CN116942815A - The use of proton pump inhibitors in tumor immunotherapy - Google Patents

Abstract

The present invention provides the use of proton pump inhibitors in tumor immunotherapy.

Description

The use of proton pump inhibitors in tumor immunotherapy

Technical field

The present invention belongs to the field of medical technology, and specifically relates to the use of proton pump inhibitors in tumor immunotherapy.

Background technique

Proton pump inhibitors (PPIs) include Omeprazole, Lansoprazole, Pantoprazole, Rabeprazole, etc., due to their significant anti- Acidic and good in safety, it is widely used in clinical treatment of gastric acid-related diseases ^[1,2] . As a weakly alkaline compound, PPIs are not easily dissociated in an alkaline environment and are in an inactive state. They can enter the secretory ducts of parietal cells through the cell membrane. When encountering an acidic environment with a pH of less than 2, PPIs can be converted into sulfenic acid and Hypoxanthine amide compounds covalently react with the sulfhydryl group on the cysteine residue in H ⁺ /K ⁺ -ATPase to form a disulfide bond, inactivating H ⁺ /K ⁺ -ATPase, thereby inhibiting gastric acid of secretion.

ADP ribosylation factor 1 (ARF1), as a type of small G protein, is widely distributed in eukaryotic cells and participates in many intracellular physiological processes including material transport ^[3] . As an important molecular switch in cell signal transduction, ARF1 cycles between the inactive GDP-bound form and the active GTP-bound form. This cycle process is regulated by two regulatory factors, namely guanylate exchange factor (guanine). nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs promote the release of GDP from small G proteins, bind GTP, and transduce signals to downstream effector proteins. They are a positive regulatory factor; GAPs can enhance small G The GTPase activity of the protein promotes its hydrolysis of GTP and inactivation, and is a negative regulatory factor ^[4-6] .

In recent years, a large number of studies have shown that the high expression and abnormal activation of ARF1 are involved in the occurrence and development of tumors, such as proliferation, invasion and migration ^[7-10] . Recent studies have revealed the relationship between ARF1 and the survival of cancer stem cells ^[11,12] . Cancer stem cells are a subset of cells in tumors that are in a stem cell state and have stem cell characteristics. Cancer stem cells may lead to treatment resistance, tumor metastasis, tumor immune evasion, disease recurrence, and ultimately lead to patient death. The ultimate goal of cancer stem cell research is to find pathways that can selectively regulate this type of cells, and then target these pathways to kill this type of cells. Studies have pointed out that cancer stem cells are metabolically unique and their lipid metabolism process requires the regulation of ARF1; knocking out ARF1 in mice will lead to blocked lipid metabolism and accumulation of lipid droplets, further leading to metabolic stress, including mitochondrial defects and Endoplasmic reticulum stress, this metabolic stress can selectively kill cancer stem cells. Dying cancer stem cells can release pathogen-related molecular patterns and activate dendritic cells. The activation of dendritic cells further enhances the infiltration of T cells and Activation, thereby stimulating anti-tumor immunity ^[12] . Knockdown of the ARF1 pathway has multiple functions. It can not only kill cancer stem cells, but also activate tumor-specific immune responses, destroy solid tumors, and obtain lasting therapeutic effects ^[12] . Therefore, the development of ARF1 inhibitors has great scientific significance and broad application prospects for the treatment of various cancers.

Since the surface of the small G protein is relatively smooth, except for the guanylate binding pocket, it lacks stable and easy-to-bind pockets for small molecules ^[5,6] , and the small G protein has a picomolar affinity for guanylate, which are all limitations. led to the development of inhibitors that directly target small G proteins ^[13] . Despite this, researchers have made a lot of efforts in finding ARF1 inhibitors in recent years. Reported ARF1 inhibitors include BFA ^[14] , LM11 ^[15] , SecinH3 ^[16] , Golgicide A ^[17] , AMF-26 ^[18] , etc. Most of them interfere with the normal function of GEF or destroy ARF1 and GEF. interactions to exert the effect of inhibiting ARF1 activity. Their discovery is meaningful for studying the function of ARF1. However, so far, the mode of action of these inhibitors is relatively single, and the relevant activity research is not comprehensive enough. Their clinical The effectiveness and safety require further study. Therefore, the development of safe and effective ARF1 inhibitors has great scientific significance and broad application prospects for the treatment of various cancers.

Contents of the invention

The inventor found that proton pump inhibitors (rabeprazole, lansoprazole, omeprazole, pantoprazole, PPIs) can covalently bind to C159 of ARF1; in vitro and in vivo studies have shown that rabeprazole Azoles can significantly inhibit the activity level of ARF1 and exert anti-tumor immune effects by regulating the lipolysis pathway of cancer stem cells. Based on this, the present invention was completed.

Therefore, in one aspect, the present invention provides the use of a proton pump inhibitor in the preparation of an ARF1 inhibitor.

In the present invention, the proton pump inhibitor refers to a drug that inhibits gastric acid secretion by inhibiting the H ⁺ /K ⁺ -ATPase (proton pump) of gastric parietal cells. Examples include, but are not limited to, rabeprazole, lanso. Prazole, omeprazole, pantoprazole, etc.

In one embodiment, the proton pump inhibitor may be one or more selected from the group consisting of rabeprazole, lansoprazole, omeprazole and pantoprazole. In a preferred embodiment, the proton pump inhibitor may be rabeprazole.

In another aspect, the present invention provides the use of proton pump inhibitors in the preparation of medicaments for treating tumors.

In the present invention, the tumor refers to a tumor containing cancer stem cells.

In one embodiment, the tumor may be selected from tumors containing cancer stem cells, such as leukemia, breast cancer, colon cancer, etc.

In one embodiment, the tumor may be colon cancer.

In one embodiment, the proton pump inhibitor may be one or more selected from the group consisting of rabeprazole, lansoprazole, omeprazole and pantoprazole. In a preferred embodiment, the proton pump inhibitor may be rabeprazole.

In such uses, the proton pump inhibitor acts as an ARF1 inhibitor.

In one embodiment, the proton pump inhibitor can significantly reduce the activity level of ARF1 in colon cancer cells and significantly induce lipid droplet accumulation.

In one embodiment, the proton pump inhibitor is capable of significantly inhibiting tumor growth.

In one embodiment, the proton pump inhibitor can significantly enhance the infiltration of immune cells in tumor tissue, increase the number of killer T cells, and reduce the number of exhausted T cells.

The present invention first discovered through protein thermal migration experiments that PPIs can significantly reduce the thermal stability of ARF1 protein in a concentration-dependent manner; due to the covalent binding properties of PPIs and H ⁺ /K ⁺ -ATPase, the only cysteine C159 of ARF1 is For binding site verification, protein thermal migration experiments, primary mass spectrometry experiments, and secondary mass spectrometry experiments of wild-type ARF1 and point mutant ARF1 (C159) jointly proved that PPIs covalently bind to ARF1, and confirmed that the binding site is C159. Rabeprazole has the greatest impact on the thermal stability of ARF1 and is used in subsequent activity studies.

Next, in vitro guanylate exchange experiments found that rabeprazole significantly inhibited the guanylate exchange process of ARF1. Cell-level studies have found that rabeprazole can significantly reduce the activity level of ARF1 in CT26 colon cancer cells and significantly induce the accumulation of lipid droplets. Animal-level studies have found that rabeprazole can significantly inhibit the growth of CT26 transplanted tumors. After flow cytometry analysis of immune cell subpopulations in tumor tissues, it was found that rabeprazole has a significant immune activation effect, manifested as CD3 ⁺ CD8 ⁺ The significant up-regulation of T cells and the significant down-regulation of CD3 ⁺ CD8 ⁺ PD1 ⁺ T cells, CD3 ⁺ CD8 ⁺ TIM3 ⁺ T cells, and CD3 ⁺ CD8 ⁺ PD1 ⁺ TIM3 ⁺ T cells were consistent with the results of immunohistochemical staining.

The invention has the following beneficial effects:

1. The present invention found that proton pump inhibitors (PPIs) can covalently bind to C159 of ARF1;

2. The present invention found that rabeprazole can significantly inhibit ARF1 activity and exert anti-tumor immunity effects by regulating the lipolysis pathway of cancer stem cells, providing a new treatment method for anti-tumor immunity.

Description of the drawings

Figure 1 shows that the PPIs (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) in Example 1 were significantly reduced in the protein thermal migration experiment. Plot of thermal stability of ARF1.

Figure 2 shows the PTS experiment after adding DTT in Example 2 and the PTS experiment of the ARF1 ^C159A mutant (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) Results and summary of protein thermal migration experiment results of PPIs (e).

Figure 3 shows the effect of pre-incubation of PPIs with ARF1 ^WT (a) or ARF1/ARF1 ^C159A (b) on the molecular weight of ARF1 protein measured by Q-TOF mass spectrometry in Example 3.

Figure 4 shows the Q-Exactive mass spectrometry results (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) in Example 4, which shows PPIs modification The latter ARF1 peptide.

Figure 5 shows the inhibitory effect of rabeprazole on the ARF1 guanylate exchange process in Example 5.

Figure 6 shows the cell level activity verification results of rabeprazole in Example 6. (a) Rabeprazole can significantly reduce the activity level of ARF1 in CT26 colon cancer cells, where n.s. represents no significant difference, ** represents P<0.01, *** represents P<0.001; (b) Rabeprazole can Significantly induces lipid droplet accumulation in CT26 colon cancer cells.

Figure 7 shows the inhibitory effect of rabeprazole on the growth of transplanted tumors in CT26 mice in Example 7, where *** represents P<0.001.

Figure 8 shows the flow cytometry (a) and immunohistochemical staining (b) analysis of the effect of rabeprazole on immune cell subpopulations in CT26 mouse transplanted tumor tissue in Example 8, where * represents P<0.05, ** represents P<0.01, *** represents P<0.001.

Detailed ways

The experimental methods used in the following examples are all conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Materials and reagents

Main reagents: NaCl (SCRC, 10019318), Tris-HCl (Meilun Biotech, MB6025), anhydrous MgCl ₂ (Ourchem, 7786-30-3), SYPRO Orange dye (Sigma, S5692), DMSO (Sigma, D8418- 1L), ARF1G-LISA Activation Assay Kit (Cytoskeleton, BK132), PBS (Meilun Biotech, MA0015), immunostaining fixative (Beyotime, P0098), immunostaining permeabilization solution (Triton X-100) (Beyotime, P0096) , Nile red (MedChemExpress, HY-D0718), anti-fluorescence quenching mounting solution (containing DAPI) (Beyotime, P0131), red blood cell lysis solution (Beyotime, C3702), Fixable Viablity Stain 700 dye (BD Horizan, 564997), anti-mouse CD16/32antibody (Invitrogen, 14-0161-82), anti-CD3 antibody (Invitrogen, 11-0032-82), anti-CD8 antibody (Biolegend, 100738), anti-PD1 antibody (Biolegend, 135219), anti-TIM3 antibody (Invitrogen, 12-5870-82).

Main materials: Compounds were purchased from MedChemExpress (rabeprazole, HY-B0656; lansoprazole, HY-13662; omeprazole, HY-B0113; pantoprazole, HY-17507), DTT (MedChemExpress, HY-15917), GDP (MedChemExpress, HY-113066A), MANT-GTP (Invitrogen, M12415), 0.1mL 96-well skirtless PCR tube (low-profile tube) (DN Biotech, 5371012), heat-sealing membrane (Thermo Scientific, AB-1107), 384-well black plate (Corning, 3575), Lab-Tek 8-well plate (Thermo, 154534).

instrument

Real-time fluorescence quantitative PCR instrument (Bio-Rad), nLC-1000-Q-Exactive (ThermoFisher), Spark multifunctional microplate reader (Tecan), OLYMPUS IX73 fluorescence microscope, Bio-Rad ZE5 flow cytometer.

Example 1 Effect of PPIs on the thermal stability of ARF1 in protein thermal migration experiments

experimental method:

Protein thermal migration experiments were used to evaluate the effects of compounds on protein thermal stability, in which wild-type recombinant human Δ17ARF1 protein (a soluble truncated form lacking the first 17 amino acids, ARF1 ^WT ) was used. The reaction system is 20 μL. In the white 96-well plate, add 2.5 μM ARF1 protein, 5×SYPRO Orange (Sigma, S5692) and different concentrations (50 μM (20×), 12.5 μM (5×)) of the test compound (PPIs: Rebbe prazole, lansoprazole, omeprazole, pantoprazole), and affix Absolute qPCR Plate Seals heat-sealing film to seal the sample well, and use a real-time fluorescence quantitative PCR instrument (Bio-Rad) to collect the temperature rising from 25°C Real-time fluorescence signal to 95°C. After the experiment, the CFX Connect Evaluation System was used to analyze the fitted T _m value. The exported data were reorganized into graphs using Graphpad Prism 8.0.

Experimental results:

As shown in Figure 1a-d, rabeprazole, lansoprazole, omeprazole, and pantoprazole can significantly reduce the thermal stability of ARF1 protein.

Example 2 PTS experiment after adding DTT and PTS experiment of ARF1 ^C159A mutant.

Experimental method: Use a protein thermal migration experiment to evaluate whether DTT (a reducing agent that can destroy disulfide bonds) will affect the PTS experimental results. The method is the same as in Example 1. Add 1mM DTT to the reaction system; use a protein thermal migration experiment. To evaluate the effect of PPIs on the thermal stability of the ARF1 ^C159A mutant protein, the method was the same as in Example 1, except that the wild-type ARF1 protein was replaced by the ARF1 ^C159A protein.

Experimental results: As shown in Figure 2, after adding DTT/replacing it with ARF1 ^C159A protein, the effects of PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) on the thermal stability of ARF1 disappeared. , suggesting that PPIs may be covalently bound to cysteine 159 of ARF1.

Example 3 Q-TOF mass spectrometry measured the molecular weight of ARF1 protein before and after preincubation of PPIs and ARF1/ARF1 ^C159A .

experimental method:

This experiment is used to detect protein molecular weight and is divided into a control group and a compound group. The incubation system of the compound group is: 20mM Tris (pH 8.0), 100mM NaCl, 5mM MgCl ₂ , 100μM ARF1 ^WT /ARF1 ^C159A protein, 1mM compound (Rebella azole, lansoprazole, omeprazole, pantoprazole). DMSO was added in equal proportions to the control group. The control group and compound group were placed in a 4°C refrigerator and incubated overnight, and then sent to the mass spectrometry detection service platform of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences for protein molecular weight determination.

Experimental results:

As shown in Figure 3a-b, after pre-incubation of PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) with ARF1 ^WT , the increase in ARF1 molecular weight suggests that PPIs generate homocysteine. covalent reaction. After pre-incubation of PPIs and ARF1 ^C159A , the molecular weight of ARF1 did not change, confirming that the covalent binding site is at C159.

Example 4 Q-Exactive secondary mass spectrometry results show the ARF1 peptide modified by PPIs.

Experimental method: The sample incubation system is: 20mM Tris (pH 8.0), 100mM NaCl, 5mM MgCl ₂ , 100μMARF1 ^WT protein, 1mM compound (rabeprazole, lansoprazole, omeprazole, pantoprazole), After preparation, incubate overnight at 4°C. The detection process is as follows: 100μL of 100mM NH ₄ HCO ₃ is added to the sample, and Trypsin (10ng/μL) is enzymatically digested at 37°C for 17 hours. The next day, the sample was centrifuged at 12,000 × g for 30 minutes, and the supernatant was freeze-dried, desalted, and then freeze-dried to obtain peptide lyophilized powder. Then add 15 μL of 0.1% FA (formic acid) solution to dissolve the peptide freeze-dried powder, centrifuge at 12000×g for 5 minutes, add the supernatant to the loading bottle, detect with mass spectrometry (Q-Exactive), and search the database with MaxQuant (1.6.5.0) , control the false positive rate of protein identification to less than 1%.

Experimental results: As shown in Figure 4, the secondary mass spectrometry results further confirmed that PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) covalently modified C159 of ARF1.

Example 5 Effect of rabeprazole on the guanylate exchange process of ARF1

Experimental method: The ARF1 protein is pre-labeled with GDP to obtain ARF1 ^GDP . The experimental buffer system is 20mM HEPES pH7.5, 150mM NaCl, 1mM MgCl ₂ , 1mM DTT. The final concentrations of ARF1 ^GDP , MANT-GTP, and ARNO ^Sec7 are 20 μM and 10μM, 1μM, first use a 384-well black plate to mix ARF1 ^GDP , MANT-GTP, and compounds of different concentrations, incubate at room temperature in the dark for 15 minutes, then add ARNO ^Sec7 to start the exchange reaction, and use Spark multifunctional microplate reader (Tecan) The fluorescence signal was continuously monitored until a plateau was reached. The exported data were re-plotted using Graphpad Prism 8.0.

Experimental results: As shown in Figure 5, rabeprazole has a significant inhibitory effect on the ARF1 guanylate exchange process in a concentration-dependent manner.

Example 6 Cell-level activity verification of rabeprazole

experimental method:

GLISA experiment: Use Cytoskeleton ARF1 G-LISA Activation Assay Kit (BK132) to measure the ARF1 activity level in CT26 cells, and proceed according to the instructions.

Lipid droplet staining experiment:

When CT26 cells are growing well, they are seeded into a Thermo Lab-Tek 8-well plate one night in advance at 2×10 ⁴ cells per well. After the cells adhere to the wall the next day, compounds or DMSO are added for treatment. After processing, the staining steps are as follows:

(1) After removing the culture medium, rinse 3 times with PBS, 200 μL per well, 5 minutes each time;

(2) Fix with immunostaining fixative for 30 minutes;

(3) Wash 3 times with PBS, 200 μL per well, 5 minutes each time;

(4) Permeabilize cells with immunostaining permeabilization solution (Triton X-100) for 30 minutes;

(5) Wash 3 times with PBS, 200 μL per well, 5 minutes each time;

(6) Aspirate the PBS and prepare Nile red staining solution (2 μM) in the dark. Add 200 μL staining solution to each well and incubate in the dark for 20 minutes. During the incubation process, clean the coverslip (after 75% alcohol sterilization, use ddH ₂ O Clean three times).

(7) Under dark conditions, wash 3 times with PBS, 200 μL per well, 5 minutes each time;

(8) Use anti-fluorescence quenching sealing solution to seal the slide. Add an appropriate amount of sealing solution to the 8-well plate, then slowly flip the coverslip up without generating air bubbles. Use nail polish to cover the coverslips. Apply a circle around the edge, air-dry and store in a 4°C refrigerator. Filming will be completed on the same day using an OLYMPUS IX73 fluorescence microscope.

Experimental results: As shown in Figure 6a, rabeprazole can significantly reduce the activity level of ARF1 in CT26 colon cancer cells; as shown in Figure 6b, rabeprazole can significantly induce the accumulation of lipid droplets in CT26 colon cancer cells.

Example 7 Effect of rabeprazole on the growth of transplanted tumors in CT26 mice

Experimental methods: All animal experiments were reviewed and approved by the Experimental Animal Management Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and were conducted in accordance with the prescribed guidelines (IACUC Issue NO.2021-03-JHL-22). The experimental subjects were female Balb/c rats aged 6 to 8 weeks. Colon cancer xenograft tumor model was established by subcutaneous injection of CT26 colon cancer cells (2.5×10 ⁵ cells each). When the tumor grew to a volume of approximately ¹⁰⁰ mm, the mice were randomly divided into a control group and a drug administration group, and the mice were intraperitoneally injected with solvent blank or compound (40 mg/kg/day). After the experiment, tumor tissue from the mice was removed and analyzed.

Experimental results: As shown in Figure 7, rabeprazole has a significant inhibitory effect on the growth of transplanted tumors in CT26 mice.

Example 8 Flow cytometry and immunohistochemical staining analysis of the effect of rabeprazole on immune cell subsets in transplanted tumor tissue of CT26 mice

1. Flow cytometry experimental method:

(1) Preparation of single cell suspension of tumor tissue

Prepare the digestion solution in advance: add 0.1% collagenase, 0.001% hyaluronidase, and 0.002% DNase to 100mL RPMI1640. Since DNase is a Ca ²⁺ /Mg ²⁺ -dependent enzyme, 12 μL 1M needs to be added to the digestion solution. CaCl ₂ and 12 μL 1MMgCl ₂ .

Digestion of tumor tissue: After the mice are euthanized, peel off the tumor tissue, take about 500 mg of the non-eroded part near the outer edge and put it into an EP tube, add 300 μL of digestive juice, and cut into minced meat with scissors. Add 1 mL of digestion solution and digest in a shaking shaker at 37°C and 180 rpm for 60 minutes.

Filtration: Add 5 mL of PBS to a 15 mL centrifuge tube in advance, and filter the digested tumor tissue into the centrifuge tube with 200 mesh gauze. The resulting filtrate is a single-cell suspension. Then centrifuge it (1000 rpm, 3 minutes) and remove the supernatant. clear.

Lyse red blood cells: Add 500 μL of ammonium chloride red blood cell lysis buffer to resuspend the cells. After 1 minute, add 3 mL of PBS to stop lysis. Centrifuge and remove supernatant.

Filtration: Prepare the EP tube in advance, add 1mL PBS to the centrifuge tube from the previous step and resuspend it, filter it into the EP tube with 200 mesh gauze, centrifuge, and remove the supernatant. Add PBS and resuspend to obtain a single cell suspension of tumor tissue.

(2) Cell surface marker staining

Dead and alive staining: Take an appropriate amount of single cell suspension of tumor tissue in an EP tube, use Fixable Viablity Stain700 dye (BD Horizon, 564997) to distinguish dead and living cells, and stain on ice for 10 minutes. At the same time, take an EP tube and add 200 μL of cell sample to stain only live and dead cells. After staining, stop with PBS containing 2% FBS.

Blocking Fc receptors: This step is performed in 2% FBS in PBS. Use anti-mouse CD16/32antibody to block Fc receptors on the cell surface and incubate on ice for 10 minutes.

Staining: After blocking, directly add flow antibody Mix and incubate to stain CD3, CD8, PD-1, and TIM3 antigen molecules located on the cell surface together. After staining, centrifuge and resuspend the cells in 400 μL of 2% FBS in PBS before testing on the machine.

2. Immunohistochemical staining: The isolated fresh CT26 tumor tissue was sent to Wuhan Sevier Biotechnology Co., Ltd. for paraffin sectioning and immunohistochemical staining.

Experimental results: As shown in Figure 8 (top), flow cytometry analysis showed that rabeprazole can significantly enhance the infiltration of immune cells in mouse tumor tissues, increase the number of killer T cells, and reduce the number of exhausting T cells. It showed a significant up-regulation of CD3 ⁺ CD8 ⁺ T cells, and a significant down-regulation of CD3 ⁺ CD8 ⁺ PD1 ⁺ T cells, CD3 ⁺ CD8 ⁺ TIM3 ⁺ T cells, and CD3 ⁺ CD8 ⁺ PD1 ⁺ TIM3 ⁺ T cells. As shown in Figure 8 (bottom), the immunohistochemical staining results of CD8 and PD1 were consistent with the flow cytometry results.

The present invention found that PPIs can covalently bind to ARF1, among which rabeprazole can significantly inhibit the activity of ARF1, exert anti-tumor immunity effects by regulating the lipolysis pathway of CT26 colon cancer cells, and provide a new treatment method for anti-tumor immunity.

references

1. Malfertheiner, P., A. Kandulski, and M. Venerito, Proton-pump inhibitors: understanding the complications and risks. Nat Rev Gastroenterol Hepatol, 2017.14(12):p.697-710.

2. Spugnini, E.P. and S. Fais, Drug repurposing for anticancer therapies. Alesson from proton pump inhibitors. Expert Opin Ther Pat, 2020.30(1):p.15-25.

3. Adarska, P., L. Wong-Dilworth, and F. Bottanelli, ARF GTPases and Their Ubiquitous Role in Intracellular Trafficking Beyond the Golgi. Front Cell DevBiol, 2021.9: p.679046.

4. Cromm, P.M., et al., Direct Modulation of Small GTPase Activity and Function. Angew Chem Int Ed Engl, 2015.54(46):p.13516-37.

5. Gray, J.L., F. von Delft, and P.E. Brennan, Targeting the Small GTPaseSuperfamily through Their Regulatory Proteins. Angew Chem Int Ed Engl, 2020.59(16):p.6342-6366.

6. Toma-Fukai, S. and T. Shimizu, Structural Insights into the Regulation Mechanism of Small GTPases by GEFs. Molecules, 2019.24(18).

7.Boulay,P.-L.,et al.,ADP-ribosylation factor 1controls theactivation of the phosphatidylinositol 3-kinase pathway to regulate epidermalgrowth factor-dependent growth and migration of breast cancer cells.TheJournal of biological chemistry,2008.283(52) :p.36425-36434.

8. Lewis-Saravalli, S., S. Campbell, and A. Claing, ARF1 controls Rac1signaling to regulate migration of MDA-MB-231invasive breast cancer cells. Cell Signal, 2013.25(9):p.1813-9.

9. Boulay, P.L., et al., ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein. Oncogene, 2011.30(36):p.3846-61.

10.Tsai,M.M.,et al.,Overexpression of ADP-ribosylation factor 1inhuman gastric carcinoma and its clinicopathological significance.Cancer Sci,2012.103(6):p.1136-44.

11.Singh, S.R., et al., The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila. Nature, 2016.538(7623): p.109-113.

12. Wang, G., et al., Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice. NatCommun, 2020.11(1):p.220.

13. Nawrotek, A., M. Zeghouf, and J. Cherfils, Protein-membrane interactions in small GTPase signaling and pharmacology: perspectives from Arf GTPasesstudies. Biochem Soc Trans, 2020.48(6):p.2721-2728.

14. Mossessova, E., R.A.Corpina, and J.Goldberg, Crystal structure ofARF1*Sec7 complexed with Brefeldin A and its implications for the guaninenucleotide exchange mechanism. Mol Cell, 2003.12(6):p.1403-11.

15.Viaud,J.,et al.,Structure-based discovery of an inhibitor of Arfactivation by Sec7 domains through targeting of protein-proteincomplexes.Proc Natl Acad Sci U S A, 2007.104(25):p.10370-5.

16.Bi,

17.Sáenz,J.B.,et al.,Golgicide A reveals essential roles for GBF1 inGolgi assembly and function.Nat Chem Biol,2009.5(3):p.157-65.

18.Ohashi,Y.,et al.,AMF-26,a novel inhibitor of the Golgi system,targeting ADP-ribosylation factor 1(Arf1)with potential for cancer therapy.JBiol Chem,2012.287(6):p.3885- 97.

Claims (9)

1. Use of a proton pump inhibitor in the preparation of an ARF1 inhibitor.

2. The use according to claim 1, wherein the proton pump inhibitor is one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole.

3. The use according to claim 1, wherein the proton pump inhibitor is rabeprazole.

4. Use of a proton pump inhibitor in the manufacture of a medicament for the treatment of a tumor.

5. The use according to claim 4, wherein the tumor is a tumor containing tumor stem cells.

6. The use according to claim 4, wherein the tumor comprises leukemia, breast cancer, colon cancer.

7. The use according to any one of claims 4-6, wherein the proton pump inhibitor is one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole.

8. The use according to any one of claims 4-6, wherein the proton pump inhibitor is rabeprazole.

9. Use according to any one of claims 4-6, wherein the proton pump inhibitor is an ARF1 inhibitor.