Application of ENT-montelukast or derivative thereof in preparation of medicine for treating diseases taking TMEM164 as target point

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of ENT-montelukast or a derivative thereof in preparation of a medicine for treating diseases taking TMEM164 as a target spot. Through screening verification of a surface ion resonance (SPR) technology, the application proves that the derivatives such as ENT-montelukast or the salt thereof and the like can be strongly combined with TMEM164 protein; the biological verification shows that the TMEM164 overexpression cell line treated by the derivatives such as the ENT-montelukast or the salt of the ENT-montelukast is obvious and the sensitivity of cells to ferroptosis is effectively improved. Therefore, the ENT-montelukast or the derivatives thereof such as the salt thereof have the effect of selectively inhibiting the TMEM164, and an application technical scheme of the ENT-montelukast or the derivatives thereof in preparation of medicines for treating diseases taking the TMEM164 as a target spot is further provided. According to the scheme provided by the invention, a new way is provided for TMEM164-targeted disease treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of ENT-montelukast or its derivatives in the preparation of drugs for treating diseases targeting TMEM164. Background Art

[0002] Phospholipids are not only a protective barrier against harsh environments, but also a hub for signal and material exchange. Imbalance in lipid metabolism can lead to metabolic diseases such as obesity, fatty liver, tumors, and atherosclerosis. Lipid diversity and LAND's cycle are respectively regulated by phospholipase (PLA) 1 / 2 S and lysophospholipid acyltransferase (LPLAT) mediate the deacylation and reacylation of fatty chains, a process called phospholipid remodeling, which is closely related to development, disease progression, and treatment prognosis. At present, there is still little exploration of how phospholipases reshape phospholipids to manipulate cell fate. LPLATs (divided into AGPAT and MBOAT families) have been widely reported to participate in phospholipid remodeling and thus change cell fate (such as ferroptosis resistance). Their important feature is that they use acyl coenzyme A (acyl-CoA) as an acyl donor to catalyze acyl transfer reactions.

[0003] In 2023, an article published in Nature Chemical Biology by the laboratories of Benjamin F. Cravatt and J. Fernando Bazan detailed that TMEM164 can act as an acyltransferase, directly using C20:4-PC instead of acyl-CoA as a substrate to generate ferroptotic C20:4-ePL in multiple tumor cell lines, which means that under normal physiological conditions, TMEM164 has the function of promoting ferroptosis.

[0004] Currently, little is known about the function of TMEM164. Tao Fan's case report showed that TMEM164 was abnormally activated in patients with lung adenocarcinoma (Fan, T., Liu, Y., Liu, H., Wang, L., Tian, ​​H., Zheng, Y.,... He, J. (2022). Transmembrane Protein-Based Risk Model and H3K4me3 ModificationCharacteristics in Lung Adenocarcinoma. Front Oncol, 12, 828814.). In addition, Masashi Nishikawa reported that TMEM164 has the effect of promoting the progression of liver cancer (Nishikawa, M., Okada, H., Kawaguchi, K., Shimakami, T., Nio, K., Arai, K.,... Honda, M. (2023). Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus. Cell Mol Gastroenterol Hepatol, 16 (2), 263-286.). However, Zhou Haibo's laboratory found that overexpression of TMEM164 in the field of neurodegeneration can inhibit abnormal activation of astrocytes and protect neurons (Zhang, L., Jia, Z., Wu, Q., Bai, T., Wang, B., Hu, X., ... Zhou, H. (2023). Alleviating symptoms of neurodegenerative disorders by astrocyte-specific overexpression of TMEM164 in mice. Nat Metab, 5 (10), 1787-1802.). It is not clear how TMEM164, as an acyltransferase, confers tissue specificity in an abnormally activated state. The perturbation of TMEM164 expression level (often upregulation) mediates a variety of physiological and pathological progressions such as neurodegenerative diseases, autophagy and tumorigenesis. So far, there is no direct and effective small molecule targeted inhibition of TMEM164 in the prior art. Summary of the invention

[0005] Based on the lack of direct and effective small molecule targeted inhibition of TMEM164 in the prior art, the present invention provides an application of ENT-montelukast or its derivatives in the preparation of drugs for diseases targeting TMEM164.

[0006] Specifically, the present invention has found that S-configuration montelukast ENT-montelukast or its salts and other derivatives (ENT-montelukast or its salts and other derivatives) can be used as TMEM164 selective inhibitors. Based on this, the present application provides the use of ENT-montelukast or its derivatives in the preparation of drugs for treating diseases targeting TMEM164.

[0007] Diseases that target TMEM164 include neurodegenerative diseases, tumors, and aging. Specifically, TMEM164 is related to autophagy, which is an evolutionarily conserved process that involves the cell's own structure through the lysosome mechanism, responsible for transporting damaged organelles, misfolded proteins, and other macromolecules to the lysosome for degradation and reuse. Autophagy may occur in cells throughout the body, and diseases closely related to autophagy include neurodegenerative diseases, tumors, and aging.

[0008] More specifically, the present application study found that TMEM164 selective inhibitors can be used as therapeutic drugs for TMEM164-mediated diseases (especially tumors associated with ferroptosis), and the present application study found that ENT-montelukast or its derivatives have extremely strong affinity for TMEM164 and can increase the sensitivity of TMEM164-overexpressing cell lines to ferroptosis.

[0009] Based on this, the present application provides an application of ENT-montelukast or its derivatives in the preparation of drugs for treating diseases targeting TMEM164.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention firstly provides the use of ENT-montelukast or its derivatives in preparing drugs for treating diseases targeting TMEM164.

[0012] Furthermore, the present invention provides the use of ENT-montelukast or its derivatives in the preparation of TMEM164 targeted inhibitors. The TMEM164 targeted inhibitors of the present invention can be directly used as drugs to specifically treat diseases targeting TMEM164.

[0013] In one embodiment of the present invention, the disease targeting TMEM164 is a neurodegenerative disease.

[0014] In one embodiment of the present invention, the disease targeting TMEM164 refers to a phospholipid metabolism-related disease.

[0015] In one embodiment of the present invention, the disease targeting TMEM164 refers to a tumor.

[0016] In one embodiment of the present invention, the disease targeting TMEM164 refers to lung adenocarcinoma.

[0017] In one embodiment of the present invention, the disease targeting TMEM164 refers to liver cancer.

[0018] In a more specific embodiment of the present invention, there is provided the use of ENT-montelukast or a derivative thereof in the preparation of a medicament for treating tumors as a ferroptosis inducer.

[0019] More specifically, the present invention provides the use of ENT-montelukast or its derivatives in the preparation of a medicament for treating lung adenocarcinoma or liver cancer as a ferroptosis inducer.

[0020] Specifically, TMEM164 intervenes in lipid metabolism to induce cell resistance to ferroptosis, and treatment with ENT-montelukast or its derivatives can significantly increase the sensitivity of TMEM164-overexpressing cell lines to ferroptosis inducers.

[0021] In one embodiment of the present invention, the ENT-montelukast or its derivatives are used in the preparation of drugs for inhibiting TMEM164 protein-mediated lipid metabolism to treat diseases targeting TMEM164.

[0022] The chemical formula of ENT-montelukast of the present invention is C 35 H 36 C l NO4S, with the structure shown in Formula 1, has three branches, one branch contains a carboxyl group, which is easy to form polar contact with proteins; the other two branches contain aromatic rings, which can form pi-pi valence bonds or pi-cation stacking with adjacent amino acid residues. At present, the biological effects of ENT-montelukast have not been elucidated.

[0023]

[0024] In one embodiment of the present invention, the ENT-montelukast derivative includes a salt of ENT-montelukast, an ENT-montelukast enantiomer, an ENT-montelukast salt enantiomer, or an ENT-montelukast impurity derivative containing impurities. Preferably, the ENT-montelukast derivative is ENT-montelukast sodium.

[0025] More preferably, the ENT-montelukast derivative is selected from the following structures:

[0026]

[0027] This study found that both the absence and overexpression of TMEM164 conferred resistance to ferroptosis in cells, and overexpression of TMEM164 in tumor cell lines also triggered lipid metabolism disorders. The results once again showed that TMEM164 is a membrane lipid remodeling agent that controls ferroptosis.

[0028] The ENT-montelukast or its derivatives designed and matched based on the crystal structure of the present application can be used as selective inhibitors to inhibit the lipoacyl transfer induced by TMEM164 protease.

[0029] Specifically, based on the crystal structure of TMEM164, this application studied and analyzed the key active centers in its coordination pocket, and determined through biological verification the key amino acid residues in the pocket that enable TMEM164 to function (specifically, the ionic coordination center formed by His181, Glu106 and Tyr177 stabilizes the phospholipid substrate. Secondly, the deprotonated thiol group of Cys123 attacks the ester bond at the sn-2 of the phospholipid substrate to form an intermediate product and release the accessory substance lysophospholipid. Next, another lysophospholipid substrate enters the pocket and is stabilized by the metal ions. His181 deprotonates the hydroxyl group at the sn-2 of the lysophospholipid substrate. Finally, the resulting hydroxyl group can directly attack the thioester bond of the fatty acyl-Cys123 formed in the intermediate state to generate the final product. In addition, the steric hindrance formed by G155 and F162 determines the specificity of the lipid substrate. When these sites mutate to alanine, TMEM164 will lose its function).

[0030] In addition, this application further identified and tested 22 candidate inhibitors of TMEM164 (TMEM164i) through virtual screening and rational design. The specific method is: 1. First screen a batch of small molecules with no anti-tumor activity (all of which have been reported and patented), and then select small molecules that can significantly increase the sensitivity of TMEM164 OE cell lines to ferroptosis; 2. Take these known small molecules for structural modification to increase the affinity of small molecules to TMEM164. Based on this, 11 high-scoring derivatives were selected through virtual docking scoring, and then molecular interaction evaluation methods such as surface ion resonance (SPR) were used. ENT-Montelukast or its derivatives (ENT-Montelukast sodium salt) have a strong binding affinity (nM level molecular affinity) to TMEM164. Finally, CCK-8 cell activity test was used to evaluate the effect of ENT-montelukast or its derivatives on improving the sensitivity of TMEM164 OE cell line to ferroptosis, and it was finally determined that the effect of ENT-montelukast or its derivatives was better than montelukast.

[0031] ENT-montelukast or its derivatives can occupy the active center of TMEM164, form polar contacts or valence bonds with key amino acid residues in the active center, and then inhibit the acyl transfer reaction mediated by TMEM164. Further studies have found that ENT-montelukast or its derivatives strongly bind to TMEM164 through ionic interactions and van der Waals forces.

[0032] Among them, ENT-montelukast or its salt and other derivatives showed an affinity for TMEM164 that was 4 orders of magnitude higher than montelukast in the SPR experiment, and the CCK-8 activity test verified that ENT-montelukast or its salt and other derivatives promoted TMEM164 OE ferroptosis significantly better than montelukast. This application uses ENT-montelukast or its salt and other derivatives as small molecule inhibitors of TMEM164 acyltransferase. This application study confirms that ENT-montelukast or its derivatives bind to TMEM164 to occupy its active pocket and inhibit the enzyme-catalyzed acylation reaction.

[0033] Compared with the prior art, the advantages of this application are embodied in the following aspects:

[0034] 1. The present invention simulates the binding of ENT-montelukast or its salts and other derivatives with TMEM164 through virtual molecular docking. The results show that the carboxyl-containing branches of ENT-montelukast or its salts and other derivatives respectively form salt bridges with positively charged residues and metal ions and form polar contacts with amino acid 162 (F162) of TMEM164, and the other two branches containing aromatic rings form pi-pi or pi-cation stacks (His220) with adjacent residues, thereby binding to the ion coordination catalytic pocket in TMEM164 ( Figure 4 ).

[0035] 2. The present invention proves through CCK-8 cell activity test that ENT-montelukast or its salt and other derivatives can effectively increase the sensitivity of TMEM164 overexpressing cell lines to the ferroptosis inducer RSL3, and this effect is a dose-response response.

[0036] Based on this, the present invention proposes that ENT-montelukast or its salt and other derivatives can be used as small molecule inhibitors of TMEM164, and is expected to improve the therapeutic effect and prognosis of tumor patients resistant to ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Screening of potential inhibitors of TMEM164.

[0038] Figure 2 :SPR experiment evaluates the molecular interaction affinity (K) of ENT-montelukast or its salts and other derivatives with TMEM164 D =5.52e-10M)

[0039] Figure 3 : Trend graph of cell activity in TMEM164 tumor cell line overexpressing RSL3 co-treated with gradient concentrations of montelukast (blue), ENT-montelukast or its salt derivatives (red).

[0040] Figure 4 : Results of virtual molecular docking of ENT-montelukast or its salts and derivatives with TMEM164. The amino acid residues that coordinate with the small molecule are marked in blue (Asn-237, His-220, Phe-162, Lys-110). The yellow dotted lines represent hydrogen bonds and polar contacts, the purple represents salt bridges, and the Pi-Pi conjugation is represented by the blue dotted lines. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] The small molecule ENT-montelukast sodium salt used in the following examples of the present invention was purchased from Aladdin, CAS No.: 190078-45-6. The specific structure is as follows:

[0043]

[0044] The materials not specially described in the following examples are chemical raw materials or biological materials commonly used in the art, and the operation methods not specially described in the following examples are conventional methods in the art.

[0045] Example 1: Evaluation of affinity between ENT-montelukast sodium salt and TMEM164 molecule

[0046] Experimental materials: CM5 chip, TMEM164 protein, pH 4.0 NaAc (Cytiva), NHS (Cytiva), EDC (Cytiva), Running buffer (Cytiva), ENT-montelukast sodium salt.

[0047] Experimental methods:

[0048] The experiment was conducted using the CM5 chip amino coupling method. 400mM EDC and 100mM NHS were added to the running buffer (10mM HEPES, pH7.4, 137mM NaCl, 2.7mM KCl, 0.05% P20, 1% DMSO) at a flow rate of 10μl / min for 600 seconds to activate the CM5 chip (the surface of the chip is attached with a carboxylated dextran surface. EDC and NHS were diluted in the buffer and slowly flowed through the CM5 chip channel at a specific flow rate, so that the carboxylated dextran can capture and bind proteins without destroying its own chemical properties. This process is called activating the chip). In order to couple the target protein, TMEM164 was diluted to 10μg / ml with 10mM acetic acid solution (pH 4.0) and passed through the activated CM5 chip channel at a flow rate of 10μl / min for 300 seconds. At this time, the CM5 chip was recorded as CM5-TMEM164.

[0049] Then, the coupled CM5-TMEM164 was blocked with 1M ethanolamine at a flow rate of 10μl / min for 600 seconds. The candidate small molecule (montelukast modified derivative) was diluted from 20μM to 0.156μM in a semi-gradient manner with running buffer (10mM HEPES, pH7.4, 137mM NaCl, 2.7mM KCl, 0.05% P20, 1% DMSO). Multi-cycle kinetics were measured at a flow rate of 30μl / min (surface ion resonance experiments are refined operation experiments. Specifically, the methodology is created on the SPR software. After placing the small molecule reagent in the designated clip, the instrument will aspirate the reagent according to the set program to make it pass through the microtubule channel on the CM5 chip. You only need to enter the parameters on the computer and place the reagent. After that, the instrument will collect data). Each cycle is 180 seconds, dissociation is 180 seconds, and then the next cycle is performed to obtain the kinetic affinity data of the TMEM164 protein. The acquired data were analyzed using Biacore Insight Evaluation software (V2.0.15.12933), and the kinetic fitting was performed using a 1:1 model.

[0050] The results showed that ENT-montelukast sodium salt had a strong affinity with TMEM164 (Table 1-40728, Figure 2 ).

[0051] Table 1 Affinity results of montelukast structurally modified derivatives and TMEM164 protein

[0052]

[0053]

[0054]

[0055] In Table 1, K D is the dissociation constant. The smaller the value, the stronger the affinity with the protein.

[0056] The experimental results show that ENT-montelukast sodium salt has great potential as a TMEM164 inhibitor.

[0057] Example 2: CCK-8 method to evaluate the ferroptosis-promoting effect of TMEM164 candidate inhibitors

[0058] Experimental materials: human fibrosarcoma cell line (HT1080, ATCC, CCL-121), 15% FBS culture medium, polyethylenimine (PEI), Opti-MEM, human embryonic kidney cells (HEK293T), cell activity test kit (CCK-8), RSA selective death inducer (RSL3), deferoxamine mesylate (DFOM), TMEM164i candidate molecule library (MCE, HY-LD-000005176), ENT-montelukast sodium salt (Aladdin), etc.

[0059] Experimental methods:

[0060] The culture medium formula involved in this experimental scheme is: 15% FBS diluted in high-glucose DMEM basic, and 1% NEAA and 1% Glutamax are added. This culture medium is hereinafter referred to as 15% FBS culture medium and is used in all experiments in this example.

[0061] Virus packaging:

[0062] The calcium phosphate transfection method was used to package viruses for overexpression vectors such as TMEM164.

[0063] HEK293T cells were cultured at 2 × 10 6 The cells were seeded at a density of 100 μg / mL on a 6-well plate (containing 15% FBS culture medium, and the cell confluence after attachment was 80%), and the cells were incubated in a 5% CO2, 37°C incubator for 16 h. The cell morphology was observed and replaced with fresh conventional culture medium (10% FBS, DMEM) after the cell morphology was confirmed to be correct.

[0064] Before preparing for transfection, observe the cell morphology and adhesion under a microscope to ensure that the cells are normal and free of contamination. Replace with fresh culture medium (15% FBS, DMEM), add 1.5 mL of culture medium to the 6-well plate, and replace 3 mL of culture medium to the 6-cm dish. Calculate the amount of transfection plasmid and calcium transfer reagent.

[0065] In this embodiment, the amount of plasmid used for transfection in a 6-well plate is 2ug, and 8ug for a 6-cm plate. Prepare a calcium phosphate transfection mixture. Add water first, then add the required plasmids, including the plasmid containing the target gene (plasmid containing the puro element) and the packaging plasmid (psPAPX2, PMD.2G). Add 2M calcium chloride to the mixture and blow it 15 times with a gun to mix. Then add 2×HBS and blow it vigorously for 30 times. When absorbing the liquid in this step, the gun tip should be as deep as possible into the liquid surface, and be careful not to suck it back. After mixing, let it stand for 5 minutes. 6-well plate mixed solution system: DNA 4μg (2μg PKD-TMEM164 / EGFP-IRES2-PURO plasmid + 1.2ug psPAPX2 + 0.8ugPMD.2G), 2M CaCl2 31.25μL, 2×HBS250μL, add water to make the volume fixed to 500μL. Add the resulting mixture dropwise to the culture dish, making sure that every area in each dish is covered, and shake it slightly to mix. Observe the upper layer of the culture medium under a microscope to see if there are small black particles. These particles are calcium phosphate precipitates formed by the plasmid. If there are, it is considered normal operation. Replace the fresh culture medium 15 hours after transfection and continue to culture in the incubator. The virus can be collected 48 hours after transfection for subsequent infection.

[0066] In this embodiment, conventional stable overexpression methods rely on lentiviral infection, wherein psPAPX2 and PMD.2G are packaging plasmids, which are transfected into cells to express viral coatings for encapsulating target genes to infect cells.

[0067] Lentivirus infection of HT1080 cells: 48h virus was collected and mixed with HT1080 cell culture medium at a ratio of 1:1, and 8ug / mL polybrene was added to infect HT1080 cells inoculated for 16h. Fresh culture medium was replaced after 10h treatment, and puromycin (puromycin, an aminoglycoside antibiotic produced by Streptomyces albicans) was added after 48h for continuous screening for 72h.

[0068] TMEM164 candidate inhibitors (TMEM164i, see Figure 1 Screening of compounds): HT1080 were seeded onto a 96-well plate at a density of 5000 / well. After 24 hours of attachment, gradient concentrations of TMEM164i and 6 μM RSL3 were added for co-treatment for 12 hours (specifically, 6 μM RSL3 solution was first prepared with 15% FBS culture medium, and then 3 and 9 μM TMEM164i solutions were prepared with RSL3 solution, respectively). Subsequently, 10 μL of CCK-8 reagent was added to each well and incubated for 4 hours. The absorbance value was detected at a wavelength of 450 nM.

[0069] Screening of montelukast modified derivatives: HT1080 was seeded onto a 96-well plate at a density of 5000 / well. After 24 hours of attachment, gradient concentrations of ENT-montelukast sodium and 6 μM RSL3 were added for co-treatment for 12 hours (specifically, 6 μM RSL3 solution was first prepared with 15% FBS culture medium, and then 0, 0.01, 0.1, 0.3, 1, 3, 9, and 27 μM TMEM164i solutions were prepared with RSL3 solution, respectively). Subsequently, 10 μL of CCK-8 reagent was added to each well and incubated for 4 hours, and the absorbance value was detected at a wavelength of 450 nM.

[0070] The results showed that the TMEMi screening results showed that in this molecular library, montelukast could significantly increase the sensitivity of TMEM164 OE cell line to ferroptosis and had no significant toxicity (anti-tumor activity) to HT1080 ( Figure 1 ). Subsequently, montelukast was used as the lead compound for further modification, and 11 high-scoring derivatives were determined by virtual docking fitting scores (Table 1). ENT-montelukast sodium was determined to have the highest affinity with TMEM164 by surface ion resonance experiments (Table 1, sequence number 40728). ENT-montelukast sodium was selected for biological verification. According to the results, compared with R-montelukast, ENT-montelukast sodium (montelukast enantiomer S configuration) is more efficient in promoting the sensitivity of TMEM164 OE cell line to ferroptosis ( Figure 3 ).

[0071] The above experiments prove that ENT-montelukast or its salt and other derivatives can effectively inhibit the activity of TMEM164 protein and inhibit the enzymatic acyl transfer function of TMEM164.

[0072] As introduced in the background technology, Tao Fan's case report shows that TMEM164 is abnormally activated in patients with lung adenocarcinoma (Fan, T., Liu, Y., Liu, H., Wang, L., Tian, ​​H., Zheng, Y., ... He, J. (2022). Transmembrane Protein-Based Risk Model and H3K4me3ModificationCharacteristics in Lung Adenocarcinoma. Front Oncol, 12, 828814.), and the prior art shows that TMEM164 in patients with lung adenocarcinoma is significantly upregulated, and TMEM164 affects the treatment and prognosis of lung adenocarcinoma. The above experiments in this application can prove that ENT-montelukast or its salts and other derivatives (especially compound 40728) can effectively inhibit the activity of TMEM164 protein. Therefore, based on the research of this application and the prior art in this field, it can be confirmed that ENT-montelukast or its salts and other derivatives (especially compound 40728) can be used to prepare drugs for the treatment of lung adenocarcinoma.

[0073] Masashi Nishikawa reported that TMEM164 has the effect of promoting the progression of liver cancer (Nishikawa, M., Okada, H., Kawaguchi, K., Shimakami, T., Nio, K., Arai, K.,... Honda, M. (2023). Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus. Cell Mol Gastroenterol Hepatol, 16(2), 263-286.). 164 is significantly upregulated in human liver cancer cells, and in vitro experiments have verified that overexpression of 164 promotes cancer progression. The above experiments in this application can prove that ENT-montelukast or its salt and other derivatives (especially compound 40728) can effectively inhibit the activity of TMEM164 protein. Therefore, based on the research of this application and the existing technology in this field, it can be confirmed that ENT-montelukast or its salt and other derivatives (especially compound 40728) can be used to prepare drugs for the treatment of liver cancer.

[0074] Therefore, the above examples of the present application illustrate that ENT-montelukast or its salts and other derivatives are expected to play a therapeutic role in inhibiting TMEM164-mediated diseases.

[0075] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of ENT-montelukast or its derivatives in the preparation of drugs for treating diseases targeting TMEM164.

2. The use according to claim 1, characterized in that: ENT-montelukast or its derivatives are used in the preparation of TMEM164 targeted inhibitors, which are drugs that can specifically treat diseases targeting TMEM164.

3. The use according to claim 1, characterized in that: The disease targeting TMEM164 refers to a neurodegenerative disease.

4. The use according to claim 1, characterized in that: The diseases targeting TMEM164 refer to phospholipid metabolism-related diseases.

5. The use according to claim 1, characterized in that: The disease targeting TMEM164 refers to tumors.

6. The use according to claim 1, characterized in that: The disease targeting TMEM164 refers to lung adenocarcinoma or liver cancer.

7. The use according to claim 1, characterized in that: ENT-montelukast or its derivatives are used in the preparation of drugs for treating lung adenocarcinoma or liver cancer as ferroptosis inducers.

8. The use according to claim 1, characterized in that: The ENT-montelukast or its derivatives are used in the preparation of drugs for inhibiting TMEM164 protein-mediated phospholipid metabolism to treat diseases targeting TMEM164.

9. The use according to claim 1, characterized in that: The ENT-montelukast derivatives include salts of ENT-montelukast, and ENT-montelukast enantiomers, salt enantiomers of ENT-montelukast, or impurity derivatives of ENT-montelukast containing impurities.

10. The use according to claim 1, characterized in that: The ENT-montelukast derivative is selected from the following structures:

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