A targeted drug for treating malignant glioma, preparation method and application thereof

Abstract

The present invention belongs to the technical field of malignant glioma, and particularly relates to a targeted drug, preparation method and application for treating malignant glioma. The present invention prepares a chemical cross-linked complex of CTX and CLM, which has the potential to treat malignant glioma in a targeted manner. CTX acts as a targeted warhead to guide the complex and bind to the chloride ion channel specifically expressed by malignant glioma, and then causes the complex to enter the cell through endocytosis, thereby introducing CLM into the cell to kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of malignant glioma, and in particular relates to a targeted drug for treating malignant glioma, a preparation method and an application thereof. Background Art

[0002] Malignant gliomas, tumors originating from the neuroepithelium are collectively referred to as gliomas (gliomas), accounting for 40-50% of brain tumors and are the most common intracranial malignant tumors. The incidence of primary central nervous system malignant tumors worldwide is about 6.3 cases per 100,000 people per year, of which 3.7 are male patients and 2.6 are female patients. Although the incidence of primary central nervous system tumors is not high, its mortality rate is very high. According to statistics from the Ministry of Health, malignant tumors have become the number one cause of death in my country. Malignant gliomas are also the most common central nervous system malignant tumors in adults, and they are also one of the most difficult malignant tumors to treat. At present, the most common and traditional treatments for gliomas at home and abroad are mainly surgery and radiotherapy and chemotherapy, but due to the location of the primary lesion and the blood-brain barrier, the treatment effect is not ideal. Moreover, there are currently no drugs specifically for malignant gliomas, which makes the treatment process of malignant gliomas unsafe and has large side effects.

[0003] Studies have shown that tumor tissues of patients with malignant gliomas can express tumor-specific chloride ion channels (GCCs), which are specifically sensitive to chlorotoxin (CTX) isolated from Israeli golden scorpions. Therefore, this type of chloride channel can be used as a specific marker or target for glioma cells to treat this type of glioma.

[0004] Caliciamicins (CLM) are enediyne antitumor antibiotics isolated from the fermentation broth of the rare actinomycete Micromonospora. CLM is a potent cytotoxic agent that causes DNA double-strand breaks. Caliciamicins inhibit DNA synthesis. Caliciamicins bind to DNA in both healthy and tumor cells, leading to division and cell death. Summary of the invention

[0005] The present invention provides a targeted drug for treating malignant glioma, a preparation method and an application thereof, which solves the problem that there is no drug specifically targeting malignant glioma at present.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A targeted drug for treating malignant glioma, which is a chemically cross-linked complex of calicheamicin and chlorotoxin.

[0008] A method for preparing a targeted drug for treating malignant glioma, comprising the following steps:

[0009] S1. Construct the pGEX-4T-1-His-CTX prokaryotic expression vector to obtain the 6×His-CTX gene;

[0010] S2, prokaryotic expression and purification of GST-6×His-CTX;

[0011] S3, digestion of GST-6×His-CTX and purification of 6×His-CTX;

[0012] S4, in vitro folding and purification of CTX;

[0013] SPDP chemical labeling and purification of S5 and CTX;

[0014] S6, terminal thiol reduction and purification of CLM;

[0015] Chemical cross-linking and purification of S7, CTX and CLM.

[0016] Optionally, in step S1, a DNA primer pair of a pGEX-4T-1-His-CTX prokaryotic expression vector is constructed, the upstream sequence of the primer pair is SEQ ID NO: 1 (5′-CGCGGATCCATGCATCACCATCACCA-3′), the downstream sequence of the primer pair is SEQ ID NO: 2 (5′-CCGCTCGAGTTAACGGCACAGACACT-3′), and the DNA primer pair is used to obtain a 6×His-CTX gene by PCR;

[0017] In step S1, the 6×His-CTX gene amplified by PCR was detected by electrophoresis and gel purified, and then treated with restriction endonucleases BamHⅠ and XhoⅠ at 37°C overnight. After gel purification again, it was connected with the pGEX-4T-1 vector treated in the same way at 16°C overnight. The connection product was transformed into Escherichia coli DH12S by heat shock, spread on a plate with ampicillin resistance, and cultured at 37°C overnight. The next day, a single clone was picked in LB medium, and the bacteria were shaken at 37°C and 200 rpm overnight, and finally the plasmid was extracted.

[0018] Optionally, in step S2, the constructed pGEX-4T-1-His-CTX prokaryotic expression vector is heat-shock transformed into Escherichia coli BL21, spread on a plate with ampicillin resistance, and cultured at 37°C overnight. The next day, a single clone is picked in LB medium and cultured at 37°C and 200 rpm overnight. The next day, the bacterial solution is inoculated into TB medium at a ratio of 1:100 and cultured at 37°C and 200 rpm until the OD 600=2.5, add IPTG to a final concentration of 1 mM, then culture at 16°C, 170 rpm for 20 hours, collect the cells by centrifugation, resuspend in PBS buffer, and store at -20°C for later use;

[0019] Add PMSF, a protease inhibitor, to the resuspended cells, break the cells under pressure, and then add DNase I for digestion for 0.5 to 1 hour. After DNase I digestion is completed, add sodium sulfite with a final concentration of 0.2 M / L, react at room temperature for 0.5 hours, then add sodium tetrathionate with a final concentration of 0.15 M / L, react at room temperature for 1.5 hours, centrifuge, and filter the supernatant with a 0.42 μm membrane;

[0020] Wash Ni with loading buffer 2+ -Chelating Sepharose Fast Flow affinity column to the baseline, pass the above supernatant through the column, continue to rinse the affinity column with loading buffer to the baseline after loading, and then elute with PBS containing 30mM / L imidazole. After the baseline is flushed, elute with PBS containing 100mM / L imidazole and collect the elution peak, and finally detect the eluted sample with Tricine-SDS-PAGE.

[0021] Optionally, in step S3, the elution peak collected in step S2 is first concentrated into a volume of 5 to 10 mL by ultrafiltration, then enterokinase is added for enzyme digestion at 25°C overnight, the enzyme digestion is detected by Tricine SDS-PAGE, centrifuged and loaded onto a pre-loaded G-50 molecular sieve, and the elution peak is collected.

[0022] Optionally, in step S4, arginine is added to the eluted sample containing CTX to a final concentration of 0.5 M / L, SDS is added to a final concentration of 1 mM / L, and the pH is adjusted to 8.5, and the sample is pre-cooled in an ice bath to 0°C for about 0.5 to 1 hour. After pre-cooling, GSH and GSSG are added to the eluate successively to 1 mM / L, mixed, and folded in an ice bath for 2 hours. The folded product is adjusted to acid with TFA, centrifuged, and purified by C18 reverse phase column HPLC.

[0023] Optionally, in step S5, in a reaction system of 50 mM sodium phosphate, 150 mM NaCl, pH=8.0, recombinant CTX and SPDP are reacted at a molar ratio of 1:1 at room temperature for 1 hour, then the reaction is adjusted with TFA and purified by HPLC.

[0024] Optionally, in step S6, in a reaction system of: 50 mM sodium phosphate, 150 mM NaCl, pH=8.0, 500 mM DTT, DMSO and water in a ratio of 1:1, react at room temperature for 1 hour, adjust the acidity with TFA, and purify by HPLC.

[0025] Optionally, in step S7, the purified SPDP-labeled CTX and 2-iminothiolane hydrochloride-labeled calicheamicin are reacted at a molar ratio of about 1:1 in a reaction system: 50 mM sodium phosphate, 150 mM NaCl, pH=8.0, DMSO and water in a ratio of 1:1 at room temperature for 0.5 hours, acidified with TFA, and purified by HPLC.

[0026] Application of chemically cross-linked complexes of calicheamicin and chlorotoxin in the treatment of malignant gliomas.

[0027] After adopting the above technical solution, the beneficial effects of the present invention are:

[0028] The present invention prepares a chemical cross-linked complex of CTX and CLM (CTX-CLM), which has the potential to target and treat malignant gliomas. CTX acts as a targeted warhead to guide the complex and bind to the chloride ion channel specifically expressed by malignant gliomas, and then causes the complex to enter the cell through endocytosis, thereby introducing CLM into the cell to kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 is the pGEX-4T-1 vector map;

[0031] Figure 2 is a schematic diagram of SPDP chemical labeling of CTX;

[0032] Figure 3 is a schematic diagram of the reduction of the terminal thiol group of CLM;

[0033] Figure 4 is a schematic diagram of chemical cross-linking of CTX and CLM;

[0034] Figure 5 It is a schematic diagram of electrophoresis detection of CTX and CLM before and after cross-linking and reduction of the cross-linked product;

[0035] Figure 6 This is a schematic diagram of the CCK8 cell survival activity detection;

[0036] Figure 7 The growth status of cells after being treated with CTX-CLM and a mixture of CTX and CLM at the same molar concentration for the same period of time. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The embodiments of the present application disclose a targeted drug for treating malignant glioma, a preparation method and an application thereof.

[0039] Example

[0040] according to Figures 1 to 7 As shown, a targeted drug for treating malignant glioma, its preparation method and application.

[0041] 1. Construction of pGEX-4T-1-His-CTX prokaryotic expression vector

[0042] 1. Construct a DNA primer pair for the pGEX-4T-1-His-CTX prokaryotic expression vector, wherein the upstream sequence of the primer pair is SEQ ID NO: 1 (5′-CGCGGATCCATGCATCACCATCACCA-3′), and the downstream sequence of the primer pair is SEQ ID NO: 2 (5′-CCGCTCGAGTTAACGGCACAGACACT-3′). The DNA primer pair was used to obtain the 6×His-CTX gene by PCR;

[0043] 2. PCR reaction system

[0044] Table 1 PCR reaction system

[0045]

[0046]

[0047] 3. PCR reaction procedure

[0048] Table 2 PCR reaction conditions

[0049] project temperature time Pre-denaturation 94℃ 3min transsexual 94℃ 30s Annealing extension 58℃ 30s Annealing extension 72℃ 30s Melting curve collection 72℃ 90s Melting curve collection 4℃ save

[0050] The 6×His-CTX gene amplified by PCR was detected by electrophoresis and gel purified, and then treated with restriction endonucleases BamHI and XhoⅠ at 37℃ overnight. After gel purification again, it was combined with the pGEX-4T-1 vector (see Figure 1) and ligated overnight at 16°C. The ligation product was transformed into E. coli DH12S by heat shock, spread on a plate with ampicillin resistance, and cultured at 37°C overnight. The next day, a single clone was picked in LB medium and incubated at 37°C, 200 rpm, overnight, and finally the plasmid was extracted.

[0051] 4. Double enzyme digestion system:

[0052] Table 3 Cloning fragment restriction enzyme digestion system

[0053]

[0054]

[0055] Table 4 Cloning vector restriction enzyme system

[0056] Components Volume (μL) pGEX-4T-1 (100 ng / μL) 10 10×buffer 4 4 Bam HI 1 XhoⅠ 1 <![CDATA[ddH2O]]> 24 Total volume 40

[0057] Table 5 Connection system

[0058] Components Volume (μL) pGEX-4T-1 (50 ng / μL) after enzyme digestion and purification 7 6×His-CTX (20ng / μL) after enzyme digestion and purification 2 <![CDATA[10×T4 DNA Ligation Buffer]]> 1 <![CDATA[10× T4 DNA Ligase]]> 1 Total volume 10

[0059] 2. Prokaryotic expression of GST-6×His-CTX

[0060] 1. Heat-shock transform the constructed pGEX-4T-1-His-CTX prokaryotic expression vector into Escherichia coli BL21 (DE3), apply it to a plate with ampicillin resistance, and culture it at 37°C overnight. The next day, pick a single clone in 50 mL of LB medium and shake it at 37°C and 200 rpm overnight. The next day, inoculate the bacterial solution in TB medium at a ratio of 1:100 and shake it at 37°C and 200 rpm until OD 600 =2.5, add IPTG to a final concentration of 1mM, then culture at 16℃, 170rpm for 20 hours, centrifuge (8000rpm, 5min) to collect the cells, resuspend the cells in PBS (50mM phosphate, 0.1mMNaCl, pH=7.5), and store at -20℃ for later use.

[0061] 2. Purification of GST-6×His-CTX

[0062] Add PMSF, a protease inhibitor, to the resuspended cells, break the cells by pressure, and then add DNase I for digestion for 0.5 to 1 hour. After DNase I digestion is completed, add sodium sulfite with a final concentration of 0.2 M / L, react at room temperature for 0.5 hours, then add sodium tetrathionate with a final concentration of 0.15 M / L, react at room temperature for 1.5 hours, centrifuge (18000 rpm, 30 min), and filter the supernatant with a 0.42 μm membrane.

[0063] Wash Ni with loading buffer (PBS) 2+-Chelating Sepharose Fast Flow affinity column to the baseline, and pass the above supernatant through the column. After the sample is loaded, continue to rinse the affinity column with loading buffer (PBS) to the baseline, then elute with PBS containing 30mM / L imidazole, and after the baseline is flushed, elute with PBS containing 100mM / L imidazole and collect the elution peak. Finally, use Tricine-SDS-PAGE to detect the eluted sample.

[0064] 3. Enzyme digestion of GST-6×His-CTX and purification of 6×His-CTX

[0065] The collected elution peak was first ultrafiltered (10 kDa ultrafiltration tube, 6000 g centrifugation), concentrated to a volume of 5-10 mL, and then added with enterokinase (2500:1) for enzymatic digestion at 25°C overnight. Enzymatic digestion was detected by Tricine SDS-PAGE, centrifuged and loaded on pre-loaded G-50 molecular sieves (eluent: 50 mM Tris, pH = 8.5), and the elution peak was collected.

[0066] 4. In vitro folding and purification of CTX

[0067] Arginine was added to the eluted sample containing CTX to a final concentration of 0.5 M / L, sodium dodecyl sulfate was added to a final concentration of 1 mM / L, and the pH was adjusted to 8.5, and ice-bathed to 0°C for about 0.5 to 1 hour. After precooling, GSH (reduced glutathione) and GSSG (oxidized glutathione) were added to the eluate to 1 mM / L, mixed, and folded for 2 hours in an ice bath. The folded product was adjusted with TFA, centrifuged, and purified by C18 reverse phase HPLC.

[0068] 5. SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester) chemical labeling and purification of CTX

[0069] In the reaction system (50 mM sodium phosphate, 150 mM NaCl, pH = 8.0), recombinant CTX and SPDP were reacted at a molar ratio of 1:1 at room temperature for 1 hour, then the mixture was adjusted with TFA (trifluoroacetic acid) and purified by HPLC. Figure 2 The process of SPDP chemical labeling of CTX can be seen in Figure 2. Figure 2 In the process, SPDP first reacts with the amino group of a protein molecule via its amino group, and SPDP introduces a pyridyldisulfide group into the protein. Then, the pyridyldisulfide group, which is an activated group, easily forms a disulfide bond with a sulfhydryl-containing molecule to form a heterodimer.

[0070] 6. Terminal thiol reduction and purification of CLM

[0071] In the reaction system (50mM sodium phosphate, 150mM NaCl, pH=8.0, 500mM DTT (dithiothreitol), dimethyl sulfoxide and water ratio 1:1), react at room temperature for 1 hour, adjust the acidity with TFA, and purify by HPLC. Figure 3 The reaction principle of terminal thiol reduction of CLM can be seen in FIG. Figure 3 DTT has strong reducing properties and can open the disulfide bonds of the molecules and reduce them to thiol groups. This reaction uses the chemical property of DTT to reduce the terminal disulfide bonds of CLM to thiol groups so that it can subsequently undergo a cross-linking reaction with SPDP-labeled CTX.

[0072] 7. Chemical cross-linking and purification of CTX and CLM

[0073] The purified SPDP-labeled CTX and 2-iminothiolane hydrochloride-labeled calicheamicin were reacted at a molar ratio of about 1:1 in a reaction system (50 mM sodium phosphate, 150 mM NaCl, pH=8.0, dimethyl sulfoxide and water ratio 1:1) at room temperature for 0.5 hours, acidified with TFA, and purified by HPLC. Figure 4 The reaction principle of chemical cross-linking between CTX and CLM can be seen in the figure. Figure 4 In the process, after CTX labeled with SPDP is reduced with DTT, CLM is placed in an alkaline environment. The terminal thiol group of CLM will exchange with the activated pyridyldisulfide group in SPDP, thereby realizing the cross-linking process between CTX and CLM.

[0074] 8. Tricine-SDS-PAGE electrophoresis detection of CTX and CLM cross-linking

[0075] The cross-linking between CTX and CLM was detected by Tricine-SDS-PAGE electrophoresis. Figure 5 It can be seen that the molecular weight increases significantly after chemical cross-linking, and the molecular weight of the cross-linked product returns to the molecular weight before cross-linking after DTT chemical reduction, indicating that the CTX-CLM cross-linked complex was successfully designed and prepared.

[0076] 9. Detection of the anti-malignant glioma activity of CTX-CLM cross-linked complex

[0077] Figure 6In the experiment, CCK8 detected the growth inhibition of malignant glioma SHG-44 by CTX-CLM cross-linked complex and CTX and CLM mixture (B represents the cell growth inhibition by CTX-CLM cross-linked complex, and E represents the cell growth inhibition by CTX and CLM mixture). Through CCK8 cell survival activity detection, it was found that CTX-CLM cross-linked complex inhibited the growth of malignant glioma SHG-44 more significantly than CTX and CLM mixture, proving that CTX in the cross-linked complex had the effect of targeted adsorption of CML to promote its entry into malignant glioma.

[0078] Figure 7 The concentration of CTX and CLM mixture was 1×10 -5.5 mM / L growth of malignant glioma cells SHG-44. Figure 7 It can be seen that at the same concentration, the number of surviving cells treated with CTX-CLM crosslinkers is small, indicating that at the same molar concentration, the crosslinker can introduce more CLM into cells to cause their death, thereby increasing the IC50 (half inhibitory concentration) of CLM on CTX targeted cell growth, which also improves drug utilization, which is beneficial to increase the local concentration of the drug and reduce systemic side effects of the drug.

[0079] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a targeted drug for treating malignant glioma, characterized in that: The targeted drug is a chemically cross-linked complex of calicheamicin and chlorotoxin; the preparation method of the targeted drug specifically comprises the following steps: S1. Construct the pGEX-4T-1-His-CTX prokaryotic expression vector to obtain the 6×His-CTX gene; S2, prokaryotic expression and purification of GST-6×His-CTX; S3, digestion of GST-6×His-CTX and purification of 6×His-CTX; S4, in vitro folding and purification of CTX; SPDP chemical labeling and purification of S5 and CTX; S6, terminal thiol reduction and purification of CLM; in step S6, in a reaction system of: 50 mM sodium phosphate, 150 mM NaCl, pH = 8.0, 500 mM DTT, DMSO and water in a ratio of 1:1, react at room temperature, adjust the acidity with TFA, and purify by HPLC; S7, chemical crosslinking and purification of CTX and CLM; in step S7, the purified SPDP-labeled CTX and the DTT-reduced calicheamicin are reacted in a reaction system of 50 mM sodium phosphate, 150 mM NaCl, pH=8.0, and a DMSO to water ratio of 1:1 at a molar ratio of 1:1, reacted at room temperature, acidified with TFA, and purified by HPLC; The process of DTT reduction of CLM is as follows: 。 2. The method for preparing a targeted drug for treating malignant glioma according to claim 1, characterized in that: In step S1, a DNA primer pair of the pGEX-4T-1-His-CTX prokaryotic expression vector is constructed, the upstream sequence of the primer pair is SEQ ID NO: 1, the downstream sequence of the primer pair is SEQ ID NO: 2, and the DNA primer pair is used to obtain the 6×His-CTX gene by PCR; In step S1, the 6×His-CTX gene amplified by PCR was detected by electrophoresis and gel purified, and then treated with restriction endonucleases BamHⅠ and XhoⅠ overnight. After gel purification again, it was ligated with the pGEX-4T-1 vector that had been treated in the same way overnight. The ligation product was transformed into Escherichia coli DH12S by heat shock, spread on a plate with ampicillin resistance, and cultured at 37°C overnight. The next day, a single clone was picked in LB medium, shaken overnight, and finally the plasmid was extracted.

3. The method for preparing a targeted drug for treating malignant glioma according to claim 2, characterized in that: In step S2, the constructed pGEX-4T-1-His-CTX prokaryotic expression vector is heat-shock transformed into Escherichia coli BL21, spread on a plate with ampicillin resistance, cultured overnight, and a single clone is picked in LB medium the next day, shaken overnight, and the bacterial solution is inoculated into TB medium the next day. After shaking culture, IPTG is added, and after shaking culture again, the bacterial cells are collected by centrifugation and resuspended in PBS; Add PMSF, a protease inhibitor, to the resuspended cells, break the cells under pressure, and then add DNaseⅠ for digestion. After the DNaseⅠ digestion is completed, add sodium sulfite, react at room temperature, then add sodium tetrathionate, react at room temperature, centrifuge, and filter the supernatant with a 0.42µm membrane. Wash Ni with loading buffer 2+ -Chelating Sepharose Fast Flow affinity column to the baseline, pass the filtered supernatant through the column, continue to rinse the affinity column with loading buffer to the baseline after loading, then elute with PBS containing imidazole, elute with PBS containing imidazole after the baseline is flushed and collect the elution peak, and finally detect the eluted sample with Tricine-SDS-PAGE.

4. The method for preparing a targeted drug for treating malignant glioma according to claim 3, characterized in that: In step S3, the elution peak collected in step S2 is first concentrated into a volume of 5 to 10 mL by ultrafiltration, then enterokinase is added, and enzyme digestion is performed overnight. The enzyme digestion is detected by Tricine SDS-PAGE, centrifuged and pre-loaded with G-50 molecular sieves, and the elution peak is collected.

5. The method for preparing a targeted drug for treating malignant glioma according to claim 4, characterized in that: In step S4, arginine and sodium dodecyl sulfate are added to the eluted sample containing CTX, the pH value is adjusted to 8.5, and the sample is pre-cooled to 0°C in an ice bath. After pre-cooling, GSH and GSSG are added to the eluate in sequence, the mixture is mixed and the folding reaction is carried out in an ice bath. The folded product is acidified with TFA, centrifuged, and purified by C18 reverse phase column HPLC.

6. The method for preparing a targeted drug for treating malignant glioma according to claim 5, characterized in that: In step S5, in a reaction system of 50 mM sodium phosphate, 150 mM NaCl, pH=8.0, recombinant CTX and SPDP were reacted at a molar ratio of 1:1 at room temperature, then acidified with TFA and purified by HPLC.

7. Use of the chemically cross-linked complex of calicheamicin and chlorotoxin prepared by the preparation method according to claim 1 in preparing drugs for treating malignant glioma.

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