Inhibitor for targeting protein interaction and application thereof
Through screening and evaluation, it was found that Deslanoside can inhibit ACE2-LAT1/3 interaction in a dose-dependent manner, thereby effectively inhibiting the invasion of new coronavirus cells at lower concentrations, solving the problem of the failure of existing inhibitors in mutations.
Patent Information
- Application Number
- CN202510229922.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-04-25
AI Technical Summary
Existing inhibitors of the new coronavirus may fail in the case of mutations in the S protein, making it difficult to effectively inhibit the invasion of the new coronavirus from cells.
Through screening and evaluation, it was found that Deslanoside can inhibit ACE2-LAT1/3 interaction in a dose-dependent manner, thereby inhibiting the invasion of the new coronavirus in cells.
Deslanoside can effectively inhibit the invasion of new coronavirus cells at a lower concentration of 0.2μM, providing an effective anti-new coronavirus drug.
Smart Images
Figure CN119874798A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological and protein interaction inhibitor research, in particular to an inhibitor targeting protein interaction and application thereof. Background Art
[0002] Protein-protein interaction is an important molecular event in the life activities of organisms. It is not only related to cell signal transduction, gene expression and regulation, but also directly related to the occurrence and development of many diseases, virus-host interaction, etc. It is also an important drug target under different pathological conditions (Rabbani, G. et al. Curr Protein Pept Sc 2018, 19 (10), 948-957). Screening inhibitors targeting key protein-protein interactions can provide corresponding strategies for the treatment of diseases. For example, screening inhibitors targeting the interaction between the tumor suppressor p53 protein and the E3 ubiquitin ligase Mdm2 protein can provide strategies for the treatment of cancer (Atatreh, N. et al. Drug Des Dev Ther 2018, 12, 3741-3752.); screening inhibitors targeting the interaction between integrase and host factor p75 during HIV-1 viral integration can help HIV-1 treatment. The interaction between proteins plays an important role in the life process of organisms (Chen, MH et al. Biomaterials 2015, 48, 97-107.). Studies have found that the new coronavirus is its pathogenic factor, and the cell protein ACE2 is its receptor factor for invasion. Screening and evaluating inhibitor compounds for the interaction between the new coronavirus protein and the host factor protein can provide help for the prevention and treatment of the new coronavirus. Although several inhibitors have been reported to disrupt S-ACE2 interaction and inhibit viral entry, such as sACE2 and Bamlanivimab (LY-CoV555), mainly by occupying the RBD or ACE2 binding sites of the S protein and preventing the two from binding, mutations in the S protein may cause the inhibitors to fail.
[0003] Therefore, the present invention screened a protein interaction inhibitor for the ACE2-LAT1 / 3 interaction. After screening and evaluation, it was found that Deslanoside can inhibit the invasion of new coronavirus into cells in a dose-dependent manner. Deslanoside is an effective inhibitor that targets the ACE2-LAT1 / 3 interaction and inhibits the invasion of new coronavirus. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an inhibitor targeting protein interaction and its application.
[0005] The technical solution adopted by the present invention to solve its technical problem is:
[0006] An inhibitor targeting protein ACE2-LAT1 / 3 interaction, the inhibitor is Deslanoside, i.e. deacetyl lanolin, with a chemical formula of C 47 H 74 O 19 , the structural formula is:
[0007] Furthermore, the inhibitor is capable of inhibiting ACE2-LAT1 / 3 interaction.
[0008] Furthermore, the inhibitor can inhibit the invasion of new coronavirus into cells in a dose-dependent manner.
[0009] Furthermore, the inhibitor is able to inhibit the invasion of the new coronavirus at a lower concentration of 0.2 μM.
[0010] Furthermore, the inhibitor can inhibit the ACE2-LAT1 / 3 interaction, thereby inhibiting the invasion of the new coronavirus into cells in a dose-dependent manner.
[0011] Use of the inhibitor as described above in the preparation of a drug for inhibiting ACE2-LAT1 / 3 interaction.
[0012] Use of the inhibitor as described above in the preparation of a drug for inhibiting the invasion of the new coronavirus in living cells.
[0013] The use of the inhibitors as described above in the preparation of drugs that target ACE2-LAT1 / 3 interaction and thereby inhibit the invasion of the new coronavirus.
[0014] The advantages and positive effects achieved by the present invention are:
[0015] 1. The present invention discovered that Deslanoside inhibitors can inhibit ACE2-LAT1 / 3 interaction; moreover, Deslanoside inhibitors can inhibit the invasion of novel coronavirus into cells in a dose-dependent manner; at the same time, Deslanoside inhibitors can exert the effect of inhibiting the invasion of novel coronavirus at lower concentrations, therefore, Deslanoside inhibitors can be used as drugs against novel coronavirus.
[0016] 2. The present invention provides an inhibitor targeting the protein ACE2-LAT1 / 3 interaction - Deslanoside. The inhibitor Deslanoside can be used as an inhibitor targeting the ACE2-LAT1 / 3 interaction, and the inhibitor can inhibit the invasion process of the new coronavirus in living cells.
[0017] 3. The present invention takes ACE2-LAT1 / 3 interaction as the research object. First, through virtual screening, compounds with higher scores are screened from the compound library; then the effect of the compound on protein interaction is tested at the living cell level; finally, the effect of the compound on the invasion of new coronavirus into cells is tested at the living cell level. An inhibitor targeting protein interaction is screened, and the inhibitor is Deslanoside. An application of an inhibitor targeting protein interaction, the application process of which includes: Deslanoside inhibitor can inhibit ACE2-LAT1 / 3 interaction, and then inhibit the invasion of new coronavirus into cells in a dose-dependent manner.
[0018] 4. The present invention first identified the interaction of ACE2-LAT1 / 3 proteins, then evaluated the targeted inhibitors through virtual screening of the ACE2-LAT1 / 3 interaction region, and finally evaluated the effect of the inhibitors on the invasion of the new coronavirus at the living cell level. After screening and evaluation, we found that Deslanoside can inhibit the invasion of the new coronavirus into cells in a dose-dependent manner. Deslanoside is an effective inhibitor that targets the ACE2-LAT1 / 3 interaction and inhibits the invasion of the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a fluorescence schematic diagram of the ACE2-LAT1 / 3 protein interaction in the present invention;
[0020] Figure 2 It is a fluorescence schematic diagram of the ACE2-LAT1 / 3 protein interaction region in the present invention;
[0021] Figure 3 It is a fluorescence statistical diagram of the intensity of Deslanoside targeting ACE2-LAT1 / 3 interaction in the present invention;
[0022] Figure 4 This is a statistical chart showing that Deslanoside in the present invention inhibits the invasion of the new coronavirus. DETAILED DESCRIPTION
[0023] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.
[0024] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.
[0025] An inhibitor targeting protein ACE2-LAT1 / 3 interaction, the inhibitor is Deslanoside, i.e. deacetyl lanolin, with a chemical formula of C 47 H 74 O 19 , the structural formula is:
[0026] Preferably, the inhibitor is capable of inhibiting ACE2-LAT1 / 3 interaction.
[0027] Preferably, the inhibitor can inhibit the invasion of SARS-CoV-2 into cells in a dose-dependent manner.
[0028] Preferably, the inhibitor is able to inhibit the invasion of the new coronavirus at a lower concentration of 0.2 μM.
[0029] Preferably, the inhibitor can inhibit the ACE2-LAT1 / 3 interaction, thereby inhibiting the invasion of the new coronavirus into cells in a dose-dependent manner.
[0030] Use of the inhibitor as described above in the preparation of a drug for inhibiting ACE2-LAT1 / 3 interaction.
[0031] Use of the inhibitor as described above in the preparation of a drug for inhibiting the invasion of the new coronavirus in living cells.
[0032] The use of the inhibitors as described above in the preparation of drugs that target ACE2-LAT1 / 3 interaction and thereby inhibit the invasion of the new coronavirus.
[0033] Specifically, the relevant preparation and detection are as follows:
[0034] Embodiment 1:
[0035] An inhibitor targeting protein interaction is obtained according to the following steps:
[0036] (1) Identification of ACE2-LAT1 / 3 interaction: The interaction between ACE2 and LAT1 and LAT3 was identified by the developed miRFP670nano3 bimolecular fluorescence complementation system. Specifically, ACE2 was fused with the miR3C108 fragment split by miRFP670nano3, and LAT1 / 3 was fused with the miR3N107 fragment split by miRFP670nano3; then they were co-transfected into HEK 293T cells, respectively. Figure 1 , the generation of miRFP670nano3 reconstructed fluorescence was detected, proving that ACE2 interacts with both LAT1 and LAT3.
[0037] (2) Identification of ACE2-LAT1 / 3 interaction regions: The miRFP670nano3 bimolecular fluorescence complementation system was developed to identify the regions where ACE2 interacts with LAT1 and LAT3. Specifically, the different split regions of ACE2 were fused with the miR3C108 fragment split by miRFP670nano3, and LAT1 / 3 were fused with the miR3N107 fragment split by miRFP670nano3; then they were co-transfected into HEK 293T cells, such as Figure 2 The PD domain of ACE2 did not detect the production of mi RFP670nano3 reconstructed fluorescence with LAT1 and LAT3, while the CLD domain of ACE2 detected the production of miRFP670nano3 reconstructed fluorescence with LAT1 and LAT3, proving that it is mainly the CLD domain of ACE2 that mediates the interaction with LAT1 and LAT3.
[0038] (3) Virtual screening of inhibitors targeting ACE2-LAT1 / 3 interaction: Experiments have shown that LAT1 / 3 mainly interacts with the CLD region of ACE2. Inhibitor compounds targeting the CLD region were screened virtually and ranked according to binding strength. The software used for virtual screening is Maestro 12.8, 3D drawing software is PyMol.
[0039] Evaluation of inhibitors targeting ACE2-LAT1 / 3 interaction: ACE2 was fused with the miR3C108 fragment split by miRFP670nano3, and LAT1 / 3 was fused with the miR3N107 fragment split by miRFP670nano3, respectively; then they were co-transfected into HEK 293T cells, and 4 hours after transfection, the medium was changed and 20 μM of Deslanoside (deacetylated lanolin, chemical formula C) obtained by virtual screening was added 47 H 74 O 19 , the structural formula is: ), and the control was treated with DMSO. After 24 hours of transfection, the intensity of the reconstructed fluorescence of miRFP670nano3 was detected, and the fluorescence intensity of each group was counted. Figure 3 As shown, the inhibitor Deslanoside effectively inhibited the interaction between ACE2 and LAT1 and LAT3.
[0040] (4) Inhibitor inhibition of novel coronavirus invasion into cells: Inhibitors of different concentrations (0, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM) were added to Caco-2 cells. After 6 hours, the culture medium was discarded and the cells were washed once with PBS. 2 mL of novel coronavirus solution (virus titer was 1 × 10 5 TU / mL), incubated on ice for 30 min to allow the virus to adhere to the cell surface, then incubated at 37°C for 2 h, collected and lysed the cells, and RNA was extracted for qPCR detection of the amount of virus invasion in the cells. Figure 4 As shown, Deslanoside can inhibit the invasion of the new coronavirus at a lower concentration of 0.2μM.
[0041] The excitation channel selected for EGFP green fluorescence is 488nm, the excitation channel selected for miRFP670nano3 near-infrared fluorescence is 640nm, and the excitation channel selected for Hoechst is 405nm.
[0042] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.
Claims
1. An inhibitor targeting protein ACE2-LAT1 / 3 interaction, characterized in that: The inhibitor is Deslanoside, i.e. deacetyl lanolin, with a chemical formula of C 47 H 74 O 19 , the structural formula is:
2. The inhibitor according to claim 1, characterized in that: The inhibitor is capable of inhibiting ACE2-LAT1 / 3 interaction.
3. The inhibitor according to claim 1, characterized in that: The inhibitor can inhibit the invasion of new coronavirus into cells in a dose-dependent manner.
4. The inhibitor according to claim 1, characterized in that: The inhibitor is able to inhibit the invasion of the new coronavirus at a lower concentration of 0.2 μM.
5. The inhibitor according to any one of claims 1 to 4, characterized in that: The inhibitor can inhibit the ACE2-LAT1 / 3 interaction, thereby inhibiting the invasion of the new coronavirus into cells in a dose-dependent manner.
6. Use of the inhibitor according to any one of claims 1 to 5 in the preparation of a drug for inhibiting ACE2-LAT1 / 3 interaction.
7. Use of the inhibitor as described in any one of claims 1 to 5 in the preparation of a drug for inhibiting the invasion of the new coronavirus in living cells.
8. Use of the inhibitor as described in any one of claims 1 to 5 in the preparation of a drug that targets the ACE2-LAT1 / 3 interaction and thereby inhibits the invasion of the new coronavirus.