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Antiviral agent


Title: Antiviral agent.
Abstract: An object of the present invention is to provide a viral disease control agent which has a mechanism of action different from conventional one as a substitute for existing viral disease control methods and is used in more practical and safer manners. The present invention utilizes a compound having inhibitory activity on the binding of a substance α to a PTGS suppressor protein, wherein the substance α has a property of inducing PTGS and a property of binding to the PTGS suppressor protein and shows a decrease in the property of inducing PTGS upon binding to the PTGS suppressor protein. ...

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USPTO Applicaton #: #20100216981 - Class: $ApplicationNatlClass (USPTO) -
Inventors: Shinsuke Sano, Takako Fukagawa, Hirokazu Yamada, Chikara Masuta, Hanako Shimura



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The Patent Description & Claims data below is from USPTO Patent Application 20100216981, Antiviral agent.

TECHNICAL FIELD

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The present invention relates to a PTGS suppressor protein binding inhibitor and an antiviral agent having a novel mechanism of action.

BACKGROUND ART

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A very large number of pathogenic viruses have spread on earth, and their infection has caused suffering or economic losses in various organisms such as animals including humans and useful plants (e.g., crops). Examples of viral diseases caused by a pathogenic virus infection include rice dwarf disease caused by rice dwarf virus infection, tomato mosaic disease caused by tobacco mosaic virus infection, human influenza caused by influenza virus infection, human hepatitis B caused by hepatitis B virus infection, and acquired immune deficiency syndrome caused by human immunodeficiency virus (HIV) infection.

Host organisms such as plants or animals, when infected with pathogenic viruses, develop various biological defense mechanisms against the infection to inhibit the growth of the viruses or to relieve the disease symptom. One of these defense mechanisms is post-transcriptional gene silencing (hereinafter, abbreviated to PTGS). PTGS is a phenomenon induced by double-stranded RNAs (dsRNAs) of viruses or the like, wherein transcribed messenger RNAs (mRNAs) are degraded in a sequence-specific manner. PTGS is a mechanism conserved not only in higher organisms such as plants or animals but also in various other organism species from protozoans to fungi and is thought to be a particularly important defense mechanism for plants which do not have an immune system, unlike animals. Specifically, in this mechanism, PTGS-inducing dsRNAs are degraded by intracellular nuclease Dicer or an enzyme analogous thereto into short RNAs of approximately 21 to 24 bases called small interfering RNAs (siRNAs), and the siRNA is further incorporated into a nuclease complex called an RNA-induced silencing complex (RISC), which in turn cleaves mRNA homologous to the siRNA sequence, thereby inhibiting the expression of the target protein such as viruses.

It has recently been revealed that many pathogenic viruses encode, as a counter against the PTGS mechanism of host organisms, a suppressor protein that inhibits this PTGS (PTGS suppressor protein; hereinafter, abbreviated to PTGS-SP) (e.g., Patent Document 1). It has further been reported that the majority of these PTGS-SPs inhibit PTGS through the direct binding to siRNAs (e.g., Non-Patent Document 1).

PTGS-SPs expressed by plant viruses have been reported, for example, HC-Pro of viruses of the genus Potyvirus (see Non-Patent Document 2), 2b of viruses of the genus Cucumovirus (see Non-Patent Document 3), p25 of viruses of the genus Potexvirus (see Non-Patent Document 4), p19 of viruses of the genus Tombusvirus (see Non-Patent Document 5), and coat proteins of viruses of the genus Carmovirus (see Non-Patent Document 6).

Many attempts have been made to develop preventive or therapeutic agents for the viral diseases for reducing damages caused by the viral diseases. For example, M2 ion-channel inhibitors (e.g., amantadine) and neuraminidase inhibitors (e.g., zanamivir phosphate and oseltamivir) are known as effective therapeutic agents for influenza. These M2 ion-channel and neuraminidase inhibitors probably exert therapeutic effects on influenza by preventing the influenza viruses from growing or infecting other cells. Moreover, known effective therapeutic agents for acquired immune deficiency syndrome are broadly classified into reverse transcriptase inhibitors (e.g., azidothymidine and didanosine) and protease inhibitors (e.g., ritonavir and indinavir). Multi-drug therapy using these agents exerts remarkable effects, which drastically reduces the number of deaths in advanced countries.

However, these therapeutic agents for influenza or acquired immune deficiency syndrome are also known to have side effects, and it is believed that drug resistance viruses will inevitably appear due to the variability of the viruses. Therefore, the development of a novel antiviral agent having a different mechanism of action has been demanded. Furthermore, agents against viruses, except for some agents structurally similar to nucleic acids, are only applicable to a target viral disease. Moreover, vaccination, albeit effective, must be performed before infection and has problems such as time taken to develop antibodies or the easily variable antigenic site of viruses. Furthermore, the therapeutic agents or vaccines are only applicable to a target viral disease. Therefore, therapeutic agents for viral diseases had to be developed for each type of virus.

On the other hand, various control methods have been developed for viral diseases in plants. Examples thereof include selective breeding of resistant varieties to viral diseases, raising of virus-free plants by stem tip culture or heat treatment, inhibition of pathogenic virus infection by treatment with selected attenuated viruses, and use of plants having virus resistance imparted by transformation. Moreover, examples of the control methods using agricultural chemicals include use of a fungicide that induces the resistance of plants or an insecticide that targets insect vectors or the like for viruses.

However, the breeding of resistant varieties requires a long period, and resistant strains of viruses inevitably appear. The raising of virus-free plants by stem tip culture or the like is not perfect. The use of attenuated viruses is highly effective for viral disease control. However, attenuated viruses are difficult to stably prepare and are effective only for viruses of the same species or related species. Plant defense activators have unstable effects. Spraying large amounts of insecticide that preventively controls insect vectors for viruses may lead to environmental pollution and cannot be expected to have therapeutic effects on virus-infected plants.

On the other hand, urgency and markets for antiviral agents for viral diseases in plants are much smaller than those for antiviral agents for viral diseases in humans. Therefore, it is highly possible that the cost of developing the antiviral agents for viral diseases in plants cannot be recovered even if they are developed at cost much lower than the cost of developing the antiviral agents for viral diseases in humans, for example, anti-HIV drugs or anti-influenza drugs. In addition, the existing vaccines or antiviral agents are expected, as described above, to be effective only for the target viruses, and resistant strains of viruses inevitably appear. Therefore, the development of antiviral agents for viral diseases in plants has hardly proceeded so far. Patent Document 1: Japanese Laid-Open Patent Application No. 2004-344110 Non-Patent Document 1: Goto K., et al., Plant Cell Physiol. 2007, 48, 1050-60 Non-Patent Document 2: Anandalakshmi R., et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 13079-13084 Non-Patent Document 3: Brigneti G., et al., EMBO J., 1998, 17, 6739-6746 Non-Patent Document 4: Voinnet O., et al., Cell, 2000, 103, 157-167 Non-Patent Document 5: Baulcombe D C., et al., Trends Biochem Sci. 2004, 29, 279-81 Non-Patent Document 6: Qu F., et al., J. Virol. 2003, 77, 511-522

DISCLOSURE OF THE INVENTION

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Object to be Solved by the Invention

It has been demanded to provide a control method which has a mechanism of action different from conventional one as a substitute for the existing viral disease control methods described above and is used in more practical and safer manners. Thus, an object of the present invention is to provide a viral disease control agent having a mechanism of action different from conventional one.

Means to Solve the Object

The present inventors have conducted diligent studies in consideration of the object and consequently found that a therapeutic effect of reducing damages caused by viral diseases or an effect of attenuating highly virulent viruses having strong pathogenicity is obtained without toxicity to or strong influence on applicable organisms by inhibiting the binding of PTGS-SP to siRNA such that the PTGS-SP function is inhibited. Furthermore, the present inventors have actually studied the effects of compounds that actually inhibit the functions of various PTGS-SPs. Based on these findings, the present invention has been completed.

Specifically, the present invention relates to:

[1] a PTGS suppressor protein binding inhibitor containing a compound having inhibitory activity on the binding of a substance α to a PTGS suppressor protein, wherein the substance α has a property of inducing PTGS and a property of binding to the PTGS suppressor protein and shows a decrease in the property of inducing PTGS upon binding to the PTGS suppressor protein;

[2] the PTGS suppressor protein binding inhibitor according to [1], wherein the substance α is siRNA; and

[3] the PTGS suppressor protein binding inhibitor according to [1] or [2], wherein the compound is at least one selected from the group consisting of cyclic ketone compounds represented by the following formulas (1) to (6):

Moreover, the present invention relates to:

[4] the PTGS suppressor protein binding inhibitor according to [1] or [2], wherein the compound is a reaction product of croconic acid with hydrogen peroxide;

[5] an antiviral agent comprising as an active ingredient a compound having inhibitory activity on the binding of a substance α to a PTGS suppressor protein, wherein the substance α has a property of inducing PTGS and a property of binding to the PTGS suppressor protein and shows a decrease in the property of inducing PTGS upon binding to the PTGS suppressor protein;

[6] the antiviral agent according to [5], wherein the substance α is siRNA; and

[7] the antiviral agent according to [5] or [6], wherein the compound is at least one selected from the group consisting of cyclic ketone compounds represented by the following formulas (1) to (6):

Furthermore, the present invention relates to:

[8] the antiviral agent according to [5] or [6], wherein the compound is a reaction product of croconic acid with hydrogen peroxide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 It is a graph showing the inhibitory activity of compound 7 of the present invention on CMV 2b according to a protoplast method.

FIG. 2 It is a photograph showing the inhibitory activity of compound 7 of the present invention on CMV 2b according to a silencing plant method.

FIG. 3 It is a graph showing the antiviral activity of compound 7 of the present invention according to a symptom test method.

BEST MODE OF CARRYING OUT THE INVENTION

A PTGS-SP binding inhibitor of the present invention is characterized by containing a compound having inhibitory activity on the binding of PTGS-SP to a substance α (hereinafter, also referred to as a “compound according to the present invention”), wherein the substance α has a property of inducing PTGS and a property of binding to the PTGS-SP and shows a decrease in the property of inducing PTGS upon binding to the PTGS-SP. The inhibition of the binding of a substance α to PTGS-SP can restore the substance α function inhibited by PTGS-SP, i.e., the function of inducing PTGS. Therefore, the use of the compound according to the present invention can reduce the inhibitory effect of viruses on PTGS, resulting in, for example, the reduced toxicity or inhibited growth of the viruses. As a result, an antiviral effect is obtained.

(PTGS-SP Protein)

The PTGS-SP targeted by the PTGS-SP binding inhibitor according to the present invention is not particularly limited as long as it is a protein that is derived from any kind of virus and has the ability to inhibit the PTGS of one or more organisms. Preferable examples of the organisms can include plants and animals. The plants can be exemplified more preferably by: food crops such as cereals (e.g., rice, wheat, and corn), pulse crops (e.g., soybean and peanut), tubers (e.g., potato and sweet potato), vegetables (e.g., Japanese radish, carrot, cabbage, lettuce, eggplant, cucumber, and tomato), and fruit trees (e.g., apple, pear, and citrus); and nonfood crops such as flowers and ornamental plants (e.g., rose, carnation, and chrysanthemum), foliage plants (e.g., ivy and maidenhair), and trees (e.g., pine and cherry). More preferable examples of the animals can include birds and mammals such as human, monkey, chimpanzee, cow, horse, pig, sheep, rabbit, dog, cat, rat, mouse, and guinea pig.

Specifically, the PTGS-SP targeted by the PTGS-SP binding inhibitor according to the present invention can be exemplified preferably by: PTGS-SPs derived from viruses whose natural host is a plant, such as HC-Pro of viruses of the genus Potyvirus (e.g., TuMV: turnip mosaic virus), 2b of viruses of the genus Cucumovirus (e.g., CMV: cucumber mosaic virus), p25 of viruses of the genus Potexvirus (e.g., PVX: potato virus X), p19 of viruses of the genus Tombusvirus (e.g., TBSV: tomato bushy stunt virus), and coat proteins of viruses of the genus Carmovirus (e.g., CarMV: carnation mottle virus); and PTGS-SPs derived from viruses whose natural host is an animal (particularly, a mammal), such as tat (Genbank Accession No. NC—001802 or NC 001722) of viruses of the genus Lentivirus (e.g., HIV: human immunodeficiency virus), NS1 (Genbank Accession No. NC 002020) of viruses of the genus Influenzavirus A (e.g., FLUAV: influenza A virus), NS1 (Genbank Accession No. NC 002211) of viruses of the genus Influenzavirus B (e.g., FLUBV: influenza B virus), and NS1 of viruses of the genus Influenzavirus C (e.g., FLUCV: influenza C virus).

In addition to the above-exemplified PTGS-SPs known in the art, any protein that functions as PTGS-SP can be targeted by the PTGS-SP binding inhibitor of the present invention.

Whether a certain protein is a PTGS-SP can be confirmed by: for example, artificially inducing PTGS of an arbitrary gene in a cell; causing the cell to express the protein simultaneously with a protein encoded by the arbitrary gene; and examining the recovery of the arbitrary gene from PTGS, compared with the cell not expressing the protein. The decrease in PTGS can be confirmed by detecting a decrease in the accumulation level of mRNAs of the gene targeted by PTGS or a decrease in the accumulation level of its target protein itself.

For example, whether a certain protein is a PTGS-SP can be confirmed by an agroinfiltration method which involves: injecting a GFP (green fluorescence protein) gene into the intercellular space of a plant leaf using Agrobacterium; and transiently inducing PTGS of the GFP gene. A virus gene encoding a PTGS-SP candidate is expressed simultaneously with the GFP gene, and the protein encoded by this virus gene is identified as PTGS-SP when light emitted by GFP is observed. Alternatively, it can also be confirmed by a protoplast method which involves transiently inducing PTGS of the GFP gene using the protoplast of a plant leaf. In this case, the GFP gene and so on are directly incorporated into the protoplast by polyethylene glycol (PEG), and PTGS-SP can be identified based on the presence or absence of light emitted by GFP.

(Substance α)

The substance α according to the present invention is not particularly limited as long as it is a substance that has property of binding to any PTGS-SP and property of inducing PTGS in the cell of any organism and shows a decrease in its own property of inducing PTGS upon binding to the PTGS-SP. The substance α is preferably a nucleic acid, more preferably virus-derived RNA (e.g., dsRNA or siRNA), and even more preferably siRNA. Specifically, the RNA used as the substance α can be exemplified by a virus-derived RNA sequence or a portion thereof and can be exemplified more preferably by virus-derived siRNA of 21 to 23 bases.

The sequence of the dsRNA or siRNA used as the substance α according to the present invention is not particularly limited and is preferably a sequence (or a partial sequence thereof) corresponding to the nucleic acid of a virus serving as a target to obtain the antiviral effect or an RNA sequence transcribed from the nucleic acid, more preferably a sequence of 20 to 27 bases corresponding to the nucleic acid of a virus serving as a target to obtain the antiviral effect or an RNA sequence transcribed from the nucleic acid, and even more preferably a sequence of 21 to 23 bases corresponding to the nucleic acid of a virus serving as a target to obtain the antiviral effect or an RNA sequence transcribed from the nucleic acid.

Whether the substance α such as dsRNA or siRNA binds to PTGS-SP can be confirmed by using a method described later such as surface plasmon resonance, single-molecule fluorescence analysis, gel shift assay, and thermal denaturation. Moreover, whether the substance α such as dsRNA or siRNA shows a decrease in its property of inducing PTGS upon binding to the PTGS-SP can be confirmed by: artificially inducing PTGS of an arbitrary gene in a cell; causing the cell to express PTGS-SP; and examining the promotion of the intracellular PTGS in the presence of the particular substance, compared with the absence of the substance.

(Inhibitor of the Present Invention)

The compound used as the inhibitor according to the present invention is not particularly limited as long as it has inhibitory activity on the binding of a substance a to PTGS-SP. The compound according to the present invention also includes compounds such as proteins (e.g., antibodies) and nucleic acids. Specifically, the compound according to the present invention can be exemplified preferably by compounds shown in Table 1 below or derivatives thereof.

These compounds are cyclic ketone compounds. The compounds of compound Nos. 7 and 8 are reaction products of croconic acid with hydrogen peroxide, synthesized in Synthesis Examples 1 and 2, respectively. The compound according to the present invention other than the compounds shown in Table 1 can be identified easily by screening using an evaluation system of the present invention (in vitro and in vivo screening methods) described later.

TABLE 1 Compound No. Structural formula


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stats Patent Info
Application #
US 20100216981 A1
Publish Date
08/26/2010
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
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