fmoc-Leu-OH, fmoc-Val-OH, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Ile-OH and Fmoc-Gly-OH are sequentially coupled by adopting Fmoc solid-phase polypeptide synthesis technology to obtain the Melite polypeptide chain-containing resin.
Arginine (Arg) is unstable at high temperature, so arginine undergoes a condensation reaction at 30 ℃. According to Fmoc-arginine: HCTU: DIPEA =3eq:2.8eq: the amino acids were condensed at a molar ratio of 6 eq. The arginine two-condensation method comprises the following specific steps: weighed Fmoc-arginine and HCTU were placed in a 5mL centrifuge tube, 4mL of DMF solution was added to the tube, and an ultrasonic cleaner ultrasonically assisted the dissolution of the solid. Then, DIPEA solution was added quickly and the centrifuge tube was immediately shaken. After the DIPEA addition, the activated solution was added to the Fmoc-removed synthesis tube. Finally, the synthesis tube was placed in a constant temperature shaker (30 ℃,120 rpm) for reaction. The reaction is carried out twice, the first time is 25min-30min, and the second time is 30min-35min. Two reactions ensure the arginine condensation reaction yield.
(9) Synthesis of linkers
After the coupling of the Melittin polypeptide chains is completed, the Fmoc group of the last amino acid Fmoc-Gly-OH on the resin is removed. Then, four amino acids in the linker were coupled with Melitin polypeptide chain in sequence according to the conventional method for Fmoc solid-phase polypeptide synthesis, fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Phe-OH, fmoc-Gly-OH.
Example 2: the coupling of methotrexate and the polypeptide chain Melittin and the cleavage of peptides thereof.
Conjugation of methotrexate to the polypeptide chain Melittin. The Fmoc protecting group of the last amino acid was removed and then condensation reaction of MTX with amino acid was performed. The method comprises the following specific steps: 10eq methotrexate and 2eq HOBt were weighed, dissolved in DMF and 2eq DIC was added for 5min activation. Then the reaction solution was added to a polypeptide synthesis tube, and the reaction was carried out twice at 30 ℃ for 1.5 hours each time.
And (5) cutting the peptide. TFA was prepared beforehand: TIPS: phenol: h 2 5mL of lysate with O = 88. Filtering, collecting filtrate with three-neck flask, and adding high purity N 2 The filtrate was concentrated to 4mL by bubbling. 40mL of precooled anhydrous ether was added to the three-necked flask and centrifuged to obtain the crude peptide. Finally, acetonitrile/water is used for dissolving the crude peptide, the crude peptide is frozen in a refrigerator at the temperature of minus 80 ℃, and a freeze dryer is used for freeze drying the frozen crude peptide to obtain the polypeptide coupling medicament A, wherein the crude product of the polypeptide coupling medicament A is 101mg. The polypeptide coupled drug A has the following structure:
101mg of the crude product of the polypeptide coupling medicament A is obtained by synthesis, and the crude product is separated and purified by semi-preparative RP-HPLC and dried by a freeze dryer to obtain the pure solid polypeptide product of the polypeptide coupling medicament A.
Example 3: synthesizing crude Melitin, MTX-GFLG-Melitin and MTX-GFLG-Melitin, and analyzing, separating and purifying Melitin and MTX-GFLG-Melitin.
The crude peptide sample obtained was added with a small amount of acetonitrile, and then added to deionized water to dissolve the crude peptide sample. And (3) carrying out ultrasonic treatment on the MTX-GFLG-Melitin crude peptide sample by using an ultrasonic cleaner to promote the dissolution of the crude peptide sample. The resulting crude peptide solution was used for subsequent analysis and separation, purification. Analysis of the crude peptide was done using analytical RP-HPLC and isolation, purification of the crude peptide was done using semi-preparative RP-HPLC.
Analytical RP-HPLC using a C18 reverse phase column (4.6X 250mm, 5-. Mu.m particle size) at a flow rate of 1mL/min. Semi-preparative RP-HPLC used a C18 reverse phase column (10X 250mm, 10-. Mu.m particle size) at a flow rate of 4mL/min. We monitored the entire analysis and purification process using two wavelengths, 214nm and 254 nm. In the analytical reverse phase high-performance liquid phase and the semi-preparative reverse phase high-performance liquid phase, the gradient of acetonitrile solution (containing 0.1 percent TFA) used in the analysis, separation and purification of the polypeptide Melitin is 20 to 30 to 2 to 30 to 70 to 30min, and the gradient of acetonitrile solution (containing 0.1 percent TFA) used in the analysis, separation and purification of MTX-GFLG-Melitin is 30 to 40 to 2 to 40 to 80 to 30min.
FIGS. 4 and 5 are analytical reversed-phase HPLC analysis spectra of Melittin and MTX-GFLG-Melittin crude and purified peptides, respectively. As is obvious from the figure, the impurities contained in the purified Melitin and MTX-GFLF-Melitin are obviously reduced, and the samples with higher purity are obtained. The purity of the sample required in the anti-tumor cell assay is greater than 95%.
The resulting solution was frozen in a freezer at-80 ℃ overnight. Then, drying by using a freeze dryer to obtain pure Melittin and MTX-GFLG-Melittin in solid forms.
The molecular weight of MTX-GFLG-Melitin was determined by electrospray tandem mass spectrometry (ESI-MS) which was recorded on a LTQ Orbitrap XL spectrometer (Thermo Scientific, USA).
FIGS. 2 and 3 are mass spectrometry (ESI-MS) analysis spectra of pure Melitin and MTX-GFLG-Melitin, respectively. Determination of molecular weight of Melitin ([ M +3H ]] 3+ = 949.6) is 2845.8Da, the theoretical molecular weight of Melittin is 2846.5Da, the determined molecular weight is consistent with the theoretical molecular weight, and this shows the correctness of the structure of the synthesized Melittin polypeptide. Determination of molecular weight of MTX-GFLF-Melitin ([ M + 5H)] 5+ = 732.44) is 3657.2Da, the theoretical molecular weight of MTX-GFLG-Melitin is 3657.4Da, and the determined molecular weight is consistent with the theoretical molecular weight, which shows the correctness of the synthesized MTX-GFLG-Melitin structure.
Example 4: MTX-GFLG-Melitin anchorage-resistant tumor cell test.
(1) HepG2, hepa1-6 and ES-2 adherent tumor cells were treated with 10 4 Cells/well were seeded in 96-well plates at 100. Mu.L/well and incubated overnight for 24h.
(2) To a 96-well plate, 50. Mu.L of DMEM complete medium containing MTX-GFLG-Melitin at concentrations of 0.3, 0.9, 3, 9, 30 and 90. Mu.M was added and incubated in a cell incubator for four hours.
(3) mu.L of MTT solution containing 5mg/L was added and incubated for 4h in a cell incubator.
(4) The cell culture medium in the 96-well plate was aspirated, 150. Mu.L of DMSO was added to each well, and the cells were incubated in an incubator for 1 hour.
(5) The 96-well plate was shaken and the absorbance values were recorded at 492 nm.
(6) The control group was not dosed with drug, and the zero-adjusted group was dosed with medium without cells, which was identical to the experimental group in operation.
(7) Anti-adherent tumor cell activity = (a 492nm control-a 492nm experiment)/(a 492nm control-a 492nm zeroing).
The anti-adherent tumor cell activity of MTX-GFLG-Melittin is calculated, the activity data are expressed as mean + -SEM, the cell experiment is repeated three times (n = 3), and SPSS software counts the activity data, and the statistical results are shown in Table 1.
Example 5: MTX anti-adherent tumor cell assay.
(1) HepG2, hepa1-6 and ES-2 adherent tumor cells were treated with 10 4 Cells/well were seeded in 96-well plates at 100. Mu.L/well and incubated overnight for 24h.
(2) To a 96-well plate, 50. Mu.L of DMEM complete medium containing MTX at concentrations of 3, 9, 30, 90, 30 and 900. Mu.M was added and incubated for four hours in a cell incubator.
(3) mu.L of MTT solution containing 5mg/L was added and incubated in a cell incubator for 4h.
(4) The cell culture medium in the 96-well plate was aspirated, 150. Mu.L of DMSO was added to each well, and the cells were incubated in an incubator for 1 hour.
(5) The 96-well plate was shaken and absorbance values were recorded at 492 nm.
(6) The control group was not dosed with drug, and the zero-adjusted group was dosed with medium without cells, which was identical to the experimental group in operation.
(7) Anti-adherent tumor cell activity = (a 492nm control-a 492nm experiment)/(a 492nm control-a 492nm zeroing).
The anti-adherent tumor cell activity of MTX was calculated and the activity data were expressed as mean ± SEM, the cell experiment was repeated three times (n = 3), the SPSS software counted the activity data and the results are shown in table 1.
Table 1: antitumor activity of different drugs on three adherent tumor cells
The statistics of MTX and MTX-GFLG anti-adherent tumor cells (HepG 2, hepa1-6 and ES-2) are shown in Table 1, FIG. 6 and FIG. 8. IC for three adherent tumor cells 50 Are all larger than 300. Mu.M, which indicates that the anti-adherent tumor effect of MTX is less than ideal. For MTX-GFLG-Melitin drugs, IC thereof 50 All are less than 5 mu M, which indicates that MTX-GFLG-Melitin has stronger anti-adherent tumor activity. Compared with MTX alone, the effect of MTX-GFLG-Melitin on anti-adherent tumor cells is improved by dozens of times. The results demonstrate that Melittin as a lead compound can increase the anti-adherent tumor cell activity of MTX.
Example 6: MTX-GFLG-Melittin anti-suspension tumor cell test.
(1) Suspending U937, daudi, COC1 and A20 tumor cells in 10 4 Cells/well were seeded in 96-well plates at 100. Mu.L/well and incubated overnight for 24h.
(2) To a 96-well plate, 50. Mu.L of DMEM complete medium containing MTX-GFLG-Melitin at concentrations of 0.3, 0.9, 3, 9, 30 and 90. Mu.M was added, and incubated in a cell incubator for four hours.
(3) After the incubation, 15. Mu.L of CCK8 working solution was added to each well, the cells were incubated in a cell incubator for 3 hours, and the absorbance was measured using a microplate reader at 450 nm.
(4) Anti-suspension tumor cell activity = (a 450nm control-a 450nm experiment)/(a 450nm control-a 450nm zeroing).
(5) The anti-adherent tumor cell activity of MTX-GFLG-Melitin was calculated, the activity data are expressed as mean + -SEM, the cell experiment was repeated three times (n = 3), the SPSS software counted the activity data, and the statistical results are shown in Table 2.
Example 7: MTX anti-suspension tumor cell assay.
(1) Suspending U937, daudi, COC1 and A20 tumor cells in 10 4 Cells/well were seeded in 96-well plates at 100. Mu.L/well and incubated overnight for 24h.
(2) To a 96-well plate, 50. Mu.L of DMEM complete medium containing MTX concentrations of 3, 9, 30, 90, 300 and 900. Mu.M was added and incubated in a cell incubator for four hours.
(3) After the incubation, 15. Mu.L of CCK8 working solution was added to each well, the cells were incubated in a cell incubator for 3 hours, and the absorbance was measured using a microplate reader at 450 nm.
(4) Anti-suspension tumor cell activity = (a 450nm control-a 450nm experiment)/(a 450nm control-a 450nm zeroing).
(5) The anti-adherent tumor cell activity of MTX was calculated and the activity data are expressed as mean ± SEM, the cell experiment was repeated three times (n = 3), the SPSS software counted the activity data and the results are shown in table 2.
Table 2: antitumor activity of different drugs on four kinds of suspended tumor cells
Statistics of MTX and MTX-GFLG-Melitin anti-suspension tumor cells (U937, daudi, COC1 and A20) are shown in Table 2, FIG. 7 and FIG. 9. For MXT alone, IC thereof 50 IC of more than 300 mu M and MTX-GFLG-Melitin 50 Are all lower than 3 mu M, compared with MTX-GFLG-Melitin, the anti-suspension tumor cell effect is improved by at least 100 times. This shows that the Melittin polypeptide as a lead compound can effectively improve the anti-suspension tumor effect of MTX.
The result of the antitumor activity shows that MTX-GFLG-Melittin has strong killing effect on various tumor cells. This confirms the broad spectrum of MTX-GFLG-Melitin antitumor properties. Most importantly, compared with the single MTX anti-tumor effect, the MTX-GFLG-Melitin has a great improvement on the killing effect on seven tumor cells, and thus, revelation and thinking are provided for the development of subsequent PDC anti-tumor drugs.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all the modifications made by the present invention or directly or indirectly applied to the related technical field are included in the scope of the present invention.
Claims (6)
1. The preparation of polypeptide coupled medicine and its application as antitumor medicine features that Melittin and MTX cytotoxic medicine are connected via connector GFLG.
2. The polypeptide-conjugated drug of claim 1, wherein the polypeptide-conjugated drug has a structure represented by formula a:
3. the Peptide of claim 2, wherein said Peptide is Melittin, and the amino acid sequence thereof is: gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln.
4. The polypeptide-conjugated drug of claim 2, wherein the cytotoxic drug is Methotrexate (MTX), a tumor chemotherapeutic drug.
5. The polypeptide-conjugated drug of claim 2, wherein the linker is glycine-phenylalanine-leucine-glycine, gly-Phe-Leu-Gly.
6. The use of claim 1 as an anti-tumor agent, wherein the polypeptide conjugate is used in the treatment of cancers including liver cancer, ovarian cancer and lymph cancer.
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