CN111329858B - Application of small molecule inhibitor in inhibiting virus silencing inhibitory protein - Google Patents

Abstract

The invention provides an application of a small-molecule inhibitor in inhibiting a virus silence suppressor protein. The small molecule inhibitor is a compound shown as the following formula (I), or a derivative, a racemate, a stereoisomer, a geometric isomer, a tautomer, a solvate and a pharmaceutically acceptable salt thereof:

the small molecule inhibitor has good inhibition effect on virus silencing inhibitory proteins including P19 and 2b, and provides a foundation for the development of novel antiviral preparations.

Description

Application of a small molecule inhibitor in inhibiting viral silencing inhibitory protein

technical field

The invention relates to the field of molecular therapy, in particular to the application of a small molecule inhibitor in inhibiting viral silencing inhibitory proteins.

Background technique

Viruses are tiny parasitic objects with a size of about 0.02 to 0.3 μm, and are mainly composed of a protein shell (capsid) and nucleic acid (RNA or DNA) inside the shell. For viruses, their replication is completely dependent on cells, and they are first adsorbed to host cells and invaded into cells. After that, DNA and RNA are released (uncoated) in the cell and then replicated, and a specific enzyme is required for this process. A virus-infected host cell fails to function properly and usually dies, and new virus released from the host cell infects other host cells.

In recent years, an increasing number of deaths caused by viral infections such as SARS (Severe Acute Respiratory Syndrome), norovirus and avian influenza have been reported. The prospect of a pandemic now poses a global threat due to increasingly developed transportation networks and mutating viruses. The emergence of new influenza viruses is also an urgent issue requiring immediate action. While the development of antiviral vaccines is one solution to these problems and has been ongoing, due to the specificity of vaccines, they are only effective in inhibiting infection by specific viruses. Vaccines take a considerable amount of time to develop specificity, and they are only preventive, with limited efficacy in patients who have already been infected with viral diseases. Since the virus infects the host, it is fully dependent on the host's metabolism for proliferation, and it is difficult to specifically eliminate the virus without affecting the normal metabolic function of the host, so the prevention and treatment of viral diseases is extremely difficult.

In addition to the rapid decline of the therapeutic effect of the current antiviral drugs with the increase of virus resistance, the side effects on the human body are also too large. For example, the current anti-hepatitis C virus treatment method is the combination of pegylated interferon and ribavirin; but from the perspective of the sustained viral response rate, this standard treatment method is not very effective, and the clinical cure rate is about 50%. At present, the treatment time of this therapy is relatively long, and serious adverse reactions often occur, such as accompanied by mental problems, influenza-like symptoms and hematological toxicity, resulting in the successful cure rate of the existing therapy. Less than 10%. Therefore, it is particularly important to develop a new mechanism, more efficient and less toxic antiviral drugs.

The RNA silencing mechanism (RNA silencing) discovered in recent years is now considered to be the most important and important defense mechanism against viruses. In short, after the virus is infected, its own nucleic acid will cause the host to produce specific degradation of the nucleic acid, so as to achieve the purpose of eliminating the virus. But similar to the arms competition, the virus has correspondingly evolved a silencing suppressor protein (Viral RNA silencing suppressor) to resist the defense method of host RNA silencing. Its mechanism of action is mainly to interfere with the entire silencing mechanism by combining with small RNAs that play an important role in RNA silencing. Therefore, if we find an inhibitor that can effectively inhibit viral silencing inhibitory protein, it can help the host to win the arms competition and achieve the purpose of effectively treating viral diseases.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a small molecule inhibitor or its derivatives, racemates, stereoisomers, geometric isomers, tautomers, solvates, pharmaceutically acceptable salts in Inhibition of viral silencing suppressor protein applications.

The second object of the present invention is to provide a pharmaceutical composition for inhibiting viral silencing inhibitory protein.

In order to realize the above-mentioned purpose of the present invention, the following technical solutions are specially adopted:

A small molecule inhibitor represented by the following formula (I) or its derivatives, racemates, stereoisomers, geometric isomers, tautomers, solvates, pharmaceutically acceptable salts in Use in suppressing viral silencing inhibitory proteins;

Wherein, in formula (I), R ₁ -R ₂₀ are independently hydrogen, substituted or unsubstituted C ₁ -C ₁₂ straight or branched chain alkyl, C ₃ -C ₇ cycloalkyl, C ₁ -C ₅ alkoxy, C ₅ -C ₁₂ aryl, C ₅ -C ₁₂ heteroaryl, (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ aryl, or (C ₁ -C 12 aryl ₄ alkylene)-C ₅ -C ₁₂ heteroaryl;

X ₁ and X ₂ are independently chemical bonds, substituted or unsubstituted C ₁ -C ₆ linear or branched alkyl, C ₁ -C ₅ alkyleneoxy, C ₅ -C ₁₂ arylene, C ₅ -C ₁₂ heteroarylene, (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ arylene, or (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ heteroarylene base.

At the same time, the present invention also provides a pharmaceutical composition for inhibiting viral silencing inhibitory protein, which comprises the small molecule inhibitor of the present invention or its derivatives, racemates, stereoisomers, geometric isomers, tautomers Isomers, solvates, pharmaceutically acceptable salts.

Compared with the prior art, the beneficial effects of the present invention are:

In the present invention, according to the latest molecular mechanism of virus interaction, a virus silencing inhibitory protein inhibitor with good effect is provided.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art.

Fig. 1 is the fluorescence intensity ratio of the upstream and downstream bands of the gel image in Test Example 1;

Figure 2 shows the fluorescence intensity ratio of the upstream and downstream bands of the gel image in Experimental Example 2.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Viruses are pathogens that rely on the host's own factors to multiply, and usually can only be prevented by vaccination and other methods, and there is a lack of effective treatment methods. In the present invention, by screening in the small molecule library (ZINC chemical database), a class of small molecule inhibitors with the effect of inhibiting viral silencing inhibitory protein is obtained, and it is experimentally verified that it has a good inhibitory effect on P19 and 2b proteins, This also provides a basis for the development of new antiviral agents.

In some embodiments of the present invention, the provided small molecule inhibitor with the function of inhibiting viral silencing protein is a compound represented by the following formula (I), or a derivative, racemate, stereoisomer of the compound of formula (I) One of isomers, geometric isomers, tautomers, solvates, and pharmaceutically acceptable salts.

Wherein, in formula (I), R ₁ -R ₂₀ are independently hydrogen, substituted or unsubstituted C ₁ -C ₁₂ linear or branched alkyl, substituted or unsubstituted C ₃ -C ₇ ring Alkyl, substituted or unsubstituted C ₁ -C ₅ alkoxy, substituted or unsubstituted C ₅ -C ₁₂ aryl, substituted or unsubstituted C ₅ -C ₁₂ heteroaryl, substituted or unsubstituted (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ aryl, one of substituted or unsubstituted (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ heteroaryl;

X ₁ and X ₂ are each independently a chemical bond, a substituted or unsubstituted C ₁ -C ₆ linear or branched alkylene group, a substituted or unsubstituted C ₁ -C ₅ alkyleneoxy group, a substituted or unsubstituted C 1 -C 5 alkyleneoxy group, Substituted C ₅ -C ₁₂ arylene, substituted or unsubstituted C ₅ -C ₁₂ heteroarylene, substituted or unsubstituted (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ arylene , or a substituted or unsubstituted (C ₁ -C ₄ alkylene)-C ₅ -C ₁₂ heteroarylene.

In the above and subsequent group definitions of the present invention, the "substituted" means that one or more substitutable hydrogen atoms in the given structure are substituted by a specific substituent, and a substituted group may have a substitution A group is substituted at each substitutable position of a group, and when more than one position in a given formula can be substituted by one or more substituents of a particular group, the substituents may be substituted identically or differently at each position. Specifically, when the substituent is present, then the substituent is a C ₁ -C ₆ alkyl group (eg methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl etc.), or a _C1 - _C5 alkoxy group (eg, methoxy, ethoxy, propoxy, butoxy, etc.).

Meanwhile, in the above and subsequent group definitions in the present invention, "C _a -C _b alkyl" represents a straight-chain or branched saturated alkyl group containing a to b carbon atoms, such as methyl, ethyl, Propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, etc., such as "C ₁ -C ₅ alkyl" means a linear or branched saturated alkyl group containing 1 to 5 carbon atoms ; "C ₃ -C ₇ cycloalkyl" represents a cyclic alkyl group containing only 3 to 7 carbon atoms containing only two elements of carbon and hydrogen, such as cyclopropyl, 2-methylcyclopropyl, cyclopentane base, etc.; "C ₁ -C ₅ alkoxy" means a group containing 1 to 5 carbon atoms and one oxygen atom, such as methoxy, ethoxy, propoxy, isopropoxy, etc.; ""C ₅ -C ₁₂ aryl" means an aromatic cyclic group containing 5 to 12 carbon atoms, such as benzene ring, naphthalene ring, etc.; "C ₅ -C ₁₂ heteroaryl" means containing 5 to 12 carbon atoms A cyclic group with aromatic properties, such as pyrrolidinyl, pyridinyl, etc. ; "C _n -C _m alkylene" means an alkyl group containing n or m methylene groups, such as CH ₂ , (CH ₂ ) ₂ and the like.

In some embodiments of the present invention, in the compound represented by the structure of the above formula (I), R ₁ -R ₂₀ are independently hydrogen, substituted or unsubstituted C ₁ -C ₆ straight or branched chain alkyl, Substituted or unsubstituted C ₃ -C ₇ cycloalkyl, or substituted or unsubstituted C ₁ -C ₅ alkoxy;

X ₁ and X ₂ are independently chemical bonds, substituted or unsubstituted C ₁ -C ₃ linear or branched alkylene (methylene, ethylene, propylene, isopropylidene, etc.), Or C ₁ -C ₃ alkyleneoxy (methyleneoxy, ethyleneoxy, propyleneoxy, etc.).

In some preferred embodiments of the present invention, the provided small-molecule inhibitor is a compound whose structure is shown in the following formula (II), or a derivative, racemate, stereoisomer, geometrically isomeric compound of the compound of formula (II) One of isomers, tautomers, solvates and pharmaceutically acceptable salts.

In formula (II), R ₁ -R ₂₀ are independently hydrogen, substituted or unsubstituted C ₁ -C ₃ linear or branched alkyl, or substituted or unsubstituted C ₁ -C ₃ alkoxy base;

X ₃ is a chemical bond, or a substituted or unsubstituted C ₁ -C ₃ straight-chain alkylene group (methylene, ethylene, propylene, isopropylene, etc.).

In some more preferred embodiments of the present invention, the provided small-molecule inhibitor is a compound represented by the following formula (III), or a derivative, racemate, stereoisomer, geometric isomer of the compound of formula (III) One of isomers, tautomers, solvates, and pharmaceutically acceptable salts.

In formula (III), R ₄ and R ₁₀ are independently hydrogen, C ₁ -C ₃ straight-chain or branched-chain alkyl (methyl, ethyl, propyl, isopropyl, etc.).

In some of the most preferred embodiments of the present invention, the provided small molecule inhibitor is a compound represented by the following formula (IV), or a derivative, racemate, stereoisomer, geometric isomer of the compound of formula (IV) One of isomers, tautomers, solvates, and pharmaceutically acceptable salts.

The compound of formula (IV) above is a commercially available compound (for details, please refer to the ZINC compound database, ID: ZINC79196213).

The small molecule inhibitor with the above structure of the present invention has a good inhibitory effect on virus silencing inhibitory proteins, especially for virus silencing inhibitory proteins including P19 and 2b, has obvious inhibitory effect.

In view of the function of inhibiting viral silencing inhibitory protein provided by the small molecule inhibitor of the above structure provided by the present invention, the present invention can also provide a pharmaceutical composition based on the small molecule inhibitor of the present invention, which also has inhibitory effect on The role of viral silencing inhibitory proteins.

Specifically, the pharmaceutical composition provided by the present invention includes, in addition to the small molecule inhibitor of the present invention having any of the above structures as a functional substance, at least one of a carrier or an adjuvant. In order to achieve a compound therapeutic effect, the pharmaceutical composition of the present invention may further include a second pharmaceutical functional component in addition to the small molecule inhibitor of the present invention.

In some embodiments of the present invention, the carrier is a pharmaceutically acceptable carrier, and some examples that can be used as a pharmaceutically acceptable carrier include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum albumin ( such as human serum albumin), buffer substances (such as twin80, phosphate, glycine, sorbic acid or potassium sorbate), mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, dihydrogen phosphate sodium, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, methylcellulose , hydroxypropyl methylcellulose, lanolin, sugars (such as lactose, glucose and sucrose); starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose vegan and cellulose acetate; powdered tragacanth; malt; gelatin; talc, etc.

In some embodiments of the present invention, in order to facilitate the administration of the pharmaceutical composition, it can also be prepared into different pharmaceutical dosage forms.

Liquid dosage forms for oral administration include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to small molecule inhibitors as active substances, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate esters, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), Fatty acid esters of glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan, and mixtures thereof; and auxiliary materials such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and aromatic agents.

At the same time, corresponding injection preparations can also be prepared, and injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersing agents or wetting agents and suspending agents and other auxiliary materials. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, U.S.P. Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active substance is mixed with at least one inert pharmaceutically acceptable excipient including: a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) Binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) humectants such as glycerol, d) disintegrants such as agar-agar, calcium carbonate, Potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution blockers such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and monohard Glycerides of fatty acids, h) absorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof .

For topical application, the small molecule inhibitors of the present invention can be formulated as a suitable ointment containing the active components suspended or dissolved in one or more carriers. Adjuvants for topical administration of the pharmaceutical composition of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the small molecule inhibitors of the invention may be formulated as micronized suspensions in isotonic, pH-adjusted sterile saline, with or without preservatives such as benzalkonium chloride, or specifically, prepared as Solutions in isotonic, pH-adjusted sterile saline.

Administration of the pharmaceutical composition by nasal aerosol or inhalation is also provided. Such pharmaceutical compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Test Example 1

The structural formula of the small molecule inhibitor used for testing in Test Example 1 is shown below (ie, the compound of formula (IV) of the present invention):

The small molecule inhibitor with the above structure was used to detect the activity of inhibiting silencing suppressor protein P19 by gel electrophoretic mobility shift assay (EMSA). The specific test method is as follows:

P19 and small molecule inhibitors were mixed in binding buffer and fully reacted at room temperature for 25 minutes. The concentration of P19 is 1 μM, and the concentration of this small molecule inhibitor is 2-100 ppm. Subsequently, dsRNA with a final concentration of 25 nM was added, and the reaction was continued for 25 minutes at room temperature after thorough mixing. Then, the results were observed after electrophoresis transfer to the membrane. In the color development results, only dsRNA can show bands, and it is in the downstream position. After the silencing protein is combined with dsRNA, the electrophoresis rate of dsRNA will be slowed down and the electrophoresis distance will be shortened, so it is in the upstream position. When the compound was added and the binding of the protein to dsRNA was inhibited, the dsRNA resumed the original electrophoresis rate, so the band signal was enhanced downstream. The inhibitory activity of the small molecule inhibitor on the binding of suppressor protein to dsRNA can be determined by the ratio of the fluorescence intensity of the upstream and downstream bands.

The ratio of the fluorescence intensity of the upstream and downstream bands of the gel image is shown in Figure 1. Among them, lane 1 is a negative control, containing only dsRNA. Lane 2 is a positive control, containing dsRNA and P19. Lanes 3-8 are experimental groups, both containing dsRNA and P19, and small molecule inhibitors, with a concentration gradient of 2, 5, 10, 20, 40, and 100 mg/L.

It can be seen from the test results shown in Figure 1 that the fluorescence ratio of the negative control group is 0, which means that all dsRNAs are downstream, and there is no P19 binding to them. After the inhibitor protein was added to the positive control, part of the dsRNA was bound to the protein, and the fluorescence ratio was significantly enhanced. However, when increasing concentrations of small molecule inhibitors were added, the fluorescence ratio gradually weakened, indicating that the small molecule inhibitors could effectively prevent the binding reaction between silencing protein and dsRNA.

In conclusion, the small molecule inhibitor at a concentration of about 20 mg/L can effectively inhibit the binding of P19 at a concentration of 1 μM to dsRNA.

Test Example 2

The structural formula of the small molecule inhibitor used for testing in Test Example 1 is shown below (ie, the compound of formula (IV) of the present invention):

The small molecule inhibitor with the above structure was used to detect the activity of inhibiting silencing inhibitor 2b in a gel electrophoresis migration test. The specific test method is as follows:

2b and the small molecule inhibitor were mixed in the binding buffer and fully reacted at room temperature for 25 minutes. The concentration of 2b is 2 μM, and the concentration of this small molecule inhibitor is 2-200 ppm. Subsequently, dsRNA with a final concentration of 50 nM was added, and the reaction was continued for 25 minutes at room temperature after thorough mixing. Then, the results were observed after electrophoresis transfer to the membrane. In the color development results, only dsRNA can show bands, and it is in the downstream position. After the silencing protein is combined with dsRNA, the electrophoresis rate of dsRNA will be slowed down and the electrophoresis distance will be shortened, so it is in the upstream position. When the compound was added and the binding of the protein to dsRNA was inhibited, the dsRNA resumed the original electrophoresis rate, so the band signal was enhanced downstream. The inhibitory activity of the small molecule inhibitor on the binding of suppressor protein to dsRNA can be determined by the ratio of the fluorescence intensity of the upstream and downstream bands.

The ratio of the fluorescence intensity of the upstream and downstream bands of the gel image is shown in Figure 2, among which, lane 1 is a negative control, containing only dsRNA. Lane 2 is a positive control, containing dsRNA and 2b. Lanes 3-9 are experimental groups, all containing dsRNA and 2b, and small molecule inhibitors, with a concentration gradient of 2, 5, 10, 20, 40, 100, and 200 mg/L.

It can be seen from the test results shown in Fig. 2 that the fluorescence ratio of the negative control group is 0, which means that all dsRNAs are downstream, and there is no 2b binding to them. After the inhibitor protein was added to the positive control, part of the dsRNA was bound to the protein, and the fluorescence ratio was significantly enhanced. However, when increasing concentrations of small molecule inhibitors were added, the fluorescence ratio gradually weakened, indicating that the small molecule inhibitors could effectively prevent the binding reaction between silencing protein and dsRNA.

In conclusion, the small molecule inhibitor at a concentration of about 200 mg/L can well inhibit the binding of 2b at a concentration of 2 μM to dsRNA.

Although specific embodiments of the present invention have been illustrated and described, it should be understood that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications as fall within the scope of this invention be included in the appended claims.

Claims (1)

1. The application of a small molecule inhibitor represented by the following formula (IV) in the preparation of a medicine for inhibiting the activity of virus silencing inhibitory protein P19 or 2b;

(IV).

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