Biological preparation for inhibiting generation of migration body and application of biological preparation
By developing biological agents that inhibit Rac1 and PIP5K1α, the nucleation and maturation of the migrating organisms were suppressed, solving the problem of receptor-independent viral transmission, achieving highly efficient viral inhibition, and expanding the target range for antiviral research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-10
AI Technical Summary
In the current technology, the traditional view is that viral infection depends on receptor-mediated recognition and endocytosis. However, in some cell types, viruses may spread through non-receptor-dependent mechanisms, and there is a lack of effective means to inhibit them.
A biological agent containing the Rac1 inhibitor NSC23766 and the PIP5K1α inhibitor ISA-2011B was developed to block receptor-dependent viral transmission by inhibiting the nucleation and maturation processes of the migratory organism.
It significantly inhibits the spread of the virus through migratory organisms, reduces the concentration of the drug used, and is highly effective and low in toxicity. It expands the target range for antiviral research and has important clinical application value.
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Figure CN121622685A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to biological agents for inhibiting the generation of migrasome and the application thereof. BACKGROUND
[0002] Viral infection is an important pathogenic mechanism of many human and animal diseases. The traditional view believes that the invasion of viruses into host cells mainly depends on receptor-mediated recognition and endocytosis process, that is, viruses enter the cell interior by combining with specific receptors on the surface of host cell membrane, inducing endocytosis or membrane fusion. However, in some cell types, even if the virus lacks specific receptors, the presence of viral particles or infection markers can still be detected, suggesting that viruses may spread through other non-receptor-dependent ways.
[0003] Migrasome is a newly discovered membrane organelle in recent years, which is formed by breaking away from the cell on the contractile filament of the migration track during cell migration. Studies have shown that the generation of migrasome undergoes three stages of nucleation, maturation and detachment. In the nucleation stage, Rac1 activation triggers cytoskeleton reconstruction, playing a core regulatory role. Rac1 belongs to the Rho family of small GTPases, and its activated form can further activate the Arp2 / 3 complex by combining with the WAVE complex, inducing branched polymerization of actin, thereby driving the generation of migrasome. The maturation of migrasome depends on the regulation of membrane phospholipid signals, in which PIP5K1α (phosphatidylinositol-4-phosphate 5-kinase type Iα) plays a key role. PIP5K1α catalytically generates PI (4, 5) P2, which recruits small GTPase Rab35 and integrin Integrin α5, and promotes the aggregation of TSPAN4 in the membrane domain of migrasome, thereby driving the maturation of migrasome.
[0004] Recent studies have shown that the process of viral infection is closely related to the dynamic regulation of the cytoskeleton system of host cells, forming membrane protrusion structures including lamellipodia, filopodia and migrasome. The applicant found that migrasome not only plays a role in normal cell migration, embryonic development, tissue repair and other processes, but also can be used by viruses as a channel for transmission or invasion. Therefore, the development of new small molecule inhibitors based on the inhibition of migrasome generation and maturation can inhibit viral non-receptor pathway infection and play a broad-spectrum antiviral role. SUMMARY
[0005] In view of the above technical limitations, the present application provides a biological agent for inhibiting cell migration body formation and its application in inhibiting virus transmission. First, a series of small molecule inhibitors against Rac1 and PIP5K1a are screened from the National Cancer Institute (NCI) public compound database, and then small molecule inhibitors that can effectively inhibit the formation of migration bodies are further screened by nanoparticle tracking analysis for subsequent antiviral research. The present application overcomes the deficiencies and defects mentioned in the background art.
[0006] To achieve the above object, the present application adopts the following technical solution: The present application provides a biological agent for inhibiting cell migration body formation, which comprises a Rac1 inhibitor; preferably, the Rac1 inhibitor is NSC23766.
[0007] Optionally, the biological agent, the effective concentration of the Rac1 inhibitor is 20-50 μM, preferably 25 μM.
[0008] Optionally, the biological agent, the cell comprises one or more of brain tumor cell lines, lung cancer cell lines, cervical cancer cell lines, and leukemia myeloma cell lines.
[0009] Optionally, the biological agent, the biological agent further comprises a PIP5K1a inhibitor; preferably, the PIP5K1a inhibitor is ISA-2011B.
[0010] Optionally, the biological agent, the effective concentration of the PIP5K1a inhibitor is 10-25 μM.
[0011] The second application point of the present application is to provide a method for inhibiting cell migration body formation, which is achieved by adding the above-mentioned biological agent to the cell.
[0012] Optionally, the method, the cell is selected as a virus-infected cell.
[0013] The third application point of the present application is to provide the application of the above-mentioned biological agent in inhibiting virus transmission.
[0014] Optionally, the application, the virus transmission refers to the non-receptor-dependent transmission of viruses between cells in the form of migration bodies.
[0015] Optionally, the application, the Rac1 inhibitor reduces the formation of migration bodies by inhibiting the nuclearization of migration bodies, thereby inhibiting the transmission of viruses; the PIP5K1a inhibitor reduces the formation of migration bodies by inhibiting the maturation of migration bodies, thereby inhibiting the transmission of viruses; the virus is selected from hepatitis virus and / or adenovirus.
[0016] The mechanism of the present technology is that a mode of virus transmission which can be used as a potential target for the development of antiviral drugs is found, which is that the virus infection stimulates the cell to produce a migratory body, and the mature virus particles are enriched in the migratory body, and the virus transmission is achieved by a non-receptor-dependent pathway by using the characteristics of the migratory body.
[0017] Based on the above findings, a small molecule inhibitor for inhibiting the formation of migratory bodies to achieve an antiviral effect is provided, which is NSC23766, specifically binds to the GEF binding region on the surface of Rac1 protein, and can inhibit the Rac1 GDP / GTP exchange activity at a concentration of 20-50 μM, and inhibit the Rac1-mediated cell function.
[0018] The structure of NSC23766 is shown in formula (I).
[0019] Formula (I).
[0020] Meanwhile, based on the above findings, another small molecule inhibitor for inhibiting the maturation of migratory bodies to achieve an antiviral effect is also provided, which is ISA-2011B, specifically binds to the ATP / phospholipid binding pocket of PIP5K1α, and can significantly inhibit its lipid kinase activity at a concentration of 10-25 μM, and block its catalytic process of converting PI4P to PI (4, 5) P2, thereby inhibiting the maturation process of the migratory body.
[0021] The structure of ISA-2011B is shown in formula (II).
[0022] Formula (II).
[0023] The present application proposes a biological agent for inhibiting the generation of cell migratory bodies and its application in inhibiting virus transmission, which has the following advantages compared with the prior art: Firstly, the present application finds a new mechanism of virus transmission (and develops a biological agent based on this mechanism), which breaks through the traditional understanding that virus transmission only depends on the receptor-mediated pathway, and expands the understanding of the biological process of virus infection, which will provide a series of new targets for the research in the field of antiviral; Secondly, Rac1 activation and PIP5K1α activation are the primary steps of migratory body nucleation and maturation, respectively, and they are efficient as small molecule inhibitor targets; Thirdly, the small molecule inhibitor NSC23766 selected is a Rac1 specific inhibitor obtained by computer virtual screening and structure optimization, and its reaction route is clear and convenient to prepare; there are many studies that have applied it to inhibit cancer cell invasion and migration and achieved obvious effects, and the present application further expands the application of this small molecule inhibitor; Fourth, the selected small molecule inhibitor ISA-2011B is a PIP5K1α specific inhibitor obtained by computer virtual screening and structure optimization, which has weak cross-inhibition with β / γ homologues, good cell permeability, low toxicity and other advantages; existing studies have shown that it has significant effects in inhibiting tumor growth and metastasis, anti-inflammatory, inhibiting excessive insulin secretion, etc., and the application further expands the application of the small molecule inhibitor; Fifth, the application innovatively proposes a Rac1-PIP5K1α double-target combination strategy (double-inhibitor combination), which can regulate the generation of migrasomes at different levels and significantly reduce the drug use concentration, which has important clinical application value. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 As shown in an embodiment of the application, electron microscopy detection confirmed that the migrasomes released by virus-infected cells contained virus particles.
[0025] Figure 2 As shown in an embodiment of the application, the qPCR detection results of the infection of virus and virus-containing migrasomes to cells not expressing receptors.
[0026] Figure 3 As shown in an embodiment of the application, the quantitative PCR detection results of the NSC23766 and ISA-2011B anti-hepatitis virus infection experiment; wherein, Figure 3 A and 3C are migrasome concentration and cell survival rate detection data, Figure 3 B and 3D are hepatitis virus gene level detection data.
[0027] Figure 4 As shown in an embodiment of the application, the quantitative PCR detection results of the NSC23766 and ISA-2011B anti-adenovirus infection experiment; wherein, Figure 4 A and 4C are migrasome concentration and cell survival rate detection data, Figure 4 B and 4D are adenovirus gene level detection data.
[0028] Figure 5 As shown in an embodiment of the application, the quantitative PCR detection results of the NSC23766 and ISA-2011B combination anti-virus infection experiment; wherein, Figure 5 A and 5C are cell survival rate detection data, Figure 5 B and 5D are virus gene level detection data. DETAILED DESCRIPTION
[0029] In order to make the purpose, technical scheme and advantages of the application more clear and obvious, the application will be further described in detail below. However, it should be understood that the description here is only used to explain the application, and is not used to limit the scope of the application.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. The terms used in the description of the application herein are defined as follows unless otherwise limited: All reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be repeated herein.
[0031] For a further understanding of the application, reference will be made to the following detailed description of the application in conjunction with the best mode practising the application.
[0032] Example 1 Electron microscope detection of migration bodies released by virus-infected cells The specific experimental steps are as follows: 1. Virus infection and migration body collection: mouse hepatitis virus was used to infect mouse brain neural cell line Neuro-2a at MOI = 1, and the migration bodies containing viruses were collected according to the above infection time and differential centrifugation collection method.
[0033] 2. Electron microscope sample preparation: the separated migration bodies were resuspended with electron microscope fixing solution, fixed overnight at 4°C, rinsed with 0.1M PBS for 3-6 times, post-fixed with 1% osmium tetroxide for 2h, rinsed with ddH2O for 3-4 times, dehydrated with gradient concentration of ethanol and acetone, the sample was infiltrated with acetone and resin, embedded with pure resin, ultrathin section was made, and stained.
[0034] 3. Data analysis and processing: the specific experimental results are shown in Figure 1 The transmission electron microscope was used to observe the sample, and it can be seen that the migration bodies contain complete virus particles.
[0035] Example 2 Example of virus and virus-containing migration body infection of cells not expressing receptors The recognition of mouse hepatitis virus (MHV) surface S protein with receptor mCEACAM is the initial determinant of MHV infection of species specificity and tissue tropism. Mouse lung cancer cell line LLC is a cell with low expression of mCEACAM, which is usually difficult to be directly infected by MHV naked virus.
[0036] The specific experimental steps are as follows: 1. Virus infection and migration body collection: mouse hepatitis virus was used to infect mouse brain neural cell line Neuro-2a at MOI = 1, and the migration bodies containing viruses were collected according to the above infection time and differential centrifugation collection method.
[0037] 2. Virus and virus-containing blebs infection of LLC: LLC cells were infected with the same MOI of naked virus and virus-containing blebs, respectively. After 2h, the virus-containing medium was removed and the cells were washed twice with PBS and incubated for another 24h.
[0038] 3. Cell collection and RNA extraction: After washing twice with PBS, the cells were collected and the RNA was extracted using the Norgen FreeZol Reagent kit. The concentration of the RNA was measured and 500ng of the RNA was used for reverse transcription into cDNA using the Norgen HisyGo RT Red SuperMix for qPCR (+gDNA Wiper) kit.
[0039] Quantitative PCR method for detecting viral genome expression: The viral genome in the cells was detected by quantitative PCR using viral genome-specific primers (Table 1).
[0040] Table 1
[0041] Data analysis and processing: The specific experimental results are shown in Figure 2 According to the results in the figure, the virus-containing blebs infected the LLC cells in a dose-dependent manner, while the naked MHV virus with the same MOI could not infect the LLC cells.
[0042] Example 3 NSC23766 and ISA-2011B inhibit mouse hepatitis virus infection NSC23766 inhibits the Rac1 GDP / GTP exchange activity by binding to the GEF binding region of the Rac1 protein. The activation of Rac1 is a key molecular switch for the nuclearization of blebs, so this small molecule drug can be used as a bleb formation inhibitor.
[0043] ISA-2011B specifically inhibits the lipid kinase activity of PIP5K1a, and the PI (4, 5) P2 generated by its catalysis is a key to the recruitment of Rab35 and Integrin a5 for bleb maturation, so this small molecule drug can be used as a bleb formation inhibitor.
[0044] The mouse hepatitis virus has an envelope on the outside, with a diameter of 80-120nm, and its genetic material is positive-strand single-strand RNA, with a genome of 27-32kb.
[0045] The specific steps of the experiment are as follows: 1. Virus infection and drug treatment: N2a cells were seeded in Transwell chambers, and infected with mouse hepatitis virus at MOI = 1. After 2 h, the medium containing virus was removed, and the cells were washed twice with PBS. The Transwell chambers were then transferred to a 24-well plate seeded with LLC cells, and medium containing different concentrations of NSC23766 (0, 10, 25, 50, 75, and 100 μM) and ISA-2011B (0, 10, 20, 30, 40, and 50 μM) was added, respectively, and the cells were incubated for another 24 h.
[0046] 2. Cell collection and RNA extraction: The Transwell chambers were removed, and the LLC cells were washed twice with PBS. The cells were then lysed using a Norgen Biotek FreeZol Reagent kit to extract RNA, and the concentration was determined. 500 ng of the RNA was used to synthesize cDNA using a Norgen Biotek HisyGoRT Red SuperMix for qPCR (+gDNA Wiper) kit.
[0047] 3. Quantitative PCR method for detecting viral genome expression: The viral genome in the cells was detected by quantitative PCR using primers specific for the mouse hepatitis virus genome.
[0048] Data analysis and processing: The experimental results are shown in Figure 3 According to the results in the figure, NSC23766 significantly inhibited the formation of migratory bodies at 25 μM, inhibited the spread of the mouse hepatitis virus through the migratory bodies, and had no significant effect on cell viability. ISA-2011B significantly inhibited the formation of migratory bodies at 10 μM, inhibited the spread of the mouse hepatitis virus through the migratory bodies, and had no significant effect on cell viability. As the concentration of the drugs increased, the ability of both to inhibit the formation of migratory bodies and their antiviral ability also increased.
[0049] Example 4 NSC23766 and ISA-2011B against adenovirus infection Adenovirus is an external non-enveloped virus with a diameter of 70-90 nm, and its genetic material is linear double-stranded DNA with a genome of 30-40 kb. The experimental steps are as follows: 1. Virus infection and drug treatment: HeLa cells were seeded in Transwell chambers, and infected with adenovirus at MOI = 1. After 2 h, the medium containing virus was removed, and the cells were washed twice with PBS. The Transwell chambers were then transferred to a 24-well plate seeded with K562 cells, and medium containing different concentrations of NSC23766 (0, 10, 25, 50, 75, and 100 μM) and ISA-2011B (0, 10, 20, 30, 40, and 50 μM) was added, respectively, and the cells were incubated for another 24 h.
[0050] 2. Collect cells and extract RNA: Remove the Transwell chamber, wash the K562 cells with PBS twice, then extract the cell RNA using the Norgen FreeZol Reagent kit, measure the concentration, and take 500 ng of each sample for reverse transcription into cDNA using the Norgen HisyGoRT Red SuperMix for qPCR (+gDNA Wiper) kit.
[0051] 3. Quantitative PCR method for detecting viral genome expression: Use adenovirus genome-specific primers to perform quantitative PCR detection of viral genomes in cells.
[0052]
[0053] Data analysis and processing: The specific experimental results are shown in Figure 4 According to the results in the figure, NSC23766 significantly inhibited the formation of migratory bodies at 25 μM, inhibited the spread of adenovirus through migratory bodies, and had no significant effect on cell viability; ISA-2011B significantly inhibited the formation of migratory bodies at 10 μM, inhibited the spread of adenovirus through migratory bodies, and had no significant effect on cell viability; as the drug concentration increased, the ability of both to inhibit migratory body formation and their antiviral ability also increased.
[0054] Example 5 NSC23766+ISA-2011B antiviral infection example The specific experimental steps are as follows: 1. Virus infection and drug treatment: Seed N2a cells in a Transwell chamber, infect N2a cells with mouse hepatitis virus at MOI = 1, remove the virus-containing medium after 2 h, wash with PBS twice, transfer the Transwell chamber to a 24-well plate seeded with LLC cells, and add medium containing 25 μM NSC23766, 10 μM ISA-2011B, and both drugs, respectively, and continue to culture for 24 h.
[0055] Seed HeLa cells in a Transwell chamber, infect HeLa cells with adenovirus at MOI = 1, remove the virus-containing medium after 2 h, wash with PBS twice, transfer the Transwell chamber to a 24-well plate seeded with K562 cells, and add medium containing 25 μM NSC23766, 10 μM ISA-2011B, and both drugs, respectively, and continue to culture for 24 h.
[0056] 2. Collect cells and extract RNA: Remove the Transwell chamber, LLC cells and K562 cells are washed with PBS for 2 times, then use the Norgen FreeZol Reagent kit to extract cell RNA, measure the concentration, and use 500 ng of Norgen HisyGo RT Red SuperMix for qPCR (+gDNA Wiper) kit to reverse transcribe into cDNA.
[0057] 3. Quantitative PCR method to detect viral genome expression: Using viral genome-specific primers, the viral genome in the cells is detected by quantitative PCR.
[0058] Data analysis and processing: The specific experimental results are shown in Figure 5 According to the results in the figure, when 25 μM NSC23766 and 10 μM ISA-2011B are used together, the antiviral ability is stronger than using one drug alone, and there is no significant effect on cell viability.
[0059] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A biological agent for inhibiting the formation of cell migration bodies, characterized in that, The biological preparation comprises a Rac1 inhibitor; preferably, the Rac1 inhibitor is NSC23766.
2. The biological preparation of claim 1, wherein, The effective concentration of the Rac1 inhibitor is 20-50 μM.
3. The biological preparation of claim 2, wherein, The cells comprise one or more of brain neuroblastoma cell lines, lung cancer cell lines, cervical cancer cell lines, leukemia myeloid cell lines.
4. The biological preparation according to any one of claims 1 to 3, characterized in that, The biological preparation further comprises a PIP5K1α inhibitor; preferably, the PIP5K1α inhibitor is ISA-2011B.
5. The biological preparation of claim 4, wherein, The effective concentration of the PIP5K1α inhibitor is 10-25 μM.
6. A method of inhibiting migration cell formation, characterized by, The addition of the biological preparation to the cells is achieved by adding the biological preparation as claimed in any one of claims 1-5.
7. The method of claim 6, wherein, The cells are selected from virus-infected cells.
8. Use of the biological preparation as claimed in any one of claims 1-5 in inhibiting virus transmission.
9. Use according to claim 8, characterized in that, The virus transmission refers to non-receptor-dependent transmission of viruses between cells via migratory bodies.
10. Use according to claim 9, characterized in that, The Rac1 inhibitor reduces the formation of migratory bodies by inhibiting the nucleation of migratory bodies, thereby achieving the inhibition of virus transmission; the PIP5K1α inhibitor reduces the formation of migratory bodies by inhibiting the maturation of migratory bodies, thereby achieving the inhibition of virus transmission; and the virus is selected from hepatitis viruses and / or adenoviruses.