CN107441094B - Nilotinib as medicine for treating dengue virus infection and pharmaceutical application thereof - Google Patents
Nilotinib as medicine for treating dengue virus infection and pharmaceutical application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
本发明涉及尼罗替尼作为治疗登革病毒感染的药物及其制药用途，具体公开了尼洛替尼在制备治疗或预防登革病毒感染的药物中的用途。本发明通过对DENV的抑制实验证实了尼洛替尼对DENV2具有良好的抑制作用，从本质上证明了该药物在治疗DENV2感染疾病中具有广范的应用前景。该发明能够为临床治疗由DENV2引起的疾病提供良好的候选药物，具有十分良好的应用前景。
The invention relates to nilotinib as a drug for treating dengue virus infection and its pharmaceutical use, and specifically discloses the use of nilotinib in preparing a drug for treating or preventing dengue virus infection. The present invention confirms that nilotinib has a good inhibitory effect on DENV2 through the DENV inhibition experiment, which essentially proves that the drug has a wide range of application prospects in the treatment of DENV2 infection diseases. The invention can provide a good candidate drug for clinical treatment of diseases caused by DENV2, and has a very good application prospect.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of nilotinib in resisting dengue viruses.
Background
Dengue virus (DENV) genus Flaviviridae, Flaviviridae (Flaviviridae), is a most widespread insect-borne virus in the world today, and dengue is mainly caused by transmission of biting aedes aegypti and aedes albopictus, among which dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is more severe. The virus has four serotypes, serotype 2 is an epidemic strain dengue (dengueflever) which is an acute infection transmitted by mosquito vectors and is mainly epidemic in tropical and subtropical regions. Dengue epidemics were developed in succession around the world since the first discovery of dengue in 1779. According to WHO estimates that about 2/5 of the world's population is currently threatened by dengue fever, and billions of people are infected with the virus each year. In China, except for Taiwan regions, the epidemic situation is mainly concentrated in Guangdong and is characterized by outbreak and importation epidemic, and known 4 serotype dengue viruses are discovered in China and tend to spread and increase. At present, the dengue virus infection mechanism is unclear, and because effective cross protection is lacked among serotypes and an antibody dependence enhancing effect exists, so far, safe and effective vaccines are not available, and no effective treatment medicine exists clinically. Although many active molecules against the main proteins of dengue virus are discovered, no drug against dengue virus exists for various reasons (such as toxic and side effects), so that the discovery of antiviral active molecules against dengue virus has important biological research significance and practical significance, and provides support for developing effective drugs against dengue virus.
Disclosure of Invention
The invention aims to provide the application of nilotinib serving as an anti-tumor drug on the market in preparing a drug for treating or preventing dengue virus infection, wherein the inhibition rate of nilotinib to dengue virus type 2 at a concentration of 10.0M is 90%. Meanwhile, the survival rate of the nilotinib in 10M condition on BHK21 cells is 95%. Nilotinib can effectively inhibit the proliferation of dengue fever viruses, has low cytotoxicity, can be further developed into a drug for treating dengue fever virus infection, and has wide application prospect.
The nilotinib structure is shown below:
one aspect of the present invention provides the use of nilotinib in the manufacture of a medicament for the treatment or prevention of a flavivirus infection.
In another aspect, the present invention provides the use of nilotinib for the preparation of a medicament for the treatment or prevention of dengue virus infection.
In the technical scheme of the invention, the dengue virus is dengue virus 1-4.
In the technical scheme of the invention, the dengue virus is dengue virus type 2.
In a further aspect, the invention provides the use of nilotinib in the preparation of a medicament for inhibiting mRNA of NS1 and E proteins in dengue virus.
In a further aspect, the invention provides the use of nilotinib in the preparation of a medicament for inhibiting protein expression of NS1 and E proteins in dengue virus.
In a further aspect of the present invention, there is provided a pharmaceutical composition for the treatment of dengue virus infection comprising nilotinib as an active substance.
In the technical scheme of the invention, the pharmaceutical composition is an injection preparation, an oral preparation or an external preparation.
In the technical scheme of the invention, the pharmaceutical composition is tablets, capsules, powder, pills, granules, injection or emulsion.
The nilotinib serving as a medicine is prepared by improving the molecular structure of imatinib, has stronger selectivity on BCR-ABL kinase activity, has 30 times stronger inhibition effect on tyrosine kinase than imatinib, and can inhibit the kinase activity of imatinib-resistant BCR-ABL mutant. Meanwhile, the inhibitor can also inhibit the activities of KIT and PDGFR kinases, is a powerful and accurate second-generation tyrosine kinase inhibitor clinically, and effectively treats patients with drug-resistant or intolerant chronic myelogenous leukemia. The inventor discovers the antiviral effect of nilotinib by screening the existing marketed drugs, and discloses the use of nilotinib in the preparation of drugs for treating or preventing dengue virus type 2 infection for the first time, wherein the nilotinib is a marketed drug, has good pharmacokinetic characteristics and good inhibitory activity against dengue virus type 2, has no possibility of any revelation due to other compounds, has outstanding substantive characteristics, and obviously has significant progress in the treatment and prevention of dengue virus infection, and is very likely to be developed into a novel drug for resisting viral infection.
Drawings
FIG. 1 is a graph of the effects of dengue virus-induced cytopathic effects;
FIG. 2 is a graph showing the results of Western blotting;
FIG. 3 is a graph showing the result of the plaque assay.
Detailed Description
Example 1 toxicity test of nilotinib on BHK-21 cells
BHK-21 cells (baby hamster kidney cells) are susceptible cells to DENV 2. BHK-21 cells in the experiment were owned by the same laboratory; MTT was purchased from bio-technical institute in bi yun tian; fetal bovine serum was purchased from GIBICO, USA; cell culture plates were purchased from corning, usa; RPMI 1640 medium was purchased from GIBICO, USA.
The experimental procedure was as follows:
1) inoculation of BHK-21 cells: preparing single cell suspension by using RPMI 1640 culture medium containing 10% (V/V) fetal bovine serum, and inoculating 10000 cells per hole to a 96-hole cell culture plate;
2) culturing BHK-21 cells: at 37 ℃ 5% (V/V) CO2Culturing for 24 hours under the culture condition;
3) adding nilotinib: removing the culture medium from each well, adding 100. mu.l of nilotinib diluted to the corresponding concentration with 10% (V/V) fetal bovine serum in RPMI 1640 medium to each well, and adding 100. mu.l of drug-free 10% (V/V) fetal bovine serum in RPMI 1640 medium to the control wells;
4) color generation: after culturing for 48 hours, observing under a common optical microscope to find that the Formazan is completely dissolved;
5) measurement and calculation: the absorbance was measured at 570nm and the viability of cells at different concentrations of nilotinib was determined as the absorbance of cells at that concentration compared to the absorbance of the upper control well, multiplied by 100%.
TABLE 1 toxicity test of nilotinib at various concentrations on BHK-21 cells
Results of the experiment
BHK-21, a Baby Hamster Kidney cell (Baby Hamster Syrian Kidney), is infected with dengue virus type 2 (DENV2) and is one of the most used cells for dengue virus research. The experiment firstly detects the cytotoxicity of nilotinib on BHK-21 cells, and nilotinib with different concentrations acts on the BHK-21 cells to know the maximum using concentration of nilotinib which survives more than 90% on the cells, so that reference data is provided for the subsequent experiment of the inhibition effect of nilotinib on DENV 2.
Example 2 inhibition of DENV2 infection of BHK-21 cells by nilotinib:
BHK-21 was used as a cell for culturing the virus DENV2, 10TCID50, and the experimental procedure was as follows:
1) BHK-21 is inoculated into a cell culture plate, after 24 hours, the cells grow to a monolayer, the area of the cell covering the bottom of a hole is about 80% -90%, the culture medium is sucked out, PBS is washed for 1 time, a virus sample is inoculated into 200 mu l, and the virus sample is adsorbed for 1 hour at 37 ℃. After completion of the adsorption, the virus solution in each well was aspirated and washed 1 time with PBS. Adding nilotinib at a specified concentration diluted in RPMI 1640 medium containing 10% (V/V) fetal bovine serum at 37 deg.C with 5% (V/V) CO2Culturing under the culture condition. After 96 hours, after the cells had developed significant cytopathic effects, the cytopathic effects were observed under a microscope. See fig. 1.
2) Collecting supernatant, and measuring the content of Lactate Dehydrogenase (LDH) released by cells in the supernatant; or culturing for 48 hours, collecting cell supernatant, and performing virus plaque experiment; or after culturing for 48 hours, collecting total protein total mRNA of the cells, and detecting the content of virus protein in the cells by a protein immunoblotting method and a real-time fluorescent quantitative PCR method respectively.
3) Measuring the content of lactate dehydrogenase released by cells in the supernatant: 120 μ l of cell supernatant of a 96-well plate was aspirated, and the activity of lactate dehydrogenase released during Cytotoxicity due to DENV was detected using a lactate dehydrogenase Cytotoxicity detection kit (LDH cytoxicity Assay Ki, Beyotime).
TABLE 2 Effect of different concentrations of nilotinib on LDH release from DENV 2-infected BHK-21 cells
The experimental results are as follows:
as shown in figure 1, nilotinib (10 μ M) treatment significantly reduced DENV 2-induced cytopathic effects. And when nilotinib (10, 5 and 2.5 mu M) at different concentrations acts on BHK-21 cells infected with DENV2, the LDH release level is obviously reduced, so that the inhibition efficiency of the nilotinib on viruses is obtained.
Example 3 inhibition assay of nilotinib against the key proteins NS3 and E of DENV2
1) Collecting total cellular protein: BHK-21 cells were treated with drug and dengue virus (DENV2), and were collected in 1.5ml Eppendorf tubes for 48h to extract protein.
2) Detecting the protein concentration of the sample: diluting the protein standard substance with double distilled water to gradient concentration of 0, 0.0008, 0.0016, 0.0032, 0.004, 0.006 and 0.008 mg/ml; the protein concentration in the sample was calculated from the standard curve. The protein concentration was made uniform for each sample.
3) Western blotting detection of the expression difference of dengue virus protein after gradient concentration nilotinib treatment: adding various reagents in sequence, carrying out electrophoresis at a constant voltage of 80V at the beginning of electrophoresis, changing the voltage to 120V after the front edge of the dye enters the separation gel, and determining the electrophoresis time for 75min according to the separation degree of the pre-dyed protein Marker and the molecular weight of the target protein.
4) Film transfer: the gel was removed from the glass plate, sheared and soaked in methanol for 1min, followed by a 5min soak in transfer buffer (1 × tris/glycine buffer, Biorad). The gel, filter paper, PVDF membrane were soaked in the transfer solution. And (3) placing the membrane rotating device in a membrane rotating box, confirming that the electrodes are correct, adding the membrane rotating liquid to cover the whole membrane rotating device, covering the membrane rotating box and the upper part of the membrane rotating box with ice blocks, switching on a power supply, rotating the membrane at a constant voltage of 100V for 70min, and taking out the PVDF membrane after the operation is finished.
5) Protein blocking: and (3) placing the PVDF membrane in a sealing solution, and soaking for 1h in a shaking table at room temperature to seal the nonspecific antigen. After blocking, the membrane was washed 3 times with TBS/TT shaking at room temperature, 5min each time followed by incubation of one antibody.
6) Primary antibody hybridization: the PVDF membrane protein face up was placed in a small box, and 5% (w/v) skim milk buffer in TBS/T formulation containing a Dengue virus Envelope protein (GeneTex) and nonstructural protein 1 (Dengue virus NS3glycoprotein antibody, abcam) primary antibody was added and shaken overnight at 4 ℃. PBST room temperature shaking washing membrane six times, each time for 5 min.
7) And (3) hybridization of a second antibody: the PVDF membrane protein face up was placed in a small box, and 5% (w/v) skim milk buffer in TBS/T formulation containing secondary antibody (rabbit antibody or mouse antibody, Proteitech) was added and shaken for 1 hour at room temperature. PBST room temperature shaking washing membrane six times, each time for 5 min.
8) And (3) chemiluminescence development: and uniformly adding ECL developing solution A/B (CST) according to a proportion, soaking the PVDF film in a luminescent solution, and reacting for 2 min. Taking out the PVDF film, placing in a cassette, exposing in a dark room, placing an X-ray negative film, exposing, taking out for development, airing the film, recording and storing.
The DENV2 virus contains structural protein and non-structural protein, and structural protein E protein and non-structural protein NS3 protein play an important role in the propagation and amplification of the DENV2 virus, so that the mRNA inhibition levels of NS3 and E protein are detected by nilotinib at different concentrations.
The experimental results are as follows:
referring to fig. 2, the inhibition level of the protein of the structural protein E and the protein of the non-structural protein NS3 is gradually increased with the increase of the amount of nilotinib, thereby further confirming the virus inhibition effect of nilotinib.
Example 4 plaque inhibition assay of nilotinib against DENV2 production of progeny virus
1) The cells were plated at C6/36 in a 24-well plate, and after 24 hours, the cells grew to a monolayer (the area of the bottom of the well covered by the cells was about 90% to 100%), the medium was aspirated from each well, washed 1 time with PBS, and 200. mu.l of a virus sample diluted with PBS was plated and adsorbed at 37 ℃ for 1 hour.
2) After completion of adsorption, the supernatant from each well was discarded, and the unadsorbed virus was washed away with PBS. Adding RPMI 1640 medium containing 1.2% methylcellulose 2% (V/V) fetal bovine serum at 37 deg.C and 5% (V/V) CO2The culture is carried out for 96-120 hours in the incubator.
3) After the formation of plaques, the methylcellulose overlay medium was blotted and fixed with 4% paraformaldehyde (bosch biosystems), stained with 1% (w/v) crystal violet, washed off the crystal violet with running water after 2 hours, air dried, scanned through a 24-well plate, and the titer of the virus (PFU/ml, PFU: a plaque formation unit). PFU ═ virus dilution × P/V, (P: number of plaques; V: inoculum size).
Results of the experiment
Referring to FIG. 3, the plaque inhibition assay is an important indicator of the ability to detect viral titer and infection, and after viral infection of cells, the released virus can only spread from the initially infected cells to the periphery due to the restriction of the solid medium. Over several cycles of proliferation, a localized diseased cell area, known as a viral plaque, is formed. After staining and washing, the viral plaques were not stained with color, thus appearing as a blank, with the remaining cells intact in blue. After taking out the supernatant of the BHK-21 cells treated by the nilotinib, performing a virus plaque experiment on the supernatant, thereby further confirming the virus content, titer and infectivity level in the cell supernatant after the nilotinib treatment. More accurate determination of the anti-DENV 2 progeny virus effect of nilotinib. According to the experimental results, the plaque formed gradually decreases with the increase of the nilotinib concentration.
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