CN118121602A - Application of chloroquinaldol in preparation of GPX4 protein degradation products - Google Patents

Application of chloroquinaldol in preparation of GPX4 protein degradation products Download PDF

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Publication number
CN118121602A
CN118121602A CN202410034291.XA CN202410034291A CN118121602A CN 118121602 A CN118121602 A CN 118121602A CN 202410034291 A CN202410034291 A CN 202410034291A CN 118121602 A CN118121602 A CN 118121602A
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gpx4
chloroquinaldo
iron death
chlorquinaldo
death inducer
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CN202410034291.XA
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陆翠霞
赵熙
宋佳兴
马瑜浓
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Guangxi University
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Guangxi University
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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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  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses application of chlorquinaldo in preparation of a product for degrading GPX4 protein. Experiments show that the degradation of GPX4 protein in colorectal cancer cells can be induced by using the chlorquinaldo, and the expression level of GPX4 in cancer cells can be down-regulated by enhancing the GPX4 protein lysosome pathway. The combination of chlorquinaldol and an iron death inducer Erastin can enhance the down regulation of GPX4 level and effectively increase the killing effect on tumors. Thus, chloroquinaldo can be used to degrade GPX4 protein, or in combination with iron death inducers for anti-tumor use.

Description

Application of chloroquinaldol in preparation of GPX4 protein degradation products
Technical Field
The invention relates to the technical field of medicine application, in particular to application of chloroquinaldo in preparation of a product for degrading GPX4 protein.
Background
GPX4, also known as phospholipidic glutathione peroxide peroxidase (PHGPx), is the fourth member of the selenium-containing GPX family. The protein is considered as an important regulator of iron death, has the capability of scavenging membrane lipid hydrogen peroxide products and preventing oxidative stress.
Since GPX4 has a high expression level in tumor tissue and is closely related to poor prognosis for cancer patients, targeting GPX4 is also a strategy to combat cancer. Inhibition of GPX4 activity affects GPX 4's ability to scavenge lipid peroxides, ultimately leading to the occurrence of cellular iron death.
Various inhibitors targeting GPX4 are currently found, such as RSL3, ML162, erastin, etc., however no GPX4 inhibitors have been reported to enter the stage clear of clinical research. And the drugs with the functions of inhibiting GPX4 correlation or the like are searched from the approved drugs on the market, and the potential application value of the drugs is explored, so that the drugs are an excellent way for obtaining the GPX4 inhibitor with application prospect.
The chloroquinaldo has good antibacterial effect as an antibacterial drug, but until now, no related report on the use of the chloroquinaldo for degrading the GPX4 protein of tumor cells exists.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide the application of the chloroquinaldo in preparing the products for degrading GPX4 protein.
It is another object of the present invention to provide the use of chloroquinaldo in combination with an iron death inducer in the preparation of a product for degradation of GPX4 protein.
It is a further object of the present invention to provide the use of chloroquinaldo in the preparation of iron death drug sensitizers.
The aim of the invention is achieved by the following technical scheme:
Use of chloroquinaldo in the preparation of a product for degrading GPX4 protein.
The GPX4 protein degradation induced by the chlorquinaldo can be inhibited by a lysosomal inhibitor.
The lysosomal inhibitor is preferably Bafilomycin A < 1 >.
The products include medicines, kits and the like.
The medicine comprises degradation agents and the like.
Use of chloroquinaldo in combination with an iron death inducer in the preparation of a product for degrading GPX4 protein.
The combination of the iron death inducer and the chloroquinaldo can enhance the down regulation of the GPX4 level, namely the combination of the iron death inducer and the chloroquinaldo can enhance the degradation effect of GPX4 protein.
The iron death inducer is preferably Erastin.
The products include medicines, kits and the like.
The medicine comprises degradation agents and the like.
The application of chlorquinaldo in preparing iron death drug sensitizer.
The iron death medicament is an iron death inducer; preferably an iron death inducer Erastin.
The application of the combination of the chloroquinaldo and the iron death inducer in preparing the anti-tumor drugs can be used for degrading GPX4 protein and effectively increasing the tumor killing effect of the iron death inducer.
The iron death inducer is preferably Erastin.
The tumor includes colorectal cancer and the like.
Use of chloroquinaldo in combination with an iron death inducer for the manufacture of a medicament for inhibiting proliferation and/or growth of colorectal cancer cells.
The colorectal cancer cells are preferably at least one of colorectal cancer DLD-1 cells and HCT116 cells.
The effective concentration of the chloroquinaldo is 3.75 to 60. Mu. Mol/L (preferably 10 to 50. Mu. Mol/L; more preferably 30. Mu.M), and the effective concentration of the iron death inducer is 20. Mu. Mol/L.
The medicament may also contain one or at least two pharmaceutically acceptable carriers.
The carrier is at least one of a slow release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption promoter, a surfactant and a lubricant.
The medicine can be prepared into various dosage forms by adopting a conventional method in the field, including powder, injection (powder injection), tablet, capsule, powder, granule, electuary, oral liquid, syrup and the like.
Compared with the prior art, the invention has the following advantages and effects:
1. The invention discovers that the chlorquinaldo is used for inducing the degradation of GPX4 protein in colorectal cancer cells for the first time, can down regulate the level of GPX4 in cancer cells (through a way of enhancing GPX4 protein lysosomes), and provides a new strategy for applying the chlorquinaldo to colorectal cancer treatment.
2. The invention also finds that the lysosome inhibitor (Bafilomycin A <1 >) can inhibit the degradation of GPX4 protein by the chloroquinaldo, and that the combination of the chloroquinaldo and the iron death inducer can enhance the down-regulation of GPX4 level and enhance the generation of active oxygen of cells, so that the chloroquinaldo can effectively increase the killing effect of the iron death inducer on tumors, and can be used as an iron death drug sensitizer or used in anti-tumor aspect in combination with the iron death inducer.
Drawings
FIG. 1 is a line drawing of the growth line of chloroquinaldol in inhibiting colorectal cancer cells (DLD-1, HCT 116).
Figure 2 is a graph showing the effect of chloroquinaldo in inhibiting GPX4 protein in colorectal cancer cells.
Figure 3 is a graph of GPX4 transcript levels in chloroquinaldo-regulated colorectal cancer cells.
Fig. 4 is a graph of MG132 inhibiting degradation of GPX4 by chloroquinaldo.
Figure 5 is a graph of Bafilomycin A1 inhibiting the degradation of GPX4 by chloroquinaldo.
Fig. 6 is a graph of the iron death inhibitor Fer-1 inhibiting the degradation of GPX4 by chloroquinaldo.
Fig. 7 is a graph showing the degradation of GPX4 by iron death inducer Erastin in combination with chloroquinaldo.
Fig. 8 is a graph showing the induction of ROS production by iron death inducer Erastin in combination with chlorquinaldo.
Fig. 9 is a graph showing enhanced inhibition of colorectal cancer cell growth by iron death inducer Erastin in combination with chloroquinaldo.
FIG. 10 is a graph of tissue HE staining and Ki67 staining sections of subcutaneous tumor-bearing model mice treated with chloroquinaldo.
Fig. 11 is a graph of tissue GPX4, 4-HNE histochemical section of a subcutaneous tumor-bearing model mouse after treatment with chloroquinaldo.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The experimental methods of the specific experimental conditions are not noted in the following examples, and generally follow the conventional experimental conditions. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Colorectal cancer cell lines (DLD-1, HCT116 cells) referred to in the examples of the present invention were purchased from Wohan Seville Biotechnology Co.
The chloroquinaldo referred to in the examples of the present invention was purchased from a ceramic surgery organism (catalog number: T0908).
The accession number of the GPX4 protein in the embodiment of the invention in the UniProt database is: p36969 (https:// www.uniprot.org/uniprotkb/P36969/entry).
Example 1 chloroquinaldol inhibits the growth and proliferation of colorectal cancer cells
DLD-1 and HCT116 cells were inoculated into 96-well cell culture plates, respectively, and cultured overnight at 5X 10 4 cells/well. Then, chlorquinaldo (CQD) was added at various concentrations (3.75, 7.5, 15, 30, 60. Mu.M) for 24 hours and 48 hours, respectively, and cell viability was measured using the MTT method. The experimental setup was repeated three times.
The results are shown in FIG. 1: it can be seen from the figure that chlorquinaldo can inhibit the growth and proliferation of DLD-1 and HCT116 cells.
Example 2 chloroquinaldo degrades GPX4 protein in colorectal cancer cells
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 5 cells/well. Then, chlorquinaldo (CQD) was added at various concentrations (final concentrations of 10, 20, 30, 40, 50. Mu.M) and treated for 24 hours, and samples were collected, and GPX4 expression levels in the cells were detected by Westernblot using cells without chlorquinaldo as a control (Conrtol). The experimental setup was repeated three times.
The results are shown in FIG. 2: it can be seen from the figure that as the concentration of chloroquinaldo increases, chloroquinaldo can degrade GPX4 protein in DLD-1, HCT116 cells.
Example 3 chloroquinaldo regulates GPX4 transcript levels in colorectal cancer cells
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 5 cells/well. Then, samples were collected by subjecting the samples to 24 hours with or without the addition of 30. Mu.M final chlorquinate, and the total RNA of the cells was extracted by TransZol method and subjected to fluorescent quantitative PCR assay to detect the transcription level of GPX4 gene. The experimental setup was repeated three times. Among them, the following ：GPX4-F:5′-GAGGCAAGACCGAAGTAAACTAC-3′;GPX4-R:5′-CCGAACTGGTTAC ACGGGAA;GAPDH-F:5′-CCAGAACATCATCCCTGCCTCTACT-3′;GAPDH-R:5′-GGTTTTTCTAGACGGCAGGTCAGGT-3′; primer sequences were used for PCR detection and were all submitted for Shanghai industrial synthesis. The results are shown in FIG. 3.
Example 4MG132 inhibits degradation of GPX4 by chloroquinaldo
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 4 cells/well. Then, the cells were treated with or without Chloroquinaldo (CQD) at a final concentration of 30. Mu.M for 24 hours, and MG132 (proteasome inhibitor, available from the pottery-type organism, catalog number: T2154) at a final concentration of 10. Mu.M was added or not added 8 hours before collection, respectively, and the expression level of GPX4 in the cells was detected by Westernblot. The experimental setup was repeated three times.
The results are shown in fig. 4, which demonstrate that proteasome inhibitors are not effective in inhibiting GPX4 protein degradation induced by chloroquinaldo.
EXAMPLE 5Bafilomycin A1 inhibits the degradation of GPX4 by chloroquinaldo
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 5 cells/well. Then, chlorquinaldo (CQD) treatment was performed with or without adding 30. Mu.M final concentration for 24 hours, and then, bafilomycin A1 (Bavlosin A1, abbreviated as BafA1, a lysosome inhibitor, available from selleck, catalog number S1413) with or without adding 0.2. Mu.M final concentration was performed 6 hours before sample collection, and the expression level of GPX4 in the cells was detected by Westernblot. The experimental setup was repeated three times.
The results are shown in fig. 5, demonstrating that lysosomal inhibitors are able to inhibit the degradation of GPX4 protein induced by chloroquinaldo, i.e., GPX4 degradation induced by chloroquinaldo is mediated primarily by the lysosomal pathway.
Example 6Fer-1 inhibits the degradation of GPX4 by chloroquinaldo
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 5 cells/well. The cells were then treated for 24 hours with or without Chloroquinaldo (CQD) at a final concentration of 30. Mu.M, with or without Fer-1 (Ferrostatin-1, iron death inhibitor, available from selleck, catalog number S7243) at a final concentration of 1. Mu.M, and the expression level of GPX4 was detected by Westernblot. The experimental setup was repeated three times.
The results are shown in FIG. 6, which illustrates that Fer-1 is not effective in inhibiting GPX4 degradation induced by chloroquinaldo.
Example 7 iron death inducer Erastin in combination with chloroquinaldo degradation of GPX4
DLD-1 and HCT116 cells were inoculated into 6-well cell culture plates, respectively, and cultured overnight at 6X 10 5 cells/well. The cells were then treated for 24 hours with or without Chloroquinaldo (CQD) at a final concentration of 30. Mu.M, with or without Erastin (iron death inducer, available from Michelin under the catalogue number E872563) at a final concentration of 20. Mu.M, and the expression level of GPX4 was detected by Westernblot. The experimental setup was repeated three times.
The results are shown in fig. 7, demonstrating that chlorquinaldo can down-regulate the expression level of GPX4, while Erastin in combination with chlorquinaldo can enhance down-regulation of GPX4 levels.
Example 8 iron death inducer Erastin in combination with chlorquinaldo induces ROS production
DLD-1 and HCT116 cells were individually inoculated into 6-well cell culture plates and cultured overnight at 6X 10 5 cells/well. Then, chlorquinaldo (CQD) was added or not added at a final concentration of 30. Mu.M, erastin (iron death inducer) was added or not added at a final concentration of 20. Mu.M, or Fer-1 (iron death inhibitor) was added or not added at a final concentration of 1. Mu.M, respectively, for 24 hours, and then, the cellular ROS production was detected using DCF-DA (ROS probe). The experimental setup was repeated three times.
The results are shown in FIG. 8, which illustrates that Erastin in combination with chloroquinaldol enhances the production of cellular reactive oxygen species.
Example 9 is the enhanced inhibition of colorectal cancer cell growth by iron death inducer Erastin in combination with chloroquinaldo
DLD-1 and HCT116 cells were inoculated into 96-well cell culture plates, respectively, and cultured overnight at 5X 10 3 cells/well. The cells were then assayed for viability using the MTT method with or without the addition of Chloroquinaldo (CQD) at a final concentration of 30. Mu.M, and with or without the addition of Erastin (iron death inducer) at a final concentration of 20. Mu.M, for 24 hours. The experimental setup was repeated three times.
As a result, as shown in fig. 9, it can be seen that iron death inducer Erastin in combination with chloroquinaldo can suppress the growth of colorectal cancer cells better than chloroquinaldo or iron death inducer, i.e., chloroquinaldo can increase the tumor killing effect of iron death inducer.
Example 10 detection of HE staining of tumor tissue following treatment at the Chlorquinaldo animal level
A nude mice subcutaneous tumor-bearing model was constructed by injecting 100. Mu.l DLD-1 cells (containing 4X 10 6 cells) into the lower right dorsal part of female BALB/c-nu athymic nude mice (5 weeks old, purchased from university of medical laboratory animal center, guangxi). Subsequent experiments were performed when the tumor volume reached 100mm 3. Model mice were divided equally into two groups (6 each) and treated by tail vein injection of Chloroquinaldo (CQD) (5 mg/kg) once every two days for two weeks with equal volumes of saline as controls. After completion, the tumor tissue was removed and fixed with 4% paraformaldehyde solution, and section staining (HE, ki67 staining) was prepared. The results are shown in FIG. 10.
Example 11 detection of GPX4, 4-HNE levels in tumor tissues after treatment of Chlorquinaldo animal levels
After the end of the treatment of the model mice of example 10, a portion of the tumor tissues of each group was sectioned in paraffin, and the expression levels of GPX4, 4-HNE in the tumor tissues were detected by staining with an immunohistochemical antibody (GPX 4 antibody, available from Wohan Seville Corp., 4-HNE antibody, available from Abcam). The experimental setup was repeated three times. The results are shown in FIG. 11.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. Use of chloroquinaldo in the preparation of a product for degrading GPX4 protein.
2. The use according to claim 1, characterized in that: the degradation of GPX4 protein induced by chlorquinaldo can be inhibited by lysosomal inhibitors;
The lysosomal inhibitor is Bafilomycin A < 1 >.
3. Use of chloroquinaldo in combination with an iron death inducer in the preparation of a product for degrading GPX4 protein.
4. A use according to claim 3, characterized in that:
the combination of the iron death inducer and the chlorquinaldo can enhance the down regulation of GPX4 level;
The iron death inducer is Erastin.
5. Use according to claim 1 or 3, characterized in that: the product comprises a medicament or a kit.
6. The application of chlorquinaldo in preparing iron death drug sensitizer.
7. The use according to claim 6, characterized in that: the iron death medicament is an iron death inducer Erastin.
8. The application of chlorquinaldo and an iron death inducer in preparing antitumor drugs.
9. The use according to claim 8, characterized in that:
the iron death inducer is Erastin;
The tumor is colorectal cancer.
10. Use of chloroquinaldo in combination with an iron death inducer for the manufacture of a medicament for inhibiting proliferation and/or growth of colorectal cancer cells.
CN202410034291.XA 2024-01-10 2024-01-10 Application of chloroquinaldol in preparation of GPX4 protein degradation products Pending CN118121602A (en)

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