CN109602749B

CN109602749B - Britinib as medicine for treating dengue virus infection and pharmaceutical application thereof

Info

Publication number: CN109602749B
Application number: CN201910110917.XA
Authority: CN
Inventors: 姚新刚; 万毅虹; 唐晓东; 刘叔文
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2019-02-12
Filing date: 2019-02-12
Publication date: 2021-03-12
Anticipated expiration: 2039-02-12
Also published as: CN109602749A

Abstract

The invention relates to a medicament for treating dengue virus infection and pharmaceutical application thereof, and particularly discloses a compound shown as a formula I or a medicament thereofUse of a receiving salt in a medicament for treating or preventing a flavivirus infection

Description

Britinib as medicine for treating dengue virus infection and pharmaceutical application thereof

Technical Field

The invention relates to the field of medicines, in particular to application of a compound in preparing a medicine for treating flaviviruses.

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 fever (dengue farm), is an acute infectious disease transmitted by mosquito vectors, and is mainly prevalent in tropical and subtropical regions as shown in figure I. 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, due to various reasons (such as toxic and side effects and the like), no drug or vaccine against dengue virus exists at present, so that the discovery of the antiviral active molecules against dengue virus has important biological research significance and practical significance, and provides support for developing effective drugs against dengue virus.

At present, the dengue virus infection mechanism is still unclear, and because effective cross protection is lacked among serotypes and an antibody-dependent enhancement 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 have been discovered, no drug against dengue virus is currently on the market for a variety of reasons (e.g., toxic side effects).

The compound Brivanib Alaninate of the present invention is an ATP-competitive VEGFR 2inhibitor [ Bhide, Rajeev s., et al, "Discovery and clinical standards of (R) -1- (4- (4-fluoro-2-methyl-1H-indole-5-yloxy) -5-methylpyrrolo2,1-f1,2, 4-triazine-6-yloxy) propan-2-ol (BMS-540215), an in vivo active patent VEGFR-2 Chemistry, Journal of medical Chemistry 49.7(2006):2143-6 ], published in 2006. The use of Brivanib Alaninate in the preparation of a drug for treating or preventing dengue virus type 2 infection is disclosed for the first time, and the framework type belongs to a brand-new framework type, and the Brivanib Alaninate has good effect on dengue virus type 2 inhibition activity, so that the possibility that other compounds give any revelation does not exist, the Brivanib Alaninate has outstanding substantive characteristics, and meanwhile, the Brivanib Alaninate for treating and preventing dengue virus infection obviously has remarkable progress, and the Brivanib Alaninate is very likely to be developed into a novel drug for resisting dengue virus infection.

Disclosure of Invention

The invention aims to provide Brivanib Alaninate, namely a compound shown in formula I, which has the inhibition rate of 107.64% on dengue virus type 2 at the concentration of 10.0 mu M and is used for preparing a medicament for treating or preventing dengue virus infection. While the survival rate of the Brivanib Alaninate on BHK-21 cells under the condition of 10.0 mu M is 92.74%. The Brivanib Alaninate can effectively inhibit the proliferation of dengue fever viruses, has low cytotoxicity alone, can be further developed into a drug for treating dengue fever virus infection, and has wide application prospect.

The Brivanib Alaninate compound has a structure shown in a formula I:

the compound Brivanib Alaninate is an ATP competitive VEGFR 2inhibitor and is a prodrug of Brivanib. The invention discloses the use of Brivanib Alaninate in the preparation of a medicine for treating or preventing dengue virus type 2 infection for the first time, has good effect on dengue virus type 2 inhibitory activity, does not have the possibility of giving any revelation due to other compounds, has outstanding substantive characteristics, obviously has remarkable progress in treating and preventing dengue virus infection, and is very likely to be developed into a novel medicine for resisting dengue virus infection.

One aspect of the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a flavivirus infection

In another aspect, the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture 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 a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting mRNA of NS3 and E proteins in dengue virus.

In a further aspect, the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting protein expression of NS3 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, which comprises as an active substance a compound of formula I or a pharmaceutically acceptable salt thereof.

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.

Drawings

FIG. 1 shows the inhibitory effect of Brivanib Alaninate on BHK-21 intracellular viral RNA following DENV2 infection.

FIG. 2 shows the inhibitory effect of Brivanib Alaninate on the synthesis of viral proteins in BHK-21 cells following DENV2 infection.

FIG. 3 is a graph showing the inhibitory effect of Brivanib Alaninate on the production of progeny virus by BHK-21 cells after infection with DENV 2.

Detailed Description

Example 1 toxicity assay of Brivanib Alaninate 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: prepare single cell suspension with RPMI 1640 medium containing 10% (V/V) fetal bovine serum, inoculate 96-well cell culture plate with 10000 cells per well, inoculate volume 100 μ l per well

2 culturing BHK-21 cells: at 37 ℃ 5% (V/V) CO₂Culturing for 24 hours under the culture condition;

3 adding Brivanib Alaninate: removing the medium from each well, adding 100. mu.l of Brivanib Alaninate 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 48 hours of incubation, 10. mu.l of 5mg/ml MTT solution was added to each well at 37 ℃ and 5% (V/V) CO₂Continuously culturing for 4 hours under the culture condition, then adding DMSO, and observing under a common optical microscope until the Formazan is completely dissolved;

5, measurement and calculation: the absorbance was measured at 570nm and the viability of cells at different concentrations of Brivanib Alaninate was determined by comparing the absorbance of the cells at that concentration to the absorbance of the upper control well and multiplying by 100%.

TABLE 1 toxicity test of different concentrations of Brivanib Alaninate on BHK-21 cells

The experimental results show that different concentrations of Brivanib Alaninate have substantially no effect on the survival rate of BHK-21, indicating that the toxicity of Brivanib Alaninate is low.

Example 2 inhibition experiment of Brivanib Alaninate on DENV 2:

BHK-21 was used as a cell for culturing the virus DENV2, 10TCID₅₀The 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 percent, 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 Brivanib Alaninate diluted in RPMI 1640 medium containing 10% (V/V) fetal bovine serum at a specified concentration at 37 deg.C and 5% (V/V) CO₂Culturing under the culture condition. After 96 hours, after obvious cytopathic effect appears in the cells, collecting the supernatant, and measuring the content of Lactate Dehydrogenase (LDH) released by the 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 and the content of virus RNA in the cells by a protein immunoblotting method and real-time fluorescent quantitative PCR respectively.

2 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 Brivanib Alaninate on LDH release from DENV 2-infected BHK-21 cells

The experimental results are as follows: the experimental result shows that at the concentration of 10 mu M, Brivanib Alaninate has a strong protective effect on BHK-21 cell death caused by DENV2 infection, and the inhibition effect reaches 107.64%. At this concentration, BHK-21 cells were approximately 92.74% viable.

Example 3 mRNA inhibition assay of Brivanib Alaninate on the NS3 and E proteins, key proteins of DENV2

1 collecting total RNA of cells: adding 1ml of Trizol reagent (ambion) into each well of a 6-well cell culture plate, standing at room temperature for 5 minutes, and then sucking supernatant into a 1.5ml Eppendorf tube; adding 0.2ml of chloroform into each tube, shaking, incubating for 15 minutes at room temperature, centrifuging for 15 minutes at 4 ℃ at 12000rpm, and absorbing the upper-layer liquid phase to transfer into another 1.5ml Eppendorf tube; adding 0.5ml of isopropanol, shaking, incubating for 10 minutes at room temperature, and centrifuging for 10 minutes at 4 ℃ and 12000 rpm; removing supernatant by suction, adding 1ml of 75% (V/V) ethanol, washing precipitate, centrifuging at 4 ℃ and 12000rpm for 10 minutes, and removing supernatant by suction; drying at room temperature to make it transparent; adding DEPC (diethyldithiocarbamate) treatment double distilled water 20 mu l to dissolve RNA, and storing at-80 ℃ for later use; and detecting the 260/280 absorbance ratio by an ultraviolet spectrophotometer to calculate the RNA concentration.

Reverse transcription of RNA into cDNA: reaction system: RNA 1mg, 5 XPrimeScript RT MasterMix (TAKARA) 4. mu.l, DEPC treated triple distilled water to a total volume of 20. mu.l. Carefully mix and mix for 15min at 37 ℃ and 5 sec at 85 ℃.

3, real-time fluorescent quantitative PCR (polymerase chain reaction) detection of the RNA level of the virus envelope protein and the non-structural protein 1 of each sample: reaction system (10. mu.l): mu.l of the cDNA template was used,

qPCR Master Mix (Promega) 5. mu.l, forward and reverse primers 0.4. mu.l, DEPC treated triple distilled water 3.2. mu.l. After mixing, the mixture was pre-denatured at 95 ℃ for 10 minutes, at 95 ℃ for 15 seconds and at 60 ℃ for 1 minute (40 cycles).

TABLE 3 Forward and reverse primers for respective target RNAs

4, calculating: and calculating the RNA level of each sample relative to the control group by using a 2-delta C T-T method according to the CT value of each sample.

The experimental results are as follows: referring to figure 1, mRNA levels of NS3 and E, key proteins of DENV2, were significantly reduced and concentration dependent after Brivanib Alaninate treatment. At concentrations of 5 and 10 μ M, mRNA levels of NS3 and E protein were significantly reduced. Showing its ability to inhibit the replication and amplification of dengue virus.

Example 4 protein expression inhibition assay of Brivanib Alaninate on the DENV2 viral Key proteins NS3 and E protein

1 collection of total cellular protein: after BHK-21 cells were treated with drug and dengue virus (DENV2), protein was extracted 48 h: the cells were washed with cold PBS, 100. mu.l of RIPA lysate (Kjeans) plus phosphatase inhibitor (Kjeans) plus protease inhibitor (Kjeans)) was added to each well of the 6-well plate, collected by scraping with a spatula in a 1.5ml Eppendorf tube, centrifuged at 12000rcf at 4 ℃ for 15min, and the supernatant was transferred to a fresh Eppendorf tube.

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; adding 2.5 mul of each of the protein standard substance and the protein sample into a 24-pore plate, adding 1.5ml of 1 XG 250 working solution into each pore, enabling the final volume of each pore to be 2.5ml, shaking for 3 minutes at room temperature, and measuring the absorption of 570nm in each pore by using an enzyme-labeling instrument; the protein concentration in the sample was calculated from the standard curve. Data were processed and samples were added to corresponding volumes of RIPA lysate and loading buffer to make consistent protein concentrations for each sample. Denaturing at 100 deg.c for 5min and storing at-20 deg.c for further use.

3 protein immunoblotting to detect the expression difference of dengue virus protein after treatment with a gradient concentration of Brivanib Alaninate: preparation of 10% SDS polyacrylamide gel: adding various reagents in sequence, preparing 10% separation gel, rapidly filling in glass space, flowing out the time (about 0.8cm of the lower edge of the comb) required for filling concentration gel, rapidly covering a layer of anhydrous ethanol on the separation gel, pouring off the anhydrous ethanol layer after polymerization of the separation gel, sucking with filter paper, continuously filling the concentration gel, and inserting the comb. Electrophoresis: and switching on a power supply, performing electrophoresis at a constant voltage of 80V initially, changing the voltage to 120V after the front edge of the dye enters the separation gel, and determining the electrophoresis time according to the separation degree of the pre-dyed protein Marker and the molecular weight of the target protein, wherein the electrophoresis time generally runs for about 75 min.

4, film turning: 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). Soaking the gel, the filter paper and the PVDF membrane in the transfer liquid, and installing a membrane transfer device in the following sequence: the negative electrode, the cotton pad, the filter paper, the gel, the PVDF film, the filter paper, the cotton pad and the positive electrode are stacked one by one and accurately aligned. 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 about 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.

And 7, 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, 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 experimental results are as follows: referring to figure 2, the expression levels of NS3 and E, key proteins of DENV2, were significantly reduced and concentration dependent after Brivanib Alaninate treatment. At concentrations of 5 and 10 μ M, the expression levels of NS3 and E protein were significantly reduced, consistent with the results at the gene level. It is shown that it indeed has the effect of inhibiting the expression of key proteins of dengue virus.

Example 5 plaque inhibition assay of Brivanib Alaninate on DENV2 Virus

1 cells of C6/36 were plated in 24-well plates, and after 24 hours the cells grew to a monolayer (the area of the bottom of the wells covered by the cells was about 90% -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 adsorption, the supernatant from each well was discarded and PBS was used to wash away unadsorbed virus. 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 plaque is formed, removing the methylcellulose covering culture medium by sucking, fixing the methylcellulose covering culture medium by using 4% paraformaldehyde (doctor's organism), dyeing by using 1% (w/v) crystal violet, flushing the crystal violet by using running water after 2 hours, scanning a 24-hole plate after air drying, and judging the inhibition effect of Mol-5 on the generation of progeny virus of the BHK-21 cells infected by DENV2 according to the number of the plaque.

The experimental results are as follows: referring to FIG. 3, plaque assay of the virus determines the number of progeny virus and infectivity after virus infection. The results show that after treatment, the number of progeny viruses and the infection capacity of the progeny viruses generated after the DENV2 virus infects BHK-21 cells are obviously reduced.

SEQUENCE LISTING

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<120> Britinib used as medicine for treating dengue virus infection and pharmaceutical application thereof

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Claims

1. Use of compound shown as formula I or pharmaceutically acceptable salt thereof in preparation of medicine for treating or preventing dengue virus infection

2. The use according to claim 1, wherein the dengue virus is dengue virus type 1-4.

3. Use according to claim 1, wherein the dengue virus is dengue virus type 2.

4. Application of compound shown as formula I or pharmaceutically acceptable salt thereof in preparing medicine for inhibiting mRNA of NS3 and E protein in dengue virus

5. Use of compound of formula I or its pharmaceutically acceptable salt in preparation of medicine for inhibiting protein expression of NS3 and E proteins in dengue virus