Application of loperamide in preparation of targeted inhibitor of LMCD1 protein

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to loperamide and application of pharmaceutically acceptable salt thereof in preparation of an LMCD1 targeted inhibitor. Molecular docking and trace thermophoresis experiments prove that loperamide can be specifically bound to a PET structural domain of LMCD1 protein for the first time, so that the activity of LMCD1 is inhibited. In an in-vivo animal model, loperamide can obviously delay the process of c-Met / AKT-induced mouse hepatocellular carcinoma, which shows that the number and volume of tumors are reduced, the specific gravity of liver and spleen is reduced, and liver function indexes (ALT and AST) are improved. The invention provides a brand-new targeted treatment strategy with high safety for the treatment of hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of loperamide in preparation of a targeted inhibitor of LMCD1 protein. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the main type of primary liver cancer, most cases are discovered in the late stage, and is one of the main causes of cancer-related deaths worldwide. At present, the treatment means of HCC is limited, and for patients in the late stage, chemotherapy and targeted therapy are the main choices, but there are problems such as strong drug resistance and large toxic side effects. Therefore, it is urgent to develop new, efficient and low-toxicity treatment targets and drugs.

[0003] LIM and cysteine-rich domain protein 1 (LMCD1) is a member of the LIM protein family, and is considered to be a putative metastatic gene in human HCC. Studies have shown that LMCD1 plays a role as a transcription factor in epithelial-mesenchymal transition (EMT), mediates the progression of EMT, and thus promotes the invasion and metastasis of tumor cells. In the TCGA database and clinical samples, the expression of LMCD1 in HCC tissues is significantly higher than that in normal tissues adjacent to cancer, and its high expression is related to the poor prognosis of patients. Therefore, LMCD1 is a new target with great potential for the treatment of HCC. However, there is no specific inhibitor targeting LMCD1 protein reported so far. SUMMARY

[0004] Therefore, the purpose of the present application is to overcome the shortcomings of the prior art, and for the first time, it is disclosed that loperamide is a new LMCD1 targeted inhibitor, and a new application thereof in the treatment of hepatocellular carcinoma is provided.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, the present application provides an application of loperamide or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof in the preparation of a targeted inhibitor of LMCD1 protein.

[0006] Loperamide (Loperamide, LOP) is a peripheral opioid receptor agonist, which has been widely used to control and relieve acute and chronic diarrhea since its marketing in 1973, and its safety has been fully verified, and it is even sold as an over-the-counter drug. In recent years, studies have found that loperamide has certain anti-tumor activity, such as inducing tumor cell apoptosis and inhibiting angiogenesis. However, these studies have not revealed its specific molecular target, and the mechanism of its anti-tumor effect is still unclear. So far, no prior art discloses or suggests that loperamide can specifically target LMCD1 protein and be used for targeted therapy of hepatocellular carcinoma. However, the present application discloses for the first time that loperamide is a new LMCD1 targeted inhibitor.

[0007] Furthermore, the targeting of the LMCD1 protein refers to the binding of loperamide to the PET domain of the LMCD1 protein.

[0008] In a second aspect, the invention provides the use of loperamide or a pharmaceutically acceptable salt, ester, prodrug, or metabolite thereof in the preparation of a medicament for the prevention and / or treatment of hepatocellular carcinoma.

[0009] Furthermore, the hepatocellular carcinoma is associated with the expression or activity of the LMCD1 protein; specifically, the expression of the LMCD1 protein in the hepatocellular carcinoma is higher than that in normal tissue.

[0010] Furthermore, the drug is administered by injection and / or orally.

[0011] Furthermore, the dosage of the loperamide or its pharmaceutically acceptable salt, ester, prodrug, or metabolite is 1-2 mg / kg / day.

[0012] In a third aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of hepatocellular carcinoma, the pharmaceutical composition comprising a therapeutically effective amount of loperamide or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0013] Furthermore, the composition is configured for administration by intraperitoneal injection.

[0014] The present invention also provides a method for screening LMCD1 targeting inhibitors, characterized by comprising the following steps: using molecular docking technology to test the binding ability of candidate compounds to the PET domain of LMCD1 protein; and / or using micro-thermophoresis to determine the affinity constant KD of candidate compounds to LMCD1 protein.

[0015] The beneficial effects of this invention include at least the following: 1. Clear target: This invention is the first to demonstrate through molecular docking and micro-thermophoresis experiments that loperamide is a direct target inhibitor of LMCD1, clarifying its molecular mechanism of anti-liver cancer and providing a solid theoretical basis for "repurposing old drugs".

[0016] 2. Significant therapeutic effect: In the c-Met / AKT-induced mouse HCC model, loperamide treatment can significantly reduce tumor volume and number, improve liver function indicators, and show good anti-hepatocellular carcinoma effect.

[0017] 3. High safety: Loperamide has been on the market for decades, with sufficient human safety data and few toxic side effects. Its reuse in the treatment of liver cancer can greatly shorten the new drug development cycle and reduce the risks of clinical development.

[0018] 4. Novel Strategy: This invention provides a novel targeted therapy strategy for hepatocellular carcinoma, especially for patients with hepatocellular carcinoma who highly express LMCD1. Attached Figure Description

[0019] Figure 1 The procedure for establishing a c-Met / AKT-induced mouse HCC model and the gross and liver tissue changes in FVB / N mice are shown.

[0020] Figure 2 The following is a validation diagram of LMCD1 high expression in HCC: (A) Mouse transcriptome sequencing differential gene heatmap / volcano plot shows LMCD1 upregulation, and (B) TCGA database analysis shows LMCD1 high expression in human HCC tissues.

[0021] Figure 3 The diagram shows the direct binding of LOP and LMCD1. (A) Molecular docking of LMCD1 and LOP; (B) Thermophoretic kinetics experiment to detect the change in affinity constant of LMCD1 after the addition of LOP; (C) The change in thermal melting curve of LMCD1 after the addition of LOP.

[0022] Figure 4 To delay the progression of c-Met / AKT-induced HCC by LOP, (A) experimental procedure; (B) gross and liver tissue changes in FVB / N mice; (C) liver specific gravity; (D) spleen specific gravity; (E) serum ALT; (F) serum AST. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0025] The following specific embodiments illustrate the solution proposed in this invention:

[0026] Example 1: Establishment of a mouse hepatocellular carcinoma (HCC) model (1) Using a plasmid extraction kit (purchased from Vazyme, DC202-01), sufficient amounts and appropriate concentrations of AKT-PT3EF1a plasmid, c-Met-PT3EF1a plasmid, and Sleeping Beauty transposon plasmid (plasmid provided by Hubei University of Traditional Chinese Medicine) were extracted. The extracted plasmids were transfected into cells using Lipo8000 (purchased from Beyotime, C0533), and the expression of the plasmids in cells was verified by Western blot experiment. (2) The AKT-PT3EF1a plasmid, c-Met-PT3EF1a plasmid, and Sleeping Beauty transposon plasmid were dissolved in 2 mL of 0.9% saline at a ratio of 12.5:12.5:1 to obtain the plasmid dilution. 4-6 week old FVB / N mice weighing approximately 20 g were selected, and approximately 2 mL of the plasmid dilution was injected into the mice via the tail vein using a syringe at a rapid injection time of 5-7 seconds. According to the experimental design requirements, the mouse models were randomly divided into three groups: a control group (injected only with saline), a positive control group (injected with plasmid dilution), and a LOP (purchased from MCE) treatment group (injected with plasmid dilution first, followed by drug treatment). (3) Record the weight of the mice weekly and observe the changes in their abdominal circumference; (4) If the mice show signs of abdominal bloating and weight gain after about 3 weeks, the model is considered to be successful.

[0027] (5) Mice were anesthetized and euthanized with isoflurane (purchased from EZVET, G45993). The liver and spleen were removed, weighed, and photographed. The mouse liver tissue was placed in a labeled cryovial and quickly placed in liquid nitrogen for subsequent sequencing and other experiments.

[0028] The results showed that after 3-6 weeks, the abdomen of mice in the c-Met / AKT group bulged outward, and a significant mass was observed in the liver. Compared with the saline control group, the gross liver tissue of mice in the c-Met / AKT group showed increased liver volume and weight, and the liver surface transformed into a tumor morphology with diffuse fatty degeneration, distributed in various liver lobes, indicating the successful establishment of the FVB / N mouse NAFLD-HCC model. Figure 1 ).

[0029] Example 2: Transcriptome sequencing reveals and validates the key target LMCD1 (1) Total RNA was extracted using VeZol reagent (purchased from Vazyme, R411-01) according to the manufacturer’s instructions.

[0030] (2) RNA purity was detected and quantitatively analyzed using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 bioanalyzer.

[0031] (3) Libraries were constructed using the TruSeq Stranded mRNA LT sample preparation kit. Transcriptome sequencing and subsequent analysis were performed by Shanghai Ouyi Biotechnology Co., Ltd., and library sequencing was performed on the Illumina HiSeq X Ten platform.

[0032] (4) The expression differences of LMCD1 in human hepatocellular carcinoma tissues were analyzed using the TCGA database.

[0033] The results showed that differential gene heatmap analysis revealed that LMCD1 was highly expressed in hepatocellular carcinoma tissues of a mouse HCC model. Analysis of this gene in the TCGA database showed that LMCD1 expression was increased in human hepatocellular carcinoma and significantly different from the control group. Figure 2 ).

[0034] Example 3: Verification of the direct binding of loperamide to LMCD1 3.1 Molecular docking Using the mouse-derived LMCD1 protein sequence (UniProt ID: Q8VEE1), its three-dimensional structure was predicted de novo using AlphaFold3. The model's validity was verified using visualization software, and a potential binding site identified by cavity analysis within the region was selected for subsequent docking.

[0035] The structure of the small molecule ligand loperamide was obtained from the PubChem database, and hydrogenation and energy optimization were performed under physiological pH conditions. Docking was performed using AutoDock Vina. The docking results were sorted according to binding free energy, and key binding modes such as hydrogen bonds, hydrophobic interactions, and cation-π interactions in the optimal binding conformation were analyzed using molecular visualization and interaction analysis software.

[0036] 3.2 Micro-thermophoresis (MST) Experiment (1) Preparation of protein samples: Take cells transfected with fluorescent plasmids, wash twice with pre-cooled PBS to remove the culture medium, add an appropriate amount of RIPA lysis buffer (PI-free) (purchased from Beyotime, P0013C), and lyse on ice for 30 min. Centrifuge at 4℃ and 12000 r / min for 15 min, and transfer the supernatant to a new centrifuge tube for later use; (2) Prepare the following intermediate samples: Dilute the target compound GFP-LMCD1 (10 μmol / L) to 40 nmol / L for later use. Dilute the ligand LOP (50 mmol / L) to 5 mmol / L. Add 20 μL of ligand buffer to 180 μL of detection buffer. Use the diluted ligand buffer to perform serial dilutions. Note that the 10 μL sample with the lowest concentration in tube 16 should be discarded to ensure that all sample volumes are 10 μL. (3) Ligand binding to target: Add 10 μL of 40 nmol / L GFP-LMCD1 to each of tubes 16 to 1 and mix well with a pipette; (4) On-machine testing: Immerse the Monolith NT.115 capillary tubes into tubes 1 to 16 in sequence, and then place the capillary tubes into positions 1 to 16 on the instrument tray to start the measurement.

[0037] 3.3 CETSA Experiment (1) Digest an appropriate amount of transfected cells, resuspend the cells in PBS (containing PI) (purchased from Servicebio, G0002), and divide them into two equal portions and place them into EP tubes; (2) Add equal amounts of DMSO (purchased from PanReac AppliChem, A3672) and LOP to the EP tube respectively, and let it stand at room temperature for 2 h; (3) Divide the two groups of cell suspensions into 200 μL PCR tubes, 200 μL per tube, and divide them into 7 groups; (4) Set the metal bath temperature to 40, 45, 50, 55, 60, 65 and 70℃, heat the 7 groups of PCR tubes for 3 min, and then cool them down to 25℃ and place them for 3 min. (5) After the PCR tube is taken out and cooled to room temperature, it is placed in liquid nitrogen and repeatedly frozen and thawed 3 times, and then centrifuged at 20000 g for 20 min. (6) Transfer the supernatant to a new EP tube, add 5× loading buffer, and perform WB detection directly.

[0038] The results are as follows Figure 3 As shown, homology modeling was performed using AlphaFold3 based on the mouse-derived LMCD1 protein sequence (UniProt ID: Q8VEE1), and its three-dimensional structure was visualized and analyzed. Previous literature indicates that this type of protein typically contains a PET domain responsible for protein-protein interactions and two binding domains. The LIM domain. This invention found that these known functional regions spatially overlap with the predicted structural cavity, thus speculating that small molecule compounds may influence protein-protein interactions of LMCD1 by binding to this cavity.

[0039] Through active cavity prediction and analysis, a potential binding pocket was identified, mainly composed of residues such as Gln20, Lys68, Arg67, Arg71, Arg170, and Ser169. Molecular docking results showed that Arg170, Arg71, and Lys68 may participate in ligand binding through cation-π interactions, while Gln20 may form hydrogen bonds with the compound. Therefore, it is inferred that LOP may specifically bind to the PET domain pocket of the LMCD1 protein, thereby competitively inhibiting its interactions with other proteins and further blocking downstream signaling pathways, ultimately inhibiting cancer cell growth. Further MST and CETSA experiments showed that the affinity constant KD of LMCD1 changed after the addition of LOP, indicating that LOP interacts with LMCD1.

[0040] Example 4: Pharmacodynamic evaluation of loperamide in delaying the progression of HCC in vivo (1) Three weeks after model establishment, mice were administered drugs via intraperitoneal injection. Mice were divided into three groups: saline control group, c-Met / AKT model group, and c-Met / AKT + loperamide treatment group. The treatment groups were given loperamide at doses of 1 mg / kg / day and 2 mg / kg / day, respectively, for 3 consecutive weeks.

[0041] (2) Six weeks later, mice were anesthetized with isoflurane, and about 1 mL of blood was collected through the heart using a heparin-soaked 1 mL syringe. The blood was centrifuged at 3000 r / min for 10 min at room temperature, and 300-400 μL of pale yellow serum was collected for ALT and AST detection. The liver and spleen were taken, weighed, and photographed.

[0042] The results showed that, compared with the model group, the number and volume of tumor nodules in the livers of mice treated with loperamide (especially the 2 mg / kg / day group) were significantly reduced. Figure 4 B); at the same time, the liver weight and spleen weight ( Figure 4 C, D) and serum levels of liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (C, D) Figure 4 E and F were also significantly reduced. These results fully demonstrate that loperamide can effectively inhibit c-Met / AKT-induced HCC progression.

[0043] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0044] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of loperamide in the preparation of drugs for inhibiting LMCD1 protein activity.

2. The application according to claim 1, characterized in that, Loperamide binds to the PET domain of the LMCD1 protein.

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