CN114432454B - Application of PLK1 inhibitor in preparation of medicines for treating Ebola virus diseases - Google Patents
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Abstract
The invention belongs to the field of biological medicine, and particularly discloses application of a PLK1 inhibitor in preparation of medicines for treating Ebola virus diseases, wherein serine/threonine kinase PLK1 is used as a target point, and the PLK1 expression is inhibited to obviously reduce the inclusion body formation of the Ebola virus and effectively inhibit proliferation of the Ebola virus in cells. The research result of the invention makes PLK1 hopefully become a potential therapeutic target for Ebola virus infection, provides a new strategy for preventing and treating acute infectious diseases caused by Ebola virus infection, and has important application value in medicine research and development and vaccine development.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of a PLK1 inhibitor in preparation of a medicament for treating Ebola virus diseases.
Background
Ebola virus (EBOV) is a single-stranded negative strand RNA virus, a filovirus that causes ebola hemorrhagic fever in humans and other primates. Along with the increase of global population mobility, and the characteristics of strong infectivity, high mortality, easy occurrence of genetic variation and the like, global public health still faces a great threat, and so far, effective antiviral drugs are not available in batches and on the market, so that the exploration of effective treatment targets and prevention and treatment strategies is necessary;
ebola virus genome size is around 19kb, which encodes seven structural proteins: nucleoprotein NP, virion protein VP35, matrix protein VP40, glycoprotein GP, VP30, VP24, RNA-dependent RNA polymerase L. Glycoprotein (GP) primarily mediates viral attachment and entry into host cells, and Nucleoprotein (NP) forms a nucleocapsid primarily with VP35, VP30 and L, VP24 and VP40 constituting the viral matrix forming the outer layer of the nucleocapsid, VP35 being an important cofactor for viral RNA polymerase, which antagonizes innate immunity and may also be involved in viral inclusion body formation. The ebola virus inclusion body is an aggregation small body formed in cytoplasm or nucleus after virus infects host cells, is round, oval or amorphous, can be used for auxiliary diagnosis of viruses and identification of certain viruses, plays an important role in the life cycle of the viruses, and researches show that the inclusion body is an important place for synthesizing and assembling RNA of the ebola viruses, and various virus structural proteins such as NP, VP35, VP30, L and the like participate in the formation of the inclusion body. In addition, viral genome replication and transcription are highly dependent on host factors, and research shows that host proteins can be hijacked into inclusion bodies to participate in regulating viral proliferation. For example, ebola virus NP can recruit host factor SMYD3 into inclusion bodies, thereby enhancing NP-VP30 interactions to promote viral transcription; ebola virus NP recruits nuclear RNA export factor 1 (NXF 1) into inclusion bodies to promote viral protein expression; the host protein BBP6 can affect ebola virus replication by disrupting the interaction between NP and VP 30; in addition, studies have found that both cellular kinases and phosphatases (SRPK 1 serine-arginine protein kinase, RUVBL1 ATPase, PP2A-B56 phosphatase, etc.) can be localized in inclusion bodies to affect ebola virus replication and transcription.
PLK1 (Polo-like kinase 1) is a serine/threonine kinase that is widely present in eukaryotes, is highly conserved in structure and function, is highly homologous at the N-terminus to the serine/threonine kinase domain (KisnaseDomain, KD), and its kinase activity is largely regulated by K82 and T210 site phosphorylation. The C-terminus has two conserved Polo cassettes (PBDs), with a flexible linker in between. Studies have shown that PLK1 plays an important regulatory role in different phases of cell mitosis, including promotion of centrosome maturation, chromosome segregation, cytokinesis, etc.; play an important role in DNA damage response, autophagy, apoptosis and cytokine signaling; the polypeptide is highly expressed in various tumor tissues and participates in regulating the occurrence and development of tumors, so that the polypeptide is considered as a new tumor treatment target with good application prospect, and reports indicate that the polypeptide can effectively inhibit the proliferation of tumor cells and induce the apoptosis of the tumor cells by blocking the expression of PLK1 by antibodies, RNA interference (RNAi) or kinase inhibitors. At present, various small molecule inhibitors targeting PLK1 enter a clinical test stage, namely imidazole triazine GSK461364 is a high-efficiency ATP competitive inhibitor, and the functions of the imidazole triazine GSK461364 are inhibited mainly by inhibiting the catalytic activity of PLK1 kinase, so that proliferation of various tumor cell lines can be effectively inhibited when half inhibition concentration (IC 50) is lower than 100nM, cell cycle arrest is caused, apoptosis is induced, and further a good tumor treatment effect is achieved. SBE13HCl is a potent and selective PLK1 inhibitor with an IC50 of 200pM, and studies have shown that SBE13HCl can also reduce cell proliferation of various cancer cell lines and cause G2/M arrest followed by apoptosis.
Although PLK1 is currently used as a hot target for tumor treatment, its application in viral infection has not been reported.
Disclosure of Invention
In order to solve the above problems, a primary object of the present invention is to provide a general therapeutic strategy for targeting PLK1 and its signaling pathway for patients infected with ebola virus, i.e. the application of PLK1 inhibitors in the preparation of drugs for treating ebola virus diseases.
The specific technical scheme of the invention comprises the following steps:
the invention provides application of a PLK1 inhibitor in preparing medicines for treating Ebola virus diseases, wherein the medicines for preventing or treating Ebola virus diseases refer to any one of the following X1) -X5):
x1) a medicament for preventing or treating ebola virus infection;
x2) a medicament for preventing or treating ebola virus disease;
x3) a drug for inhibiting ebola virus replication and proliferation;
x4) a drug for inhibiting cytopathic effects of ebola virus;
x5) ebola virus inhibitors.
As a further preferred embodiment of the present invention, the method for inhibiting PLK1 is inhibition by using a PLK 1-targeted siRNA (hereinafter referred to as PLK1 siRNA) or by using a PLK1 inhibitor.
As a further preferred embodiment of the invention, the PLK inhibitor is GSK461364 or SBE13 HCl.
The invention also provides a medicine for preventing or treating Ebola virus diseases, which is siRNA targeting PLK1 or PLK1 inhibitor.
As a further preferred embodiment of the invention, the PLK inhibitor is GSK461364 or SBE13 HCl.
To investigate whether targeting PLK1 silk/threonine kinase could effectively inhibit replication and proliferation of ebola genome, two PLK1 inhibitors GSK461364 and SBE13HCl were used in the present application, seven groups of different concentrations of drug treated HEK293 cells were set in 96-well plates for detection of cytotoxicity, and then each group of cell viability was detected using CCK8 kit, and experimental results showed that when SBE13HCl drug working concentration was below 50 μm (fig. 1A), GSK461364 drug working concentration was below 50nM with little cytotoxicity (fig. 1B). Subsequently, the inhibition of viral inclusion body formation after PLK1 was detected by immunofluorescence in the ebola minimal genome system, and a significant decrease in ebola inclusion body numbers was found after treatment with 50nMGSK461364 and 50 μmsbe13HCl compared to DMSO-added control (fig. 2A), and statistics were randomly performed on 20 cells in the field of view, as shown in the results (fig. 2B). To investigate whether targeting PLK1 regulates ebola virus replication and proliferation, the effect of PLK1siRNA and PLK1 inhibitors (GSK 461364, SBE13 HCl) on ebola virus replication and proliferation was subsequently evaluated in the ebola minimal genome system, and the results showed a significant decrease in intracellular ebola virus replication and proliferation levels, whether treated with PLK1siRNA or inhibitors (fig. 3).
In summary, the beneficial effects of the invention are as follows:
after being treated by PLK1siRNA and inhibitor, the invention verifies and discovers that the formation of the ebola virus inclusion body is obviously reduced, the virus proliferation is also obviously inhibited, and a general treatment strategy targeting PLK1 and a signal path thereof is provided for patients infected by the ebola virus. The research result of the invention shows that inhibiting host serine/threonine kinase PLK1 can obviously reduce the inclusion body formation of the ebola virus, effectively inhibit the proliferation of the ebola virus in cells, and PLK1 is expected to become a potential therapeutic target for ebola virus infection.
Drawings
FIG. 1, determination of GSK461364 and SBE13HCL cytotoxicity using CCK8 method;
FIG. 2, detection of the effect of GSK461364 and SBE13HCL on ebola virus inclusion body formation using immunofluorescence;
FIG. 3, evaluation of PLK1siRNA, GSK461364, and SBE13HCL effect on ebola virus replication and proliferation using ebola minimal genome.
Description of the embodiments
The following detailed description of the present application is provided in conjunction with the accompanying drawings, and it is to be understood that the following detailed description is merely illustrative of the application and is not to be construed as limiting the scope of the application, since numerous insubstantial modifications and adaptations of the application will be to those skilled in the art in light of the foregoing disclosure.
1. Material
1. Cell lines and plasmids
HEK293 cells are maintained in this laboratory and can also be purchased commercially from Guangzhou Yuan well Biotechnology Co.
The plasmids pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, p cis-vRNA-Rluc, pCAGGS-Tim1 and pCAGGS-T7 related to the minimum genome system of Ebola virus are described in the literature "Hoenen T, watt A, mora A, feldmann H. Modeling the lifecycle of Ebola virus under biosafety level 2 conditions with virus-like particles containing tetracistronic minigemes.J Vis exp.2014 Sep 27; (91) 52381.Doi:10.3791/52381. PMID: 25285674; PMCID PMC4828136, the relevant plasmid public can be obtained from military medical institute of the national institute of civil liberation army, and the biological material is only used for repeated relevant experiments of the invention and can not be used for other purposes.
2. Molecular biological agents and antibodies
GSK461364 (catalog number S2193), SBE13HCL (catalog number S7720) are purchased from Selleck corporation; the double luciferase assay kit was purchased from Promega corporation (catalog number T8787); CCK-8 kit (catalog number C0038) was purchased from Biyundian corporation; transfection reagents Lipofectamine2000 and Lipofectamine3000 were purchased from Thermo company; DMEM medium (catalog No. C11995500 BT), fetal bovine serum (catalog No. 16000-044), pancreatin (catalog No. 25200-056), 1XPBS (catalog No. C10010500 BT) were all purchased from GIBCO; tritonX-100 (catalog number T8787), DAPI nuclear dye (catalog number D9542) were purchased from Sigma;4% tissue fixative was purchased from Soxhobao (catalog number P1110); sheep serum albumin (catalog No. ZLI-9021) and FITC-labeled goat anti-rabbit secondary antibody (goat anti-rabbit-FITC secondary antibody, catalog No. ZF-0311) were purchased from the chinese mountain gold bridge.
2. Method of
1. The cytotoxicity of PLK1 inhibitor GSK461364 and SBE13HCL is determined by using CCK-8, and the specific experimental process is as follows:
(1) Cell culture: HEK293 cells were seeded into 96-well plates (100. Mu.l/1 well, 0.6X104 cells) and cultured for 24 hours (37 ℃,5% CO) 2 ）。
(2) And (3) adding medicines: the medium was replaced with fresh medium (100. Mu.l/1 well) containing GSK461364 (200 nM, 10nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125 nM) and SBE13HCL (200. Mu.M, 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M) with DMSO or various concentration gradients and treated for 12h.
(3) CCK-8 treatment: the original culture medium is discarded, the culture medium is replaced by a fresh culture medium (100 microliters/1 hole) containing 10% of CCK-8 solution, the culture is continued for 1-4 hours, the measurement can be carried out once every 30 minutes, and the OD value of the control group is preferably selected to be about 1.
(4) And (3) measuring: absorbance was measured using a microplate reader (measurement wavelength is 450 nm), and cell viability was calculated, cell viability (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100. Wherein, the liquid crystal display device comprises a liquid crystal display device,
a (dosing): absorbance of wells with cells, CCK solution and drug solution;
a (blank): absorbance of wells with medium and CCK solution without cells;
a (0 dosing): absorbance of wells with cells, CCK solution, and no drug solution.
As a result, as shown in FIG. 1, when the working concentration of SBE13HCl drug was less than 50. Mu.M (FIG. 1A), the cytotoxicity of GSK461364 drug was less than 50nM (FIG. 1B).
2. Immunofluorescence detects the effect of PLK1 inhibitors on ebola virus inclusion body formation, and the specific experimental procedure is as follows:
(1) Cell culture: cover slips are spread in six-hole plates, 2X 105 HEK-293 cells are inoculated, lipofectamine3000 is used for transfecting ebola virus minigemme related plasmids (pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng)) after 24 hours, GSK461364 (final concentration: 50 nM) and SBE13HC (final concentration: 50 mu M) are respectively added for 12 hours of experiment groups after transfection, equal volume DMSO treatment is added for continuous culture, and cells are harvested after 12 hours of continuous culture;
(2) Fixing: discarding the culture medium, soaking and washing with PBS for 3 times and 5min each time, and taking 1ml of 4% tissue fixing solution to fix the climbing plate for 30min;
(3) Perforating: immersing and washing the slide glass for 3 times with PBS for 3-5min each time, and taking 1ml of 0.3% Triton X-100 (prepared by PBS) and perforating for 15min at room temperature;
(4) Closing: immersing and washing the slide glass for 3 times with PBS for 3-5min each time, dripping goat serum on the slide glass, and sealing for 30min at room temperature;
(5) An antibody: immersing and washing the slide glass by PBS for 3 times, each time for 3-5min, dripping diluted Ebola NP and VP35 antibodies into each slide glass, and incubating for 1-2h at room temperature (dry sheets are avoided by frequent inspection);
(6) Fluorescent secondary antibodies: PBST soaks the climbing plate for 3 times, each time for 3-5min, and drops diluted fluorescent secondary antibody, incubate for 1-2h at room temperature (note: from adding fluorescent secondary antibody, all the following operation steps are carried out in dark);
(7) Sealing and nuclear dyeing: after PBST soaks the slide glass for 3 times, dripping a sealing piece containing DAPI onto the slide glass, covering the slide glass with the climbing piece, incubating for 5-10min in a dark place, sealing edges by nail polish after the piece is dried, and airing in a dark place for standby;
(8) And (3) image acquisition: the collected images were observed under a fluorescence microscope.
The results are shown in FIG. 2, in which the number of ebola inclusion bodies was significantly reduced after treatment with 50 nMgGSK 461364 and 50. Mu. MSBE13HCl compared to the DMSO-added control group (FIG. 2A), and the statistics were randomly performed on 20 cells in the field of view (FIG. 2B) for inhibition of viral inclusion body formation by immunofluorescence detection in the ebola minimal genome system.
3. The effect of PLK1siRNA and inhibitor on virus replication and proliferation is detected by using the Ebola minimal genome, and the specific experimental process is as follows:
(1) Construction of PLK1siRNA wherein PLK1siRNA is formed by annealing two single strands: sense (5 '-3') CCAUUAACGAGCUGCUUAATT; anti (5 '-3'): UUAAGCAGCUCGUUAAUGGTT. siRNA delegated synthesis from su Ji Ma gene stock.
(2) On day 1, virus-producing cells HEK293 (abbreviated p 0) were inoculated in 6-well plates for culture and transfected with PLK1 siRNA.
(3) Day 2, the ebola minimal genome related plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng) were transfected.
(4) On day 3, the medium was changed to 5% FBS medium, and p0 cells were harvested after further culturing for 72 hours.
(5) On day 4, virus-targeting cells HEK293 (abbreviated p 1) were inoculated into 6-well plates.
(6) On day 5, plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng) and pCAGGS-Tim1 (250 ng) were transfected.
(7) On day 6, p0 cells were harvested while p1 cell culture medium was replaced with p0 cell supernatant.
(8) On day 7, the p1 supernatant was replaced with 5% FBS medium, and treated with GSK461364 (final concentration: 50 nM) and SBE13HC (final concentration: 50. Mu.M), the control group was treated with an equal volume of DMSO, and p1 cells were harvested after further culturing for 72 hours.
The results are shown in FIG. 3, where the level of replication and proliferation of intracellular Ebola virus was significantly reduced, both after treatment with PLK1siRNA and inhibitor.
In conclusion, the invention verifies that the ebola virus inclusion body formation is obviously reduced and the virus proliferation is also obviously inhibited in a minigeme system after being treated by PLK1siRNA and an inhibitor. The research result of the invention shows that inhibiting host serine/threonine kinase PLK1 can obviously reduce the inclusion body formation of the ebola virus and effectively inhibit the proliferation of the ebola virus in cells.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that modifications can be made without departing from the spirit of the invention, which are within the scope of the invention.
Claims (2)
1. The application of PLK1 inhibitor targeting PLK1 in preparing medicines for treating Ebola virus diseases is characterized in that PLK1 inhibitor is utilized to inhibit PLK1, the purpose of inhibiting replication and proliferation of intracellular Ebola virus is achieved, and the PLK inhibitor is GSK461364 or SBE13 HCl.
2. The use of PLK1 inhibitors targeting PLK1 according to claim 1 for the preparation of a medicament for the treatment of ebola virus diseases, wherein the medicament for the treatment of ebola virus diseases refers to any one of the following X1) -X5):
x1) a medicament for treating ebola virus infection;
x2) a medicament for treating ebola virus disease;
x3) a drug for inhibiting ebola virus replication and proliferation;
x4) a drug for inhibiting cytopathic effects of ebola virus;
x5) ebola virus inhibitors.
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