TWI520734B - Use of suramin in treating chikungunya virus infection - Google Patents

Description

The use of suramin in the treatment of archavirus infection

This disclosure is about the use of suramin. More specifically, the present disclosure relates to the use of suramin for the treatment of a bowel fever virus infection.

Chikungunya (Chikungunya) virus is a virus that is spread by mosquitoes, mainly by the virus white line aegypti (Aedes albopictus), and Aedes aegypti mosquito (Aedes aegypti) like the genus Aedes (Aedes) mosquitoes to a vertebrate. The arched fever virus (family Togaviridae, genus Alphavirus) has a positive sense single stranded RNA genome. The Alpha virus sends its RNA genome into the cytoplasm by membrane-fusion caused by the endocytic uptake of the receptor and its membrane fusion, and replicates there.

The disease caused by the infection of the bowel fever virus was first confirmed in Tanzania and Uganda in 1953 as Chikungunya fever. The bow is hot in Africa, Southeast Asia, the Indian subcontinent and the Indian Ocean. 2007 In August, Italy broke out the first hot bow epidemic in continental Europe and there were 217 laboratory confirmed cases. This was the first major outbreak in a temperate climate country. Recently, more than 40 countries have confirmed the occurrence of archabolic fever virus infection.

Symptoms of the bowel fever virus infection include sudden fever, joint pain, muscle pain, headache, nausea, vomiting, and bleeding from the nose and gums. Possible but rare complications include gastrointestinal complications, cardiovascular decompensation, and meningo-ecephalitis. Usually, symptoms appear after 4 to 7 days after the infected mosquito bites. Most patients recover from days to weeks, and some patients develop chronic arthritis. The case of lethal fever virus infection is mainly caused by elderly or patients with poor immune system.

Diabetic detection reagents are available for use with the bowel fever virus infection, but antiviral drugs or certified vaccines are still lacking. To date, the treatment of Truffle fever virus infection has been treated with symptomatic therapy, including the administration of non-salicylate analgesics and non-steroids to slow or reduce the inflammatory response caused by viral infections.

In view of this, there is a need in the related art for a method for treating and/or preventing a bowel fever virus infection.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an invention The content is intended to provide a simplified summary of the disclosure to provide a basic understanding of the disclosure.

In one aspect, the disclosure is directed to a method of treating an individual caused by a disease caused by a fever virus infection.

In accordance with an embodiment of the present disclosure, the method comprises administering to the individual a therapeutically effective amount of suramin and a pharmaceutically acceptable excipient, thereby inhibiting infection or replication of the archabolic fever virus.

In various embodiments of the present disclosure, the individual can be a mouse or a human. According to various embodiments of the present disclosure, a 10% suramin solution is injected in a blood vessel or muscle manner. The therapeutically effective amount for an adult is about 1 to 2 grams per dose, about 200 mg to 1 gram for a child, and about 10 to 200 mg for an infant. The total therapeutic dose of suramin is about 5 to 20 grams in adults, about 1 to 10 grams in children, and about 50 to 2 grams in infants and young children.

According to certain embodiments of the present disclosure, infection or replication of the archabolic fever virus can be inhibited by blocking the membrane fusion reaction of the koji virus. For example, the membrane fusion can be a membrane fusion mediated by an envelope protein.

In another aspect, the disclosure is directed to a method of inhibiting fusion of a koji virus membrane in a host cell.

According to an embodiment of the present disclosure, the method comprises administering to the host cell an effective amount of suramin.

In certain embodiments, membrane fusion of the typhoid fever virus to a host cell is inhibited by blocking membrane fusion mediated by the coatment protein.

In certain embodiments, the host cell is a Sf21 cell line Or BHK-21 cell line, and the effective amount is at least 27.5 μM. For example, the effective amount is from about 27.5 μM to 1,750 μM; preferably from about 50 μM to 350 μM.

The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt;<RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; Schematic, the virus carries a bi-cistronic system that can co-express wild-type CHIKV (CHIKV or CS-WT) or TC83 mutant VEEV (VEEV-TC83 or VS-TC83) full-length structural gene (26S) (including capsid (C), E2 glycoprotein (E2) and E1 glycoprotein (E1)) and EGFP; vector as a control virus; abbreviations are as follows: PH is a polyhedrin promoter, EGFP is an enhanced green fluorescent protein gene, and Rhir is a 5'-non-translated region ribose of Rhopalosiphum padi virus (RhPV). In vivo start position (5'-UTR IRES); Figure 2 is an immunofluorescent photographs of Sf21 cells infected with recombinant virus according to Example 2 of the present disclosure; lines represent 20 μm; 4 is a third embodiment of the present disclosure Western blot analysis pictures showing the results; Figures 5 and 6 are fluorescent photos after infection and treatment of Sf21 cells with recombinant virus according to Example 4 of the present disclosure; lines represent 25 μm, Figure 7 And Fig. 8 is a result of a cell fusion test according to Example 5 of the present disclosure; Fig. 9 is a linear diagram of an in vitro antiviral test according to Example 6 of the present disclosure; The western blot analysis picture of Example 6 is disclosed; FIG. 11 is a fluorescent photograph of the Sf21 cell infected and treated with the recombinant virus according to Example 7 of the present disclosure; the line represents 20 μm; and FIG. 12 is implemented according to the present disclosure. Example 8 is a fluorescent photograph of Sf21 cells infected and treated with a recombinant virus; Figure 13 is a histogram of the cell fusion assay according to Example 7 of the present disclosure; and Figure 14 is a Western ink according to Example 9 of the present disclosure. Point analysis picture; Fig. 15 to Fig. 17 are immunofluorescence photographs of BHK-21 cells infected and treated with CHIKV virus according to Example 9 of the present disclosure; and Fig. 18 is a view of Example 10 according to the present disclosure. Suramin of said anti-CHIKV-infected cytotoxicity assays (cytotoxicity assay) test and cellular defense (cell protection assay) histogram.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

For the sake of convenience, some of the words used in the specification, examples, and accompanying claims are hereby incorporated herein. Unless otherwise defined in the specification, the scientific and technical terms used herein have the same meaning as those of ordinary skill in the art to which the invention pertains.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used. Also, words such as "at least one" and "one or more" are used herein to include one, two, three or more.

Although numerical ranges and parameter boundaries are used to define a broad range of values for the present invention, the relevant values in the specific embodiments are presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Here, "about" usually means the actual value is plus or minus 10%, 5% of a specific value or range, Within 1% or 0.5%. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains.

The term "treatment" or "treating" herein includes prophylactic (eg, prophylactic), curative or palliative treatment to produce the desired pharmacy and/or physiology. benefit. More preferably, the benefit is the partial or complete treatment or prevention of a bowel fever virus infection. In addition, the term "treatment" as used herein also refers to the administration or administration of suramin or a pharmaceutical composition thereof to a subject, wherein the individual is suffering from a fever virus infection, having a symptom of a fever virus infection, and a bow A disease or abnormality caused by a fever virus infection, or susceptible to infection by a calvaria virus, in order to partially or completely alleviate, improve, or alleviate the infection of the bowworm, or delay its occurrence, hinder its progression, and reduce its severity. Sex and/or reduce the incidence of one or more symptoms or characteristics. In general, the term "treatment", in addition to improving the symptoms or reducing the symptoms of a disease, also means stopping or slowing the progression or progression of a condition without treatment. Clinical outcomes that are beneficial or desirable include, but are not limited to, alleviating one or more symptoms, narrowing the extent of the disease, stabilizing (ie, not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or slowing the state of the disease or alleviating (regardless of All or all), whether detectable or undetectable.

The term "therapeutically effective amount" as used herein refers to an amount of a component (e.g., suramin) sufficient to produce a desired therapeutic response. A therapeutically effective amount also refers to a compound or composition whose therapeutic benefit exceeds its toxic or detrimental effects. The specific effective amount depends on a number of factors, such as the particular condition being treated, the physiological condition of the patient (eg, patient weight, age or sex), the mammal or animal species being treated, the duration of treatment, current therapy (if any) The nature of the words and the specific formula used. For example, an effective amount can be expressed as the total weight of the drug (e.g., in grams, milligrams, or micrograms) or as a ratio of drug weight to body weight (in milligrams per kilogram (mg/kg)).

In the present specification, a "pharmaceutically acceptable excipient" means a substance which is suitable for use with a main drug without causing excessive harmful side effects (such as toxicity, irritation and allergic reaction). ), with an appropriate benefit/hazard ratio. In addition, each excipient must be "acceptable", i.e., compatible with the remainder of the pharmaceutical composition. The excipient can be in the form of a solid, semi-solid, liquid diluent, cream or capsule.

The term "subject" refers to a mammal that contains humans that is treated with suramin. The term "subject" refers to both male and female individuals, unless a single gender is specifically indicated.

The present invention is based, in part, on the discovery by the inventors that suramin can be used as an active agent for the treatment of a fever virus infection. The present invention utilizes a double-effect baculovirus expression system capable of expressing EGFP and Chikungunya (CHIKV) structural proteins in Sf21 cells to select compounds which specifically inhibit the fusion of the circodian virus membrane. . As shown below, the screening results indicate that other compounds different from suramin (including heparin and polydextrose sulfate (dextran) Sulfate)), although inhibiting the membrane fusion of Alphaviruses such as Venezuelan equine encephalitis virus, does not effectively inhibit membrane fusion of the koji virus. However, suramin blocked the membrane fusion of the koji virus. Therefore, the inventors further conducted an in vitro test on suramin. The results showed that suramin inhibited the infection caused by the archabolic fever virus and/or the replication of the archabolic fever virus.

In one aspect, the disclosure is directed to a method of treating an individual caused by a disease caused by a fever virus infection.

In accordance with an embodiment of the present disclosure, the method comprises administering to the individual a therapeutically effective amount of suramin and a pharmaceutically acceptable excipient for inhibiting infection or replication of the archabolic fever virus.

In certain embodiments, the individual is a mouse. In other embodiments, the individual is a human.

According to certain embodiments of the present disclosure, infection or replication of the archabolic fever virus can be inhibited by inhibiting or blocking membrane fusion of the koji virus. For example, membrane fusion can be a membrane fusion mediated by a mantle membrane protein.

In another aspect, the disclosure is directed to a method of inhibiting fusion of a koji virus membrane in a host cell.

According to an embodiment of the present disclosure, the method comprises administering to the host cell an effective amount of suramin.

In certain embodiments, infection or replication of the archabolic fever virus can be inhibited by blocking membrane fusion of the koji virus. For example, membrane fusion can be a membrane fusion mediated by a mantle membrane protein.

In certain embodiments, the host cell is a Sf21 cell line Or BHK-21 cell line, and the effective amount is at least 50 μM. For example, the effective amount of suramin can be 50, 55, 60, 65, 70, 75, 80, 85, 88, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 μM.

In the following, a plurality of experimental examples are set forth to illustrate certain aspects of the present invention, and the present invention is not limited by the scope of the present invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

Example 1

Plasm construction, recombinant virus preparation and cell infection

The baculovirus transfer vector (pBac-) was constructed according to the method in the article "Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors" (J Virol Methods 2011, 175: 206-215) published by Kuo et al. The backbone of Rhir-E) and the pBac-CHIKV-26S-Rhir-E plasmid containing the full-length cDNA of the CHIKV 26S subgenomic RNA. The full-length cDNA of the structural protein of Venezuelan equine encephalitis virus (VEEV) attenuated strain TC-83 was cloned into the baculovirus transfer vector (pBac-Rhir-E) in the same manner to obtain pBac-VEEV-TC83-26S-Rhir-E plasmid.

The IPBL-Sf21 (Sf21) cell line was cultured in Sf-900 II insect culture medium containing 8% heat-inactivated fetal bovine serum, and cultured at 27 ° C. 1 μL of Cellfectin (Invitrogen, Carlsbad, CA, USA), 0.25 μg of linearized viral DNA Bac-N-Blue (Invitrogen, Carlsbad, CA, USA) and 0.8 μg of plastid were added to Sf21 cells ( The density of 2 x 10 ⁵ cells/culture wells was seeded in 24-well plates to co-transfection the cells. The recombinant virus was harvested from a fluorescent green microscope (Nikon, Tokyo, Japan) from a Sf21 cell culture medium that would scatter green fluorescence. Recombinant viruses (hereinafter referred to as vectors, CS-WT and VS-TC83 viruses, Figure 1) were purified using a series of three end-point dilutions. The titer of the virus was determined by endpoint dilution and fluorescence observation of the 96-well plate and was calculated based on half of the tissue culture infectious dose (TCID50). The viral DNA was sequenced to confirm the sequence of all recombinant viruses.

Viral infection was carried out as follows: IPBL-Sf21 (Sf21) cells were seeded in 6-well plates at a cell size of 2 × 10 ⁶ per well (or 10 ⁵ cells were planted in 96-well plates), and then infected in multiple numbers. The recombinant baculovirus (multiplicity of infection (MOI)) of 1 was infected in Sf-900 II insect culture medium containing 8% fetal bovine serum at pH 6.4.

Example 2

Immunofluorescence analysis

Directly staining the infected cells of Example 1 above, The staining conditions were that rabbit anti-CHIKV E2 serum or mouse anti-VEEV serum diluted 1:100 was reacted at room temperature for 30 minutes. After washing twice with ice PBS, the cells were co-localized with a second antibody for 30 minutes, wherein the second antibody was Alexa Fluor 594-conjugated (Alexa Fluor 594-conjufated) goat anti-rabbit (goat anti-rabbit) IgG or anti-mouse IgG (Invitrogen, Molecular Probes, Carlsbad, CA, USA) followed by two washes with ice PBS. The stained cells were recorded using a inverted fluorescence microscope (Model IX71, Olympus, Japan) in which CHIKV E2 staining was recorded in a red-channel.

In the combined image of Figure 2, green and red fluorescence represent the expression of EGFP and viral glycoprotein signals (indicated by arrows) after infection with Sf21 cells. Red cells without EGFP signal are dead cells. After infection by CS-WT (pictured above in Figure 2) or VS-TC83 virus (picture below Figure 2), the red signal on the cell surface circulated by the viral glycoprotein represents the viral glycoprotein of Alpha virus in the cell. surface. In contrast, cells infected with control baculovirus did not detect a red signal (right panel in Figure 2).

Example 3

Western blot analysis

Western blot analysis of CHIKV viral protein was performed on the total protein of Sf21 cells in Example 1 for two days after infection, and then subjected to 10% polydecyl sulfate-polyacrylamide gel. Electrophoresis, SDS-PAGE) was performed. After separation by SDS-PAGE, the protein was electrotransferred to a PVDF membrane (polyvinylidene difluoride; Millipore, Billerica, MA, USA). The membrane was soaked in Tris-buffered saline (TBS; 100 mM Tris (pH 7.4), 100 mM NaCl and 0.1% Tween 20) containing 5% (by volume) skim milk powder and shaken slightly at room temperature. 1 hour. Next, the membrane was placed in an anti-E2, anti-E1 or anti-CHIKV antibody (detecting the shell) diluted 1:2000 in TBS containing 5% (by volume) skim milk powder at 4 ° C. Place it until the next day. The membrane was washed three times for 5 minutes with shaking in TBS buffer at room temperature to remove unreacted antibodies. The membrane was then placed in a 1:2500 ratio of peroxidase-conjugated goat anti-Rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, CA, USA) at room temperature 1 hour. The HRP on the membrane was detected using a LumiFast Plus Chemiluminescence Detection Kit (T-Pro Biotechnology, Taiwan, R.O.C.) according to its instructions. Use the UVP AutoChemi Image System to capture and process images.

The cells infected with CS-WT virus (top panel of Figure 3) and purified CHIKV can detect protein bands corresponding to CHIKV E1, E2 and C at the colloidal positions 52, 56 and 36 kDa, respectively. The cells infected by the control virus do not have the corresponding protein bands (the second protein band in Figure 3). These results indicate that Sf21 cells infected with CS-WT will express E1, E2 and C proteins of CHIKV.

The viral protein of VEEV-TC83 was collected two days after infection. The above procedure was followed (except that the antibody was replaced with mouse anti-VEEV serum). As shown in Figure 4, these results indicated that uninfected Sf21 cells (1st protein band, negative control group) and Sf21 cells infected with control virus (2nd protein band, positive control group) were not A viral structural protein was detected. On the other hand, in the Sf21 cells infected with the VS-TC83 virus (the third protein band in Fig. 4), the viral capsid protein was detected, whereas the BHK-21 cells infected with the VEEV-TC83 virus were also The shell protein of the virus and the E1/E2 protein of the virus can be detected (Fig. 4, the fourth protein band).

Example 4

Cell fusion induced by recombinant virus

Fluorescence microscopy with a FITC channel (pictured above in Figure 5) and a bright field (figure in Figure 5) was used to observe the presence or absence of further anti-CHIKV serum infection with CS-WT virus (1) : 200) Sf21 cells treated, and Sf21 cells infected with control virus, and images were taken. In the control group, the Sf21 cells infected with the control virus did not show cell fusion (right panel of Fig. 5). Cell fusion was observed in Sf21 cells infected with CS-WT virus and not treated with anti-CHIKV serum (left panel in Figure 5; arrow indicates multinucleated cells). Anti-CHIKV serum is effective in inhibiting cell fusion caused by infection with CS-WT virus (Fig. 5).

To investigate the dose-dependency of antiserum doses and inhibition of cell fusion, Sf21 cells were infected with CS-WT virus with M.O.I.1 in Sf-900 II containing 8% FCS. 1 day after infection, The cells were cultured in growth medium, and the anti-anti-CHIKV (different anti-CHIKV) diluted in different ratios (0 (none), 1:200, 1:400, 1:800, 1:1600, and 1:3200) was added. Rabbit anti-whole CHIKV) serum, placed at 27 ° C for 1 hour. Thereafter, it was replaced with a Sf-900 II (pH 5.8) culture solution containing 2% FCS, 0.1 mg/ml cholesterol, and a corresponding diluted serum. Two days after the infection, the inverted specimens (IX71; Olympus) were used to examine and visualize the samples. As shown in the results of Figure 6, the effect of antiserum against cell fusion increases with increasing antiserum concentration.

Example 5

Screening for polysaccharides that inhibit the cell fusion caused by recombinant viruses

Sf21 cells were infected with recombinant baculovirus according to the procedure described in Example 1 above. Two days after infection, infected Sf21 cells were pretreated with growth medium containing the following polysaccharides: chondroitin sulfate (1 mg/mL), chitosan (chitosan, 2 μg/mL), poly Glucose sulfate (1 mg/mL), heparin (1 mg/mL), hyaluronic acid (1 mg/mL), and mannan (mannan, 1 mg/mL) were allowed to stand for 1 hour. The cell culture medium was replaced with Sf-900 II SFM containing the specific polysaccharide at the above concentration, 0.1 mg/ml cholesterol and 2% FCS (pH 5.8), and then allowed to stand for 2 hours. Next, the formation of cell fusion was observed using a fluorescence microscope; calculation was performed at 200 x magnification with at least 100 nuclei per field of view. The fusion index is calculated by dividing the number of cells expressing EGFP by the number of multinucleated cells, and Figure 7 summarizes the results.

As shown in Figure 7, the polysaccharides tested were as described above. At the dose, the fusion of Sf21 cells infected with CS-WT virus (CHIKV) could not be inhibited. On the other hand, only two of the six test compounds (i.e., polyglucose sulfate and heparin) significantly inhibited the fusion of Sf21 cells infected with vAc-VS-TC83-26S-Rhir-E (VS-TC83) virus. In the control group, Sf21 cells infected with the control virus (control) produced only a few cell fusions.

According to the above procedure, different concentrations of mannan, heparin and polyglucose sulfate were added to the culture medium of Sf21 cells infected with CS-WT (CHIKV) virus or VS-TC83 (VEEV-TC83) virus. Figure 8 summarizes these results with a fusion index.

As shown in Figure 8, heparin and polydextrose sulfate (1, 10, 100, and 1000 μg/ml) inhibited cell fusion induced by VS-TC83 virus, respectively, and the degree of inhibition was related to the polysaccharide compound administered. The dose is positively correlated. However, the polysaccharide compounds tested were unable to inhibit cell fusion induced by CS-WT at specific doses.

Example 6

Effect of the polysaccharides selected in Example 5 on inhibiting VEEV-TC83 infection

Another cell strain, BHK-21 cells, was also used to test the antiviral effect of polysaccharides on CHIKV or VEEV-TC83 infection. Briefly, BHK-21 cells were seeded in 6-well plates. The virus and the above concentrations of mannan, polyglucose sulfate and heparin were co-disposed on ice for 30 minutes, and then BHK-21 cells were infected with ice on the formulation for 30 minutes, then washed with growth medium and transferred. Incubate at 37 ° C for 24 hours. In the above way The plaque reduction assay was detected and calculated, and the results were summarized in Figure 9.

Colloidal electrophoresis was used to analyze the effect of polysaccharides on the production of viral capsid proteins. Briefly, according to the above procedure, BHK-21 cells were infected with CHIKV or VEEV-TC83 virus pretreated with mannan (0.32 mg/ml), polyglucose sulfate (0.625 mg/ml) or heparin (1 mg/ml). . One hour after infection, total protein was separated by 10% SDS-PAGE. Western blot analysis was performed using anti-VEEV serum or anti-CHIKV serum to detect viral capsid protein. The antibody bound to the membrane was further stripped, and the expression of tubulin was re-probing with an anti-tubulin antibody as an experimental control. The results are shown in Figure 10.

As shown in Figure 10, none of the test compounds were effective in inhibiting CHIKV infection, while heparin and polyglucose sulfate inhibited VEEV-TC83 infection in a dose-dependent manner.

Example 7

Effect of suramin on cell fusion induced by recombinant virus

In addition to the polysaccharides identified in Example 6, the recombinant virus prepared in Example 1 can also be used as a systematic screening platform for screening FDA-approved drug libraries (L1300, Selleckchem), disease resistance. Antiviral agents and synthetic peptides to identify possible agents that inhibit cell fusion caused by CS-WT infection. Most test compounds, including several known disease resistance Toxic agents (eg, arcidol hydrochloride, silymarin, carrageenan, ezetimibe, and synthetic peptides) are unable to inhibit cell fusion of infected cells (no results) ). At the time of the first screening, only one compound of suramin significantly inhibited cell fusion of infected Sf21 cells.

Specifically, 10 ⁵ of Sf21 cells were seeded in a 96-well plate, and then infected with a CS-WT virus having an MOI of 1. One day after infection, the culture medium was replaced with Sf-900 II SFM (pH 6.8 and 2% FCS) to prevent cell fusion. After 2 days of infection, the culture medium was replaced with Sf-900 II SFM (pH 5.8, 0.1 mg/mL cholesterol and 2% FCS) containing 5 mg/mL chondroitin sulfate or 5 mg/mL (350 μM) suramin. Set for 2 hours. Cell fusion was detected and photographed using an inverted fluorescence microscope (Olympus Model IX71, Tokyo, Japan), and the results are shown in Fig. 11.

CS-WT can initiate Sf21 cell fusion relative to the control virus (panel (D) of Fig. 11) (Fig. (C) of Fig. 11). Although chondroitin sulfate cannot significantly inhibit cell fusion (Fig. 11 (A)), suramin can significantly inhibit cell fusion of infected Sf21 cells (Fig. 11 (Fig. 11)).

Using the above procedure, administration of 3,500 μM, 875 μM, 219 μM or 55 μM of suramin can dose-dependently inhibit cell fusion of infected Sf21 cells. Figure 12 shows the phenomenon of cell fusion detected and photographed by inverted fluorescent microscope.

The inhibitory effect is expressed by a "fusion index", wherein a lower fusion index refers to a preferred inhibitory effect. As shown in Figure 13, the results indicate that in a specific dose range, suramin The inhibitory effect increased with increasing concentration of suramin, while the inhibitory effect of 55 μM suramin was the lowest.

Example 8

Effect of suramin on the production of structural protein of CHIKV

BHK-21 cells were infected with CHIKV to investigate whether suramin inhibited the expression of CHIKV viral proteins to further verify whether the baculovirus-insect cell screening system can be used for the screening of inhibitory drugs for CHIKV infection in the above description. Briefly, BHK-21 cells are first cultured in a 12-well plate, and then CHIKV containing 2% FCS, 1×10 ⁴ plaque-forming units (PFU) and the above-mentioned concentrations of suramin 1 mL of DMEM was added to each well. Suramin, at a final concentration of 350 μM, was added at 0, 1, 2, 3 or 4 hours after infection to perform a time-of-addition assay with a total incubation time of 20 hours. After the culture, the cells were lysed with 500 μL of 1x sample buffer. Total protein was isolated by SDS-PAGE (20 μL per well). Western blotting method is anti-CHIKV E2 serum (1:1000), free of anti-anti-CHIKV serum (1:2000) or anti-actin MAb (A4700, Sigma; 1: 500) to perform dyeing.

As shown in the results of Figure 14, suramin can dose-dependently inhibit the production of structural proteins of CHIKV. Fig. 12 and Fig. 13 also show similar results, that is, 44 μM of suramin has only a slight inhibitory effect. The results of the time course test showed that the inhibitory activity of suramin occurred in the early stage of the viral life cycle (0-2 hours after infection).

Example 9

Effect of suramin on inhibiting in vitro infection of CHIKV

BHK-21 cells were infected with CHIKV with MOI of 1 in the presence of 175 μM (2.5 mg/mL) suramin (group A) or 12.5 μg/ml ribavirin (group B); positive control was Cells that were infected with CHIKV without additional treatment (Group C); cells that were not infected with CHIKV served as a negative control (Group D). After 20 hours of infection, the cells were permeabilized with acetone, and the anti-CHIKV E2 serum diluted 1:100 was incubated for 30 minutes at room temperature. After washing twice with ice PBS, the cells were co-localized with a 1:500 dilution of the second antibody for 30 minutes, wherein the second antibody was Alexa Fluor 594-conjugated goat anti-free IgG (Invitrogen, Molecular Probes) , Carlsbad, CA, USA), followed by washing twice with ice PBS. An inverted fluorescence microscope (Model IX71, Olympus, Japan) was used to capture images of stained cells, which were recorded in red channels to record CHIKV E2 staining.

As shown in the results of Fig. 15, the addition of suramin inhibited the in vitro infection of CHIKV (Fig. 15A), and the inhibitory effect of suramin was higher than that of ribavirin (Fig. 15B).

To elucidate the dose-related effects of suramin in inhibiting CHIKV infection in vitro, BHK-21 cells were seeded in 96-well plates; 100 μL of DMEM (containing 2% FCS, 2,000 PFU of CHIKV and the above concentrations of Sue) were added to each well. Lamin). After incubation for 20 hours, immunofluorescence assays were performed according to the procedures of the above examples.

The results in Fig. 16 show that the in vitro inhibitory effect of suramin on CHIKV increases with increasing concentration of suramin. Less dose 44 μM of suramin has only a slight in vitro inhibitory effect (Figure D of Figure 16).

The time course test was to plant BHK-21 cells in 96-well plates. 50 μL of DMEM containing 2% FCS and 2,000 PFU of CHIKV was added to each well. 5 mg/mL (350 μM) of suramin was added to each cell at the designated time point after infection, and the mixture was incubated for 20 hours. Next, an immunofluorescence test was performed in accordance with the above procedure. Figure 17 shows the results of the time course test, showing that the inhibitory activity of suramin occurs early in the viral life cycle (0-2 hours after infection; Figure 17 (A) to (C)), and in the late stage Laming has almost no inhibitory effect (Fig. 17 (D) to (F)).

Example 10

Cytotoxicity test and cell defense test of suramin against CHIKV infection

10 ⁵ BHK-21 cells were seeded in 96-well plate, and cultured in 2% FCS DMEM medium. The cytotoxicity test was performed by culturing the cells in two replicates with or without different concentrations of the drug for two days. The cell defense assay was performed in two replicates with or without different concentrations of the drug, and the cells were infected with CHIKV at an MOI of 0.05 for another two days. The cells were stained with a 0.1% crystal violet solution to observe the cell monolayer gap caused by cytotoxic or cytopathic effects. The absorption wavelength of crystal violet was measured by optical density (OD) 540.

As shown in Figure 18, the addition of suramin protects BHK-21 The cells are protected from cytopathic effects caused by CHIKV infection. At a dose of 1,000 μg/ml, suramin has a small amount of cytotoxicity (Fig. 18, left panel). In addition, when the concentration of suramin was 15.6-1,000 μg/ml, it had a significant effect against the pathogenesis of CHIKV-infected cells (Fig. 18, right panel).

In summary, the foregoing example demonstrates that suramin not only inhibits cell fusion caused by infection with the recombinant virus (ie, CS-WT), but also inhibits CHIKV infection in vitro. Therefore, suramin can treat CHIKV infection in a living body. Alternatively, suramin can be used to inhibit CHIKV infection in vitro.

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

Claims (7)

A use of suramin for the preparation of a medicament for treating a fever virus infection, wherein the suramin is inhibited by an envelop protein of Chikungunya virus Membrane fusion of the media to inhibit infection or replication of the archabolic fever virus. A method of inhibiting membrane fusion mediated by a canine archavirus coat protein in a host cell in vitro, comprising administering to the host cell an effective amount of suramin. The method of claim 2, wherein the host cell is a Sf21 cell line or a BHK-21 cell line. The method of claim 2, wherein the effective amount is from 50 μM to 350 μM. A method of protecting a host cell from cytopathic effects by infection with a typhoid fever virus in vitro comprises administering to the host cell an effective amount of suramin. The method of claim 5, wherein the host cell is a Sf21 cell line or a BHK-21 cell line. The method of claim 5, wherein the effective amount is from 10 μM to 700 μM.