U.S. patent application number 12/450150 was filed with the patent office on 2010-08-26 for antiviral agent.
This patent application is currently assigned to Nippon Soda Co., Ltd.. Invention is credited to Takako Fukagawa, Chikara Masuta, Shinsuke Sano, Hanako Shimura, Hirokazu Yamada.
Application Number | 20100216981 12/450150 |
Document ID | / |
Family ID | 39788265 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216981 |
Kind Code |
A1 |
Sano; Shinsuke ; et
al. |
August 26, 2010 |
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 .alpha. to a PTGS suppressor protein,
wherein the substance .alpha. 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.
Inventors: |
Sano; Shinsuke; (Chigasaki,
JP) ; Fukagawa; Takako; (Odawara, JP) ;
Yamada; Hirokazu; (Ichihara, JP) ; Masuta;
Chikara; (Sapporo, JP) ; Shimura; Hanako;
(Sapporo, JP) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 320850
ALEXANDRIA
VA
22320-4850
US
|
Assignee: |
Nippon Soda Co., Ltd.
Tokyo
JP
Hokkaido University
Sapporo-shi
JP
|
Family ID: |
39788265 |
Appl. No.: |
12/450150 |
Filed: |
March 19, 2008 |
PCT Filed: |
March 19, 2008 |
PCT NO: |
PCT/JP2008/000655 |
371 Date: |
September 14, 2009 |
Current U.S.
Class: |
536/24.5 ;
568/327; 568/377; 568/379 |
Current CPC
Class: |
A61K 31/7105 20130101;
A61P 31/16 20180101; A61P 31/20 20180101; A61P 43/00 20180101; A61P
31/18 20180101; A61K 31/122 20130101; A61P 31/12 20180101; A61K
31/7088 20130101; A61P 31/22 20180101 |
Class at
Publication: |
536/24.5 ;
568/327; 568/377; 568/379 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C07C 49/553 20060101 C07C049/553; C07C 49/543 20060101
C07C049/543; C07C 49/537 20060101 C07C049/537 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 23, 2007 |
JP |
2007-077970 |
Claims
1. A PTGS suppressor protein binding inhibitor containing a
compound having inhibitory activity on the binding of a substance
.alpha. to a PTGS suppressor protein, wherein the substance .alpha.
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 claim
1, wherein the substance .alpha. is siRNA.
3. The PTGS suppressor protein binding inhibitor according to claim
1, wherein the compound is at least one selected from the group
consisting of cyclic ketone compounds represented by the following
formulas (1) to (6): ##STR00039##
4. The PTGS suppressor protein binding inhibitor according to claim
1, 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
.alpha. to a PTGS suppressor protein, wherein the substance .alpha.
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 claim 5, wherein the substance
.alpha. is siRNA.
7. The antiviral agent according to claim 5, wherein the compound
is at least one selected from the group consisting of cyclic ketone
compounds represented by the following formulas (1) to (6):
##STR00040##
8. The antiviral agent according to claim 5, wherein the compound
is a reaction product of croconic acid with hydrogen peroxide.
9. The PTGS suppressor protein binding inhibitor according to claim
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): ##STR00041##
10. The PTGS suppressor protein binding inhibitor according to
claim 2, wherein the compound is a reaction product of croconic
acid with hydrogen peroxide.
11. The antiviral agent according to claim 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):
##STR00042##
12. The antiviral agent according to claim 6, wherein the compound
is a reaction product of croconic acid with hydrogen peroxide.
Description
TECHNICAL FIELD
[0001] The present invention relates to a PTGS suppressor protein
binding inhibitor and an antiviral agent having a novel mechanism
of action.
BACKGROUND ART
[0002] 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.
[0003] 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.
[0004] 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).
[0005] 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).
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] Patent Document 1: Japanese Laid-Open Patent Application No.
2004-344110 [0012] Non-Patent Document 1: Goto K., et al., Plant
Cell Physiol. 2007, 48, 1050-60 [0013] Non-Patent Document 2:
Anandalakshmi R., et al., Proc. Natl. Acad. Sci. USA, 1998, 95,
13079-13084 [0014] Non-Patent Document 3: Brigneti G., et al., EMBO
J., 1998, 17, 6739-6746 [0015] Non-Patent Document 4: Voinnet O.,
et al., Cell, 2000, 103, 157-167 [0016] Non-Patent Document 5:
Baulcombe D C., et al., Trends Biochem Sci. 2004, 29, 279-81 [0017]
Non-Patent Document 6: Qu F., et al., J. Virol. 2003, 77,
511-522
DISCLOSURE OF THE INVENTION
Object to be Solved by the Invention
[0018] 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
[0019] 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.
[0020] Specifically, the present invention relates to:
[0021] [1] a PTGS suppressor protein binding inhibitor containing a
compound having inhibitory activity on the binding of a substance
.alpha. to a PTGS suppressor protein, wherein the substance .alpha.
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;
[0022] [2] the PTGS suppressor protein binding inhibitor according
to [1], wherein the substance .alpha. is siRNA; and
[0023] [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):
##STR00001##
[0024] Moreover, the present invention relates to:
[0025] [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;
[0026] [5] an antiviral agent comprising as an active ingredient a
compound having inhibitory activity on the binding of a substance
.alpha. to a PTGS suppressor protein, wherein the substance .alpha.
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;
[0027] [6] the antiviral agent according to [5], wherein the
substance .alpha. is siRNA; and
[0028] [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):
##STR00002##
[0029] Furthermore, the present invention relates to:
[0030] [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
[0031] 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.
[0032] 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.
[0033] 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
[0034] 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 .alpha. (hereinafter, also
referred to as a "compound according to the present invention"),
wherein the substance .alpha. 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 .alpha. to PTGS-SP can
restore the substance .alpha. 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)
[0035] 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.
[0036] 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.sub.--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).
[0037] 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.
[0038] 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.
[0039] 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 .alpha.)
[0040] The substance .alpha. 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 .alpha.
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 .alpha. 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.
[0041] The sequence of the dsRNA or siRNA used as the substance
.alpha. 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.
[0042] Whether the substance .alpha. 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 .alpha. 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)
[0043] 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.
[0044] 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-US-00001 TABLE 1 Compound No. Structural formula 1
##STR00003## 2 ##STR00004## 3 ##STR00005## 4 ##STR00006## 5
##STR00007## 6 ##STR00008## 7 Compound of Synthesis Example 1 8
Compound of Synthesis Example 2 9 ##STR00009## 10 ##STR00010## 11
##STR00011## 12 ##STR00012## 13 ##STR00013## 14 ##STR00014## 15
##STR00015## 16 ##STR00016## 17 ##STR00017## 18 ##STR00018## 19
##STR00019## 20 ##STR00020##
[0045] Of these compounds, the compounds of compound Nos. 5, 6, 7,
8, 12, 13, 14, and 20 are preferred.
[0046] Of the compounds of above Table 1, compound Nos. 7 and 8 are
novel compounds, and the compounds other than compound Nos. 7 and 8
are commercially available.
[0047] These compounds or derivatives thereof can be synthesized by
performing a reaction known in the art with appropriate compounds
as raw material compounds.
[0048] Particularly, the above compound Nos. 7 and 8 can be
synthesized by, for example, methods specifically described later
in Examples.
[0049] The PTGS-SP binding inhibitor of the present invention may
contain only one of the compounds according to the present
invention or may contain two or more thereof.
(Antiviral Agent of the Present Invention)
[0050] An antiviral agent of the present invention is characterized
by comprising the compound of the present invention as an active
ingredient. The use of the antiviral agent of the present invention
can restore the substance .alpha. function inhibited by PTGS-SP,
i.e., the function of inducing PTGS. Based on such a mechanism of
action, the antiviral agent of the present invention can reduce the
inhibition of PTGS caused by highly virulent viruses, resulting in,
for example, the inhibited growth or reduced toxicity of the
viruses. As a result, the antiviral agent of the present invention
exerts an antiviral effect.
[0051] The antiviral agent of the present invention may contain
only one of the compounds according to the present invention as an
active ingredient or may contain two or more thereof as active
ingredients. Moreover, it is preferred from the viewpoint of
obtaining a more excellent antiviral agent that the antiviral agent
of the present invention should further contain a substance that
promotes the general resistance of organisms to viruses unless the
PTGS-SP binding inhibitory activity of the compound according to
the present invention is inhibited. The substance that promotes the
resistance of host organisms to viruses is not particularly limited
and can be exemplified preferably by substances that promote the
resistance of plants to viruses, such as fungicides known as plant
defense activators (e.g., probenazole and tiadinil), or
isonicotinic acid, salicylic acid, and ascorbic acid).
[0052] The antiviral agent of the present invention may contain
optional additional ingredients unless the PTGS-SP binding
inhibitory activity of the compound according to the present
invention is inhibited. The optional additional ingredients can be
exemplified by diluents or excipients such as fillers, expanders,
binders, wetting agents, disintegrants, surfactants, and
lubricants.
[0053] The dosage form of the PTGS-SP binding inhibitor or the
antiviral agent of the present invention is not particularly
limited and can be selected appropriately from tablets, pills,
powders, solutions, suspensions, emulsions, granules, capsules,
suppositories, injections (solutions, suspensions, etc.),
ointments, inhalants, sprays, and so on according to an applicable
organism used. The amount of the PTGS-SP binding inhibitor or the
antiviral agent used differs depending on the mode of use, an
applicable organism used, the type of the compound of the present
invention contained therein, and so on and can however be
determined appropriately by those skilled in the art.
[0054] A method for using the PTGS-SP binding inhibitor or the
antiviral agent of the present invention is not particularly
limited and can be determined appropriately by those skilled in the
art according to the property of the compound of the present
invention contained therein, the type of an applicable organism
used, and so on. When the applicable organism used is an animal,
the use method can be exemplified preferably by oral
administration, intravenous injection, intramuscular injection,
transvaginal administration, transdermal application, and
inhalation. When the applicable organism used is a plant, the use
method can be exemplified preferably by foliage application, dip
treatment, injection into soil, seed disinfection, and smoking.
[0055] The amount of the PTGS-SP binding inhibitor or the antiviral
agent of the present invention used is not particularly limited and
can be determined appropriately by those skilled in the art
according to the type of an applicable organism used, a use method,
and so on. Particularly, when the applicable organism used is an
animal, the amount can be selected appropriately according to the
age, body weight, and condition of the applicable organism used, an
administration method, and so on.
(Screening Method)
1) In Vitro Screening Method
[0056] An in vitro screening method for the compound according to
the present invention is not particularly limited as long as it is
an evaluation method using the degree of inhibition of the binding
of a substance .alpha. to PTGS-SP as an index or an evaluation
method which involves measuring the specific binding to PTGS-SP.
Whether or not a test compound inhibits the binding of a substance
.alpha. to PTGS-SP or the degree of inhibition of the binding can
be measured quantitatively and evaluated by using, for example, a
known method such as single-molecule fluorescence analysis, surface
plasmon resonance, gel shift assay, dual polarization
interferometry, photothermal spectroscopy, and quartz crystal
microbalance. The single-molecule fluorescence analysis method
eliminates the need of sample immobilization and achieves
multi-sample treatment under conditions of a solution system close
to an in vivo environment. However, this method has the
disadvantage that it is susceptible to interference of the
autofluorescence of a compound. The surface plasmon resonance
method requires sample immobilization and is however not influenced
by the autofluorescence of a compound. The gel shift assay method
is unsuitable for multi-sample treatment and however has the
advantage that it eliminates the need of sample immobilization and
is not influenced by the autofluorescence of a compound.
[0057] The single-molecule fluorescence analysis method is an
approach which involves: detecting a change in the behavior of
labeled fluorescent molecules by measuring the number, intensity,
or the like of the labeled fluorescent molecules using a confocal
laser scanning microscope: and analyzing the intermolecular
interaction between two substances. Screening based on the
single-molecule fluorescence analysis method can be performed, for
example, by applying a sample solution comprising PTGS-SP, a
fluorescently labeled substance .alpha., and a test compound to a
measurement apparatus for single-molecule fluorescence analysis
such as Olympus MF20 to detect a change in the behavior of the
labeled substance .alpha..
[0058] The surface plasmon resonance method is an apparatus which
involves: monitoring a change in refractive index caused by, for
example, a change in the weight of molecules immobilized on a thin
gold film; and analyzing the intermolecular interaction between two
substances. Screening based on the surface plasmon resonance method
can be performed, for example, by: immobilizing a substance .alpha.
onto sensor chip surface; and applying a sample solution comprising
PTGS-SP and a test compound to a Biacore measurement apparatus or
the like to detect, as a change in the refractive index of the
solution, a change in the weight of molecules caused by the binding
of the substance .alpha. immobilized on the sensor chip to the
PTGS-SP.
[0059] The gel shift assay method is an approach which involves:
mixing two high molecular substances; subjecting the mixture to
electrophoresis; and analyzing whether the two substances are bound
with each other, from the electrophoretic band positions or
concentrations. Screening based on the gel shift assay can be
performed, for example, by subjecting a sample solution comprising
a labeled substance .alpha., PTGS-SP, and a test compound to
polyacrylamide gel electrophoresis using a universal apparatus. The
binding of the substance .alpha. to the PTGS-SP increases a
molecular weight, resulting in slow migration. By contrast, when
the binding of the substance .alpha. to the PTGS-SP is inhibited by
the compound, the migration of the substance .alpha. is faster.
Therefore, the degree of the binding can be analyzed by comparing
these electrophoretic band positions or amounts.
2) In Vivo Screening Method
[0060] An in vivo screening method for the compound according to
the present invention is not particularly limited as long as it is
an evaluation method using the degree of inhibition of the PTGS-SP
function as an index. Whether or not a test compound inhibits the
PTGS-SP function can be evaluated by, for example, an
agroinfiltration method, a protoplast method, a silencing plant
method using a change in flower color as an index, or a symptom
test method which involves examining the symptom of a viral disease
using plant seedlings.
[0061] The agroinfiltration method is a method which involves:
simultaneously injecting, for example, a GFP gene and a viral
PTGS-SP gene into the intercellular space of a plant leaf using
Agrobacterium for transient transformation; and conducting
evaluation based on the principle that the GFP fluorescence
disappears when PTGS functions while the GFP fluorescence is
observed when the viral PTGS-SP functions. Furthermore, by treating
the plant leaf with a test compound before and after the gene
injection, the test compound can be determined to inhibit the
PTGS-SP function when the GFP fluorescence disappears.
[0062] The protoplast method is a method based on the same
principle as that of the agroinfiltration method. Specifically,
this method utilizes protoplast instead of the plant leaf and
involves: directly incorporating a firefly luciferase gene and a
viral PTGS-SP gene into the protoplast by use of polyethylene
glycol; and conducting evaluation based on the principle that light
emission derived from the luciferase activity disappears when PTGS
functions while stronger light emission is observed when the viral
PTGS-SP functions. By treating the protoplast with a test compound,
the test compound can be determined to inhibit the PTGS-SP function
when light emission derived from the luciferase activity is
significantly decreased, compared with untreated protoplast.
[0063] The silencing plant method is a method which utilizes the
principle that a variety whose flower color is wholly or partially
white shows a change in the flower color when infected with a plant
virus having PTGS-SP. Specifically, this method utilizes the fact
that the expression of pigment genes is inhibited by natural PTGS
in many white flowers. The inhibition of pigment expression by PTGS
is canceled by the virus infection such that the pigment gene is
expressed. As a result, the flower exhibits a red or blue color
changed from the white color. Here, the flower is treated with a
compound, and the inhibitory effect of the compound can be
determined based on a white flower color recovered by the
inhibition of the viral PTGS-SP function.
[0064] The symptom test method using plant seedlings is a method
which involves observing the symptom of a viral disease to
determine the antiviral effect of a compound. Specifically, in this
method, the plant seedlings are treated with a compound before and
after inoculation of a plant virus whose infection causes a severe
symptom in the plants, and the antiviral effect is determined based
on the severity of the symptom.
3) Methods for Preparing PTGS-SP and Substance .alpha. for
Screening
[0065] The PTGS-SP targeted by the PTGS-SP binding inhibitor
according to the present invention can be obtained using a
universal approach, for example, PCR based on sequence information
of the GenBank Accession No. described above.
[0066] The PTGS-SP can be expressed in, for example, an expression
system using E. coli, yeast, or insect cells or a cell-free protein
synthesis system derived from wheat germs or rabbit
reticulocytes.
[0067] When E. coli is used, a tag sequence is added to a sequence
encoding the PTGS-SP of interest, and the sequence is inserted into
an arbitrary vector for E. coli expression, with which an arbitrary
E. coli strain can then be transformed to express the PTGS-SP.
Then, it is preferred that the PTGS-SP is purified according to a
standard method based on the type of the added tag. The PTGS-SP can
be purified easily by adding, for example, His-tag for TBSV p19; or
MBP (maltose-binding protein) for TAV (tomato aspermy virus) 2b
(TAV 2b) or HIV tat, and can be used without troubles in the
evaluation system.
[0068] In the present invention, instead of the PTGS-SP, a
synthetic peptide having a partial structure thereof can also be
used in the evaluation system. The synthetic peptide can be
obtained as a peptide of 20 to 40 bases comprising the portion of
two or more primarily consecutive or adjacent strongly basic amino
acids (arginines or lysines) using a usual approach, for example,
solid-phase synthesis using an Fmoc or Boc method based on the
sequence information of the PTGS-SP.
[0069] In the present invention, instead of the PTGS-SP, even a
length or sequence variant of a synthetic peptide having a partial
structure thereof can also be used in the evaluation system of
siRNA binding inhibition. The variant peptide used in the siRNA
binding test must have a sequence of three or more primarily
consecutive or adjacent strongly basic amino acids (arginines or
lysines). For example, for HIV tat, consecutive arginines or
lysines starting at N-terminal position 49 are important, and its
ability to bind to siRNA is decreased by converting even one of
arginines 52, 53, and 55 to a neutral amino acid leucine and the
ability is lost by converting two or more thereof to leucine. The
ability to bind to siRNA is maintained even if all of these
arginines are converted to another strongly basic amino acid
lysine. On the other hand, the ability to bind to siRNA is
maintained even if independently located arginine such as arginine
78 is converted to a neutral amino acid leucine.
[0070] Moreover, the nucleic acid as the substance .alpha., such as
dsRNA or siRNA, can be obtained using a universal approach, for
example, PCR.
EXAMPLES
[0071] Hereinafter, the present invention will be described more
specifically with reference to Examples. However, the technical
scope of the present invention is not intended to be limited to
these Examples.
(Synthesis of Compound)
Synthesis Example 1
Preparation of compound 7
[0072] 3.0 g of croconic acid was dissolved in 30 ml of pure water.
To the solution, 1.2 g of a 30% hydrogen peroxide solution was
added at room temperature, and the reaction solution was stirred at
20 to 30.degree. C. for 3 days. The reaction solution was
concentrated under reduced pressure to obtain 2.6 g of a compound
7. .sup.13C-NMR (D.sub.2O) .delta. 178.6, 129.5, 75.7.
Synthesis Example 2
Preparation of Compound 8
[0073] 5.0 g of croconic acid was dissolved in 50 ml of pure water.
To the solution, 2.0 g of a 30% hydrogen peroxide solution was
added at room temperature, and the reaction solution was stirred at
100.degree. C. for 24 hours. The reaction solution was concentrated
under reduced pressure to obtain 3.3 g of a compound 8.
.sup.13C-NMR (D.sub.2O) .delta. 195.3, 193.9, 180.5, 178.7, 176.7,
161.7, 151.3, 150.0, 129.7, 75.8, 73.1, 72.5, 59.9, 45.9, 39.8.
(Compound Screening)
[0074] 1. Preparation of Biotinylated siRNA
[0075] Single-stranded RNA (sense strand: SEQ ID NO: 1) consisting
of 21 bases was prepared, and this sequence was 3'-terminally
biotinylated. Next, single-stranded RNA (antisense strand: SEQ ID
NO: 2) consisting of 21 bases complementary to this sense strand
was annealed thereto. This annealing product was purified to
prepare biotinylated siRNA of 21 base pairs.
2 Preparation of PTGS-SP
1) Preparation of TBSV-p19
[0076] The PTGS-SP p19 (Genbank Accession No. NC.sub.--001554) of
TBSV used in screening was expressed and purified as follows:
constructs comprising a p19 gene and a flag-tag sequence ligated to
a vector pCold I DNA (TAKARA BIO INC.) for cold shock expression
were prepared, and E. coli BL21 strains were transformed with the
constructs. The strains were cultured at 15.degree. C. for 24 hours
in the presence of 0.1 mM IPTG to induce the expression. The
strains were collected and homogenized. After centrifugation, the
supernatant was subjected to extraction using a vector-derived
His-tag and an Ni-NTA column with 200 mM to 500 mM imidazole. The
expressed p19 was confirmed using anti-flag antibodies. The p19 in
the extraction fraction was purified.
2) Preparation of TAV-2b
[0077] The 2b (Genbank Accession No. NC.sub.--003838) of TAV used
in screening was expressed and purified as follows: constructs
pMAL-TAV2b-flag comprising a TAV-2b gene and a flag-tag sequence
ligated to a vector pMAL-c2X (BioLabs Inc.) were prepared, and E.
coli strains JM109 were transformed with the constructs and
cultured in an LB/Amp medium at 37.degree. C. The expression was
induced for 2 hours in the presence of 0.3 mM IPTG. After
centrifugation, the strains were washed and resuspended in a buffer
solution. These strains were homogenized using a homogenizer. After
centrifugation, the supernatant fraction was applied to an
amylose-lysine column, and elution with 10 mM maltose was performed
to obtain MBP-fused TAV-2b. The expressed MBP-TAV2b-flag was
confirmed by SDS-PAGE and then cleaved using Factor Xa to separate
between MBP and TAV2b-flag.
[0078] The TAV2b-flag fraction was obtained by purification using a
flag column.
3) Preparation of Synthetic Peptide
[0079] A peptide (SEQ ID NO: 3) comprising the portion of a few
consecutive or adjacent strongly basic amino acids (arginines)
contained in HIV tat was designed as a synthetic peptide having a
partial structure of the PTGS-SP based on the sequence information
of the PTGS-SP. The actual synthesis was conducted by a contract
manufacturer of peptide synthesis (Biologica Co., Ltd.) to obtain a
peptide having 98% purity.
3 Screening
1) Surface Plasmon Resonance Method
[0080] An apparatus Biacore X (manufactured by Biacore) was used. A
solution of the 3'-terminally biotinylated siRNA (10 .mu.g/ml) of
21 base pairs was injected at a rate of 5 .mu.l/min to a sensor
chip SA modified with streptavidin to immobilize the siRNA on the
sensor chip. A test compound used in screening was dissolved in
dimethylformamide (DMF) or water such that it was adjusted to 25
ppm in terms of the final compound concentration. The PTGS-SP p19
or TAV-2b wad dissolved in a buffer solution for Biacore (0.01 M
HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA) such that it was adjusted to
approximately 10 .mu.g/ml in terms of the final concentration. The
test compound was mixed with the PTGS-SP. After centrifugation,
bovine serum albumin was added to the supernatant, and the mixture
was dialyzed at 4.degree. C. for 2 hours using a dialysis membrane
(manufactured by PIERCE, 7000MWCO) and then adjusted to the
concentrations described above to obtain a sample solution of the
test compound. The Biacore X apparatus was operated according to
the manual to measure the influence of the test compound on the
binding of the siRNA immobilized on the sensor chip to the PTGS-SP.
The binding inhibition rate of the test compound was calculated
with non-biotinylated free siRNA (final concentration: 0.1 .mu.M)
as a positive control when an inhibition rate obtained without the
compound addition is defined as 0% and an inhibition rate obtained
using the positive control is defined as 100%. The binding
inhibitory activity of the compound of the present invention on the
p19 or TAV-2b is shown in Table 2.
2) Gel Shift Assay Method
[0081] A test compound used in screening was dissolved in
dimethylformamide (DMF) or water such that it was adjusted to 100
ppm in terms of the final compound concentration. A gel shift assay
kit (LightShift Chemiluminescent EMSA Kit manufactured by Pierce)
was used. The synthetic peptide of tat (500 nM) and the test
compound solution were mixed into a buffer solution supplemented
with 0.005% BSA, and the mixture was shaken at 25.degree. C. for 30
minutes. Then, the biotinylated siRNA (500 .mu.M) was mixed
therewith, and the mixture was further shaken at 25.degree. C. for
30 minutes. Each sample was electrophoresed on a 5% polyacrylamide
gel and then transferred to a nylon membrane for 1 hour. The
membrane was blocked and then treated with peroxidase-conjugated
streptavidin. A luminol-peroxide substrate solution was added
thereto for reaction, and the membrane was covered with an X-ray
film and exposed to light for approximately 2 minutes. The binding
inhibition rate of the test compound was calculated from the band
positions and concentrations of the electrophoresed free
biotinylated siRNA or the electrophoresed biotinylated siRNA bound
with the synthetic peptide. The binding inhibitory activity of the
compound of the present invention on the synthetic peptide of tat
is shown in Table 2.
TABLE-US-00002 TABLE 2 Binding inhibition rate (%) Gel Biacore
shift Compound p19 TAV2b tat 100 No. Structural formula 25 ppm 25
ppm ppm 1 ##STR00021## 80 80 10 2 ##STR00022## 75 0 -- 3
##STR00023## 0 90 -- 4 ##STR00024## 20 90 -- 5 ##STR00025## 100 100
100 6 ##STR00026## 100 100 100 7 Compound of 100 100 100 Synthesis
Example 1 Compound of 100 100 100 8 Synthesis Example 2 9
##STR00027## 95 0 0 10 ##STR00028## 70 50 -- 11 ##STR00029## 70 20
-- 12 ##STR00030## 100 95 100 13 ##STR00031## -- -- 100 14
##STR00032## -- -- 100 15 ##STR00033## 95 100 80 16 ##STR00034## 80
0 0 17 ##STR00035## 100 90 -- 18 ##STR00036## 90 80 0 19
##STR00037## 90 50 0 20 ##STR00038## 100 80 100
3) Protoplast Method
[0082] The leaf abaxial epidermis of tobacco (Nicotiana
benthamiana) seedlings was stripped off and left standing at
25.degree. C. for approximately 6 hours in an enzyme solution (2%
cellulase RS, 0.5% macerozyme R10, 0.5 M mannitol). This solution
was subjected to filtration through two-piece gauze, and mannitol
(0.4 M) addition and centrifugation (300 rpm, 2 min) were repeated
three times to obtain protoplast. This protoplast was transfected
by a PEG method with the following four nucleic acids: an
expression plasmid for firefly luciferase (Fluc) as a reporter
gene, an expression plasmid for Renilla luciferase (Rluc) as an
internal standard, dsRNA of firefly luciferase (Fluc) for inducing
silencing, and a PTGS-SP CMV 2b gene. The protoplast was cultured
for approximately 24 hours and then homogenized. The expression
level of each Fluc or Rluc protein intracellularly expressed was
measured as light emission. A test compound was added at the
predetermined concentration before the culture, and its inhibitory
activity was determined based on the Fluc/Rluc ratio in the
presence or absence of the compound.
[0083] The inhibitory activity of the compound 7 of the present
invention on the CMV-2b is shown in FIG. 1. The ordinate represents
the emission intensity of the firefly luciferase reporter gene. The
emission intensity was decreased in a manner dependent on the
concentration of the compound 7.
4) Silencing Plant Method
[0084] A petunia variety was used whose pigment gene expression was
inhibited by natural PTGS such that the flower had a white star
pattern. This petunia was inoculated and infected with a CMV-L line
to prepare a virus disease-affected petunia with completely
inhibited natural PTGS, which was then used as a test plant for the
PTGS-SP function. To eliminate individual difference, the number of
the disease-affected petunia was increased by cuttings, which were
then used in the inhibition test. A test compound used in screening
was dissolved in dimethylformamide (DMF) or water and adjusted with
an MES buffer solution to pH of 5.5 to 6.0 (final concentration:
100 to 1000 ppm). The solution of the compound of the present
invention was injected into soil around the test plant, and a
change in the flower color of the petunia starting to flower after
1 to 2 weeks was observed to determine the inhibitory activity of
the compound on the PTGS-SP function, based on the recovery of the
white star pattern.
[0085] The inhibitory activity of the compound 7 of the present
invention on the CMV-2b is shown in FIG. 2.
[0086] The CMV-infected petunia flower (upper box of FIG. 2) had no
detectable white star pattern, whereas the flower underwent the
injection of the compound 7 of the present invention into soil
(middle box of FIG. 2) recovered a white star pattern as in a
healthy flower (lower box of FIG. 2).
5) Symptom Test Method
[0087] A compound solution diluted to the predetermined
concentration was gently injected with a syringe into the abaxial
leaf epidermis of tobacco (Nicotiana benthamiana) seedlings. A dry
leaf infected with a virus (TAV) was ground with a 10-fold volume
of a 0.1 M phosphate buffer solution (pH 7.0) to prepare a crude
sap, which was then used as an inoculation sap and wound-applied
using Carborundum to the surface of the tobacco leaf to which the
compound was injected. After the inoculation, the symptom of the
viral disease was observed to determine the antiviral activity of
the compound.
[0088] The antiviral activity of the compound 7 of the present
invention is shown in FIG. 3. The progression of the symptom in the
tobacco treated with the compound 7 of the present invention was
slower than that in untreated tobacco, demonstrating the inhibition
of pathogenesis.
INDUSTRIAL APPLICABILITY
[0089] A PTGS-SP binding inhibitor of the present invention can
inhibit the binding of PTGS-SP produced by a virus to a substance
.alpha. such that the PTGS-SP function is decreased, wherein the
substance .alpha. has the property of inducing PTGS and the
property of binding to the PTGS-SP and shows a decrease in the
property of inducing PTGS upon binding to the PTGS-SP. The PTGS-SP
binding inhibitor of the present invention can decrease the PTGS
inhibitory effect involving the PTGS-SP and thereby restore the
original action of PTGS, resulting in the inhibited growth or
reduced toxicity of the virus that has infected organisms.
Therefore, the PTGS-SP binding inhibitor of the present invention
can be used as an antiviral agent. The antiviral agent of the
present invention has a different mechanism of action from that of
known antiviral agents and is therefore expected to be sufficiently
effective for, for example, viruses resistant to known antiviral
agents.
[0090] Moreover, the antiviral agent of the present invention
inhibits the binding of PTGS-SP to siRNA such that the PTGS-SP
function is inhibited. The antiviral agent of the present invention
acts on the functions of PTGS-SPs carried in common by many
pathogenic viruses and is therefore expected to be effective for a
wider range of viruses. Specifically, X-ray crystallography or the
like has revealed that many PTGS-SPs bind, at a site called a
nucleic acid-binding region, to siRNA (Non-Patent Document 5). It
was demonstrated that the PTGS-SPs bind thereto particularly at a
site where two or more strongly basic amino acids (arginines or
lysines) are consecutive or adjacent in a primary sequence or
three-dimensional positional relationship. The presence of the site
of PTGS-SP having such strongly basic amino acids with a high
frequency is the condition necessary to bind to siRNA and is a
common characteristic of the PTGS-SPs carried by pathogenic
viruses. Therefore, the antiviral agent of the present invention is
expected to be effective for a wide range of viruses.
[0091] The antiviral agent of the present invention, when used in
animals, produces therapeutic effects on viral diseases caused by
pathogenic viruses. Further, the antiviral agent of the present
invention, when used in plants, can reduce severe damage caused by
viral diseases and significantly improve the commercial value and
productivity of crops.
Sequence CWU 1
1
3121RNAArtificial SequenceSynthetic Construct - ssRNA-sense
1uugcucaaca guaugggcau u 21221RNAArtificial SequenceSynthetic
Construct - ssRNA-antisense 2ugcccauacu guugagcaau u
21334PRTArtificial SequenceSynthetic Construct - designed peptide
based on amino acid sequence in dsRNA binding motif of tat 3Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr1 5 10 15His
Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp 20 25
30Pro Thr
* * * * *