CN116139144A - Application of flunarizine targeted ZDHC 5 in preparation of glioma treatment drugs - Google Patents
Application of flunarizine targeted ZDHC 5 in preparation of glioma treatment drugs Download PDFInfo
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Abstract
The invention discloses an application of flunarizine targeted ZDHC 5 in preparation of a medicament for treating glioma, and belongs to the technical field of clinical medicine. The invention discloses an application of flunarizine in preparing medicaments for treating glioma related to ZDHC 5 targeting, and the medicament is used for treating glioma by preventing interaction between ZDHC 5 and the C end of an intracellular region of a substrate protein SSTR 5. The application provides a new application of Flunarizine in targeting high ZDHC 5 expression glioma, provides theoretical guidance for drug target design and drug selection for glioma treatment, and has important clinical application value.
Description
Technical Field
The invention belongs to the technical field of clinical medicine, and particularly relates to application of flunarizine in preparation of a medicament for treating glioma related to ZDHC 5 targeting.
Background
Gliomas (gliomas) are tumors formed by abnormal mutations of various types of glial cells (e.g., astrocytes, oligodendrocytes, etc.) in nerve tissues, and are a type of tumor with unclear boundaries with normal nerve tissues. Since glial cells are present only in the central nervous system (i.e., brain and spinal cord), gliomas occur only in the brain and spinal cord of the central nervous system, with gliomas in the spinal cord being mostly astrocytomas, and gliomas in the brain include astrocytomas, oligodendrogliomas, astrocytomas-oligodendrogliomas, and the like.
The treatment of glioma is mainly surgical treatment, but because of the invasive growth of tumor, there is no obvious boundary with brain tissue, and besides the small and proper position of early tumor, it is difficult to make complete excision, and general comprehensive treatment is claimed, i.e. after operation, radiation treatment and chemical treatment are combined, so that it can delay recurrence and prolong survival period. The drugs of choice for chemotherapy remain predominantly liposoluble, with relatively limited options. Glioma (glioma) is different from glioblastoma (gliobalastoma), which is the most malignant glioma among astrocytomas, accounting for about 15% of intracranial tumors; gliomas are the most common primary central nervous system tumors, account for about half of all intracranial primary tumors, and compared with gliomas, have high incidence, and no targeting drugs are developed for clinical use at present.
Flunarizine (Flunarizine), the chemical name is (E) -1- [ bis- (4-fluorophenyl) methyl ] -4- (2-propenyl-3-phenyl) piperazine, and the chemical structural formula is:
which is a difluorinated derivative of cinnarizine, also is a selective Ca 2+ Antagonists, which prevent excessive Ca 2+ Enters vascular smooth muscle cells to cause vasodilation, has better selectivity on cerebral vessels and poorer effect on myocardial vessels, thus having small influence on blood pressure and heart rate. Has lasting inhibiting effect on vasoconstriction caused by vasoconstrictor, and has more obvious effect on basilar artery and carotid artery. Can inhibit calcium overload caused by cerebral ischemia and anoxia, and protect cerebral tissue. For vascular endothelial cell factorThe calcium overload caused by hypoxia has preventing and treating effect, and can protect the integrity of vascular endothelial cells. Can increase intra-cochlear radiation arteriole blood flow, improve vestibular organ microcirculation, and inhibit oculars tremor and vertigo. Is mainly used for preventing and treating cerebral arterial ischemic diseases, dizziness, tinnitus, dizziness, vascular migraine, epilepsy and other diseases caused by vestibular stimulation or cerebral ischemia.
The applicant finds that flunarizine can inhibit proliferation of glioma cells by remarkably inhibiting interaction of ZDHC 5 and SSTR5, and further can be applied to preparation of medicaments for treating glioma.
Disclosure of Invention
The invention aims to provide an application of flunarizine in preparing medicaments for treating glioma related to ZDHC 5 targeting, which can inhibit proliferation of glioma cells by obviously inhibiting interaction between ZDHC 5 and SSTR5, provides theoretical guidance for designing medicament targets and selecting medicaments for glioma treatment, and has important clinical application value.
In order to achieve the above object, the present invention has the following technical scheme:
use of flunarizine for the manufacture of a medicament for the treatment of glioma in association with zdhc 5 (palmitoyl transferase 5) targeting, the flunarizine having the structure:
preferably, in the application, flunarizine inhibits proliferation of glioma cells by inhibiting the interaction of zdhc 5 (palmitoyl transferase 5) with SSTR5 (somatostatin receptor 5).
Further preferably, in the use, flunarizine inhibits proliferation of glioma cells by inhibiting interaction of the C-terminal end of the SSTR5 intracellular region with the substrate binding region of the zdhc 5 protein.
Preferably, in such applications, the flunarizine is capable of efficiently binding to the zdhc 5 substrate binding region.
Preferably, in the application, the glioma cell is selected from the group consisting of a U373 cell, a SWO38 cell, a U251 cell, a U87 cell, a U52 cell, a172 cell, a U118MGT-98G cell, further preferably a U373 cell and a SWO38 cell.
Preferably, in the use, the glioma is a high zdhc 5 expressing glioma.
Preferably, in said application, the inhibition of the interaction of flunarizine zdhc 5 with SSTR5 is determined by a micro thermophoresis assay (MST).
Further preferably, the micro thermophoresis assay comprises the steps of:
the protein of interest was expressed by BL21 (DE 3) (E.coli expression strain BL21 (DE 3)), purified by passing through a His tag nickel column (purchased from GE company), then labeled with NT-647-NHS dye (purchased from nanoTemper technologies Co., ltd.), dye-labeled ZDHC 5 protein was separated and purified by passing through a B gravity column, and after incubation with the labeled protein, the test was performed on the machine, and the analysis was performed by Initial Fluorescence Analysis program to determine the Kd value (dissociation constant) of the C-terminal of the intracellular region of SSTR5 and the binding region of ZDHC 5 substrate.
Preferably, in the application, the proliferation effect of flunarizine on glioma cells is determined by a colony formation assay.
Further preferably, the clone formation assay comprises the steps of:
culturing cells to logarithmic phase, discarding supernatant, washing once, adding enzyme for digestion, blowing down cells, centrifuging, discarding supernatant, re-suspending, adding into a blood cell counting plate, and taking under a microscope for counting; after counting, cells were spotted evenly into six well plates, and medium was added to each well for culture, and the culture was stopped when the number of cells in most of the monoclonal cells in the well was observed to be greater than 50.
(2) Washing cells with PBS for 1 time, adding paraformaldehyde, fixing at normal temperature, discarding fixing solution, dyeing, slowly and gently washing the cells with water after dyeing is completed until superfluous dye is removed, airing, photographing the bottoms of all the pore plates by using a camera, and performing clone counting by using imageJ software after uploading.
The beneficial effects of the invention are as follows:
the invention discloses a method for treating glioma by inhibiting the interaction of ZDHHC5 with the C end of the intracellular region of a substrate protein SSTR5 in a targeted manner by using Flunarizine, wherein the glioma is treated by preventing the interaction of the ZDHHC5 with the C end of the intracellular region of the substrate protein SSTR 5.
The present application carried out a micro thermophoresis (microscale thermophoresis, MST) experiment on Flunarizine and ZDHC 5 proteins to determine the Kd value, and found that the Kd value of Flunarizine and ZDHC 5 is 843nM. Microdermabrasion (microscale thermophoresis, MST) experiments were performed on the C-terminal of the SSTR5 intracellular domain and the zdhc 5 protein to determine Kd values, which differed significantly in the presence of flularizine. Meanwhile, the clone formation experiment shows that Flunarizine (10 mu M) can effectively inhibit glioma cell proliferation (U373 and SWO 38), and the inhibition effect is similar to that of siRNA transfection result of ZDHC 5.
In conclusion, the application provides a new application of Flunarizine in targeting high ZDHC 5 expression glioma, provides theoretical guidance for drug target design and drug selection for glioma treatment, and has important clinical application value.
Drawings
FIG. 1 is a graph showing experimental results of a micro thermophoresis (MST) affinity test for the ZDHC 5 protein and Flunarizine;
FIG. 2 is a graph showing the results of an affinity test under a micro thermophoresis (MST) affinity test for the ZDHC 5 protein and the C-terminal of the intracellular region of SSTR5 (solutions containing different concentrations of Flunarizine);
FIG. 3 is a graph showing the results of cloning experiments to demonstrate that siRNA and Flunarizine (10. Mu.M) inhibit proliferation of glioma cells (U373 and SWO 38).
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. The following is merely exemplary of the scope of the claimed invention and one skilled in the art can make various changes and modifications to the invention of the present application in light of the disclosure, which should also fall within the scope of the claimed invention.
The invention is further illustrated by means of the following specific examples. The various chemical reagents used in the examples of the present invention were obtained by conventional commercial means unless otherwise specified.
EXAMPLE 1 micro-thermophoresis (MST) determination of the Kd value of the ZDHC 5 substrate binding region and Flunariizine
The experimental method comprises the following steps: the Kd values of flularizine and zdhc 5 substrate binding regions (amino acids 1-219) were determined using the micro-thermophoresis (MST) technique of biomolecular analysis.
Experimental principle: microphoresis (MST) is an analytical technique for biomolecules, which is the directional movement of particles in microscopic temperature gradients. The affinity can be determined by the relative change in movement along the temperature gradient caused by the change in hydration layer due to the change in structure/conformation of the biomolecule.
Experimental materials and reagents: MO-K022 Capillites (1000 count) Monolith NT.115 Monolith TM Protein Labeling Kit RED-NHS and a microphoresis apparatus.
The experimental steps are as follows: BL21 (DE 3) is used for expressing target protein, the target protein is purified through a GE His tag nickel column, then NT-647-NHS dye is used for marking the target protein, the dye marked ZDHC 5 protein is separated and purified through a B gravity column, and after incubation, the target protein and the dye are subjected to machine test and Initial Fluorescence Analysis program analysis.
The experimental results are shown in fig. 1, and the results show that: flularizine has strong affinity to the ZDHC 5 protein substrate binding domain, and has a dissociation constant (KD) of 843. Mu.M.
Results: microphoresis (MST) experiments showed that flularizine has a strong affinity for the zdhc 5 protein, and thus flularizine can bind efficiently to the zdhc 5 substrate binding region.
EXAMPLE 2 micro-thermophoresis (MST) determination of Kd values of the ZDHHC5 substrate binding region and SSTR5 in the Presence of Flunarizine at different concentrations
The experimental method comprises the following steps: kd values of the C-terminal (307-362) and ZDHC 5 substrate binding domains (amino acids 1-219) of the intracellular domain of SSTR5 were determined by micro-thermophoresis (MST) techniques using biomolecular analysis, and 0. Mu.M, 10. Mu.M and 20. Mu.M of Flunarizine were added to PBS solution, respectively.
Experimental materials and reagents: MO-K022 Capillites (1000 count) Monolith NT.115 Monolith TM Protein LaA beling Kit RED-NHS and a microphoresis instrument.
The experimental steps are as follows: BL21 (DE 3) is used for expressing target protein, the target protein is purified through a GE His tag nickel column, then NT-647-NHS dye is used for marking the target protein, a dye marked ZDHC 5 protein substrate binding region and an SSTR5 intracellular region C end are separated and purified through a B gravity column, 0 mu M, 10 mu M and 20 mu M of Flunarizine are respectively added into PBS solution, the SSTR5 intracellular region C end and marked protein are subjected to incubation and then are subjected to machine test, and Initial Fluorescence Analysis program analysis is carried out.
The experimental results are shown in fig. 2, and the results show that: the C-terminal of the SSTR5 intracellular region has a strong affinity for the ZDHC 5 protein substrate binding region, and the dissociation constant (KD) increases significantly with increasing concentration of flularizine.
Results: microphoresis (MST) experiments showed that flularizine was effective in inhibiting the interaction of the C-terminus of the SSTR5 intracellular region with the substrate binding region of zdhc 5 protein, thus confirming that flularizine is an inhibitor of the interaction of the zdhc 5 substrate binding region with the C-terminus of the SSTR5 intracellular region.
Example 3 cloning experiments
(1) Culturing the cells to logarithmic phase, discarding the supernatant, washing with sterile PBS buffer for one time, adding pancreatin for digestion for 3min, blowing down the cells, centrifuging at 800rpm for 5min, discarding the supernatant, taking 20 mu L of the supernatant after resuspension, adding into a blood cell counting plate, and taking the blood cell counting plate to count under a microscope; after counting, 600 cells are taken from each hole, uniformly spotted into six holes, 2mL of complete culture medium is added into each hole, and the mixture is put into a incubator for culturing for 12 hours. After 3 days, a single change of fluid was performed and a microscopic observation was performed, and the suspension was stopped when the number of cells in the majority of the monoclonal cells in the wells was observed to be greater than 50. Three groups were set up, the first group being a blank control group, the second group being a siRNA group of ZDHC 5 (siRNA sequences 5 'to 3': forward: CCCACAUUAUGGGUGUGUUTT; reverse: AACACACCCAUAAUGUGGGTT), the third group being Flunarizine (10. Mu.M).
(2) Washing the cells with PBS for 1 time, adding 4% paraformaldehyde (after filling the hole bottom), and fixing at normal temperature for 30min. Removing the fixing solution, adding crystal violet dye solution (after filling the hole bottom), and dyeing for 2min. After staining was completed, the cells were gently rinsed with water several times slowly until the excess dye was cleared. Air dried, photographed with a camera at the bottom of each well plate, and clone counted with imageJ software after uploading.
The results are shown in FIG. 3, which shows that Flunarizine (10. Mu.M) can effectively inhibit glioma cell proliferation (U373 and SWO 38), and the inhibition effect is similar to that of the siRNA transfection of ZDHC 5. FIG. 1 shows that Flunarizine has a strong binding capacity (Kd=843 nM) to the substrate binding region (1-219 amino acids) of ZDHC 5, and FIG. 2 shows that Flunarizine can significantly prevent the binding of the C-terminal (307-362 amino acids) of the intracellular region of SSTR5 to the substrate binding region of ZDHC 5 at different concentrations (Kd value increases with increasing concentration of Flunarizine). The prior literature reports that activation of SSTR5 can inhibit glioma proliferation, and in combination with figures 1 and 2 demonstrates that the drug achieves inhibition of glioma cell proliferation by inhibiting the interaction of zdhc 5 with SSTR 5. That is, it was confirmed that flularizine further inhibits the proliferation of glioma by preventing zdhc 5-mediated palmitoylation of SSTR5, thereby maintaining the activity of SSTR 5.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. Use of flunarizine for the preparation of a medicament for the treatment of glioma associated with zdhc 5 targeting, said flunarizine having the structure:
2. the use according to claim 1, wherein in the use flunarizine inhibits proliferation of glioma cells by inhibiting the interaction of zdhc 5 with SSTR 5.
3. The use according to claim 1, wherein flunarizine inhibits glioma cell proliferation by inhibiting the interaction of the C-terminal end of the SSTR5 intracellular domain with the substrate binding domain of the zdhc 5 protein.
4. The use according to claim 1, wherein in said use the flunarizine is capable of efficiently binding to the zdhc 5 substrate binding domain.
5. The use according to claim 1, wherein in the use, the glioma cells are selected from the group consisting of U373 cells, SWO38 cells, U251 cells, U87 cells, U52 cells, a172 cells, U118MGT-98G cells; preferably U373 cells and SWO38 cells.
6. The use according to claim 1, wherein in the use the glioma is a high zdhc 5 expressing glioma.
7. The use according to claim 1, wherein the inhibition of the interaction of flunarizine zdhc 5 with SSTR5 is determined by a microphoresis assay.
8. The use according to claim 7, wherein the micro-thermophoresis assay comprises the steps of:
BL21 (DE 3) is used for expressing target protein, the target protein is purified through a GE His tag nickel column, then NT-647-NHS dye is used for marking the target protein, the dye marked ZDHC 5 protein is separated and purified through a B gravity column, after incubation of the Flunarizine and the marked protein, a machine test is carried out, initial Fluorescence Analysis program analysis is carried out, and the Kd value of the C end of the SSTR5 intracellular region and the ZDHC 5 substrate binding region is determined.
9. The use according to claim 1, wherein the proliferation of glioblastoma cells by flunarizine is determined by a colony formation assay.
10. The use according to claim 9, wherein the clone formation assay comprises the steps of:
culturing cells to logarithmic phase, discarding supernatant, washing once, adding enzyme for digestion, blowing down cells, centrifuging, discarding supernatant, re-suspending, adding into a blood cell counting plate, and taking under a microscope for counting; counting, uniformly counting cells into six-hole plates, adding a culture medium into each hole for culture, and stopping when the number of the cells in most of the monoclonal cells in the holes is observed to be more than 50;
(2) Washing cells with PBS for 1 time, adding paraformaldehyde, fixing at normal temperature, discarding fixing solution, dyeing, slowly and gently washing the cells with water after dyeing is completed until superfluous dye is removed, airing, photographing the bottoms of all the pore plates by using a camera, and performing clone counting by using imageJ software after uploading.
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