U.S. patent application number 11/332238 was filed with the patent office on 2006-06-08 for pre-incubation assay methods.
Invention is credited to Kenji Abe.
Application Number | 20060121453 11/332238 |
Document ID | / |
Family ID | 18619145 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060121453 |
Kind Code |
A1 |
Abe; Kenji |
June 8, 2006 |
Pre-incubation assay methods
Abstract
Pseudo-positive data can be reduced by incubating a solution
containing an enzyme in the absence of a test substance and a
substrate of the enzyme.
Inventors: |
Abe; Kenji; (Settsu-shi,
JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
18619145 |
Appl. No.: |
11/332238 |
Filed: |
January 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10149153 |
Jun 10, 2002 |
7018796 |
|
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PCT/JP01/02619 |
Mar 29, 2001 |
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11332238 |
Jan 17, 2006 |
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Current U.S.
Class: |
435/5 ;
435/6.14 |
Current CPC
Class: |
A61K 31/192 20130101;
C12Q 1/00 20130101; A61K 31/19 20130101; C07C 59/90 20130101; C12Q
1/48 20130101; G01N 33/57415 20130101; C07C 59/88 20130101; C07C
59/86 20130101; G01N 33/52 20130101; A61P 31/14 20180101; C12Q 1/18
20130101 |
Class at
Publication: |
435/005 ;
435/006 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 7, 2000 |
JP |
2000-105887 |
Claims
1. A method for an assay of an activator or an inhibitor to an
enzyme which comprises incubating a solution comprising the enzyme
in the absence of both a test substance and a substrate of the
enzyme for 15 minutes or more at 20-40.degree. C.
2. The method for an assay of an activator or an inhibitor to an
enzyme according to claim 1 which comprises: (a) incubating the
solution comprising the enzyme in the absence of both the test
substance and the substrate of the enzyme, and (b) comparing an
enzyme activity measured by mixing the test substance and the
substrate of the enzyme with the incubated solution and reacting
the enzyme with the substrate of the enzyme, with an enzyme
activity measured by mixing only the substrate of the enzyme with
the incubated solution and reacting the enzyme with the substrate
of the enzyme.
3-4. (canceled)
5. The method for an assay according to claim 1 wherein the enzyme
is viral.
6. The method for an assay according to claim 1 wherein the enzyme
is a polymerase.
7. The method for an assay according to claim 1 wherein the
solution contains a polymerase, a template, a primer and a metal
ion.
8-23. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for an assay of an
activator or an inhibitor to an enzyme, specially a polymerase, a
RNA polymerase and a RNA-dependent RNA polymerase.
BACKGROUND ART
[0002] A method for an assay of an activator or an inhibitor to an
enzyme generally comprises mixing the enzyme, a substrate of the
enzyme, anything essential to an enzyme reaction such as a metal
ion and a test substance, reacting the mixture under a condition
where the enzyme reaction can be carried out such as a buffer
condition, a reaction temperature or the like, measuring or
calculating an enzyme activity by any means and comparing it with
an enzyme activity in the absence of a test substance.
[0003] For example, an assay of an activator or an inhibitor to a
polymerase can be carried out as follows. First, a polymerase, a
labeled substrate, a template, a primer, a metal ion and a test
substance are mixed at the same time or in order for the polymerase
reaction. The labeled substrate is incorporated to a reaction
product, which is known as an incorporation. After reaction, an
amount of the labeled substrate incorporated to the reaction
product is measured. An enzyme activity calculated on the basis of
the above measured amount is compared with that in the absence of a
test substance.
[0004] Even if a result; of the above assay suggests that a test
substance may possess an activity or inhibitory activity to an
enzyme, the activity may be pseudo-positive due to nonspecifically
acting or binding to the enzyme based on the structure or character
of the test substance. Therefore, in order to prove the result of
the assay to be positive, it is necessary to confirm that the test
substance is really an activator or an inhibitor to an enzyme by
the other assay. However, the other assay with a complicated
process may not be suitable for the first assay.
[0005] Recently, a high throughput screening (referred to as HTS)
is very important on the early stage of the development of
medicine. HTS enables several hundreds to ten thousands compounds
to be assayed at the same time, but causes a problem of how to
analyze an enormous number of data.
[0006] Journal of General Virology (2000), 81, 759-767 discloses
that a pre-incubation of HCV RNA-dependent RNA polymerase in the
presence of a template and a primer enhances an enzyme activity,
without mentioning an assay of an activator or an inhibitor to an
enzyme at all. The above document only discloses a character of the
enzyme.
[0007] It is important to obtain useful information from data
obtained by HTS without pseudo-positive data. A method for an assay
of an activator or an inhibitor to an enzyme with no or little
pseudo-positive data has been desired.
DISCLOSURE OF INVENTION
[0008] The present inventor found a method for an assay of an
activator or an inhibitor to an enzyme, wherein pseudo-positive
data can be reduced by a pre-incubation, which comprises incubating
a solution containing said enzyme in the absence of a test
substance and a substrate of said enzyme.
[0009] The present invention provides the following inventions.
(1) A method for an assay of an activator or an inhibitor to an
enzyme which comprises incubating a solution containing said enzyme
in the absence of a test substance and a substrate of said
enzyme.
(2) The method for an assay of an activator or an inhibitor to an
enzyme according to the above (1) which comprises:
[0010] (a) incubating a solution containing said enzyme in the
absence of a test substance and a substrate of said enzyme, and
[0011] (b) comparing an enzyme activity measured by mixing a test
substance and a substrate of said enzyme with said solution
incubated and reacting said enzyme with said substrate of said
enzyme with an enzyme activity measured by mixing only a substrate
of said enzyme with said solution incubated and reacting said
enzyme with said substrate of said enzyme.
(3) The method for an assay according to the above (1) or (2) which
comprises incubating said solution for 0.5 hours or more.
(4) The method for an assay according to any one of the above (1)
to (3) which comprises incubating said solution at 20 to 40.degree.
C.
(5) The method for an assay according to any one of the above (1)
to (4) wherein said enzyme is of a virus.
(6) The method for an assay according to any one of the above (1)
to (5) wherein said enzyme is a polymerase.
(7) The method for an assay according to any one of the above (1)
to (6) wherein said solution contains a polymerase, a template, a
primer and a metal ion.
(8) A method for activating or inhibiting an enzyme which comprises
contacting said enzyme with a compound obtained by the method for
an assay according to any one of the above (1) to (7).
(9) A method for activating or inhibiting replication of a virus
which comprises contacting said virus with a compound obtained by
the method for an assay according to the above (5).
(10) A compound which is obtained by the method for an assay
according to any one of the above (1) to (7).
(11) A pharmaceutical composition for activating or inhibiting an
enzyme which comprises as an active ingredient a compound obtained
by the method for an assay according to any one of the above (1) to
(7).
[0012] (12) A method for an assay of an inhibitor to a virus
RNA-dependent RNA polymerase which comprises incubating a solution
containing said polymerase, a template RNA, a primer RNA and a
metal ion in the absence of a test substance and a substrate RNA of
said polymerase.
(13) A method for an assay of an inhibitor to a virus RNA-dependent
RNA polymerase according to the above (12) which comprises:
[0013] (a) incubating a solution containing said polymerase, a
template RNA, a primer RNA and a metal ion in the absence of a test
substance and a substrate RNA of said polymerase, and
[0014] (b) comparing a virus RNA-dependent RNA polymerase activity
measured by mixing a test substance and a substrate RNA of said
polymerase with said solution incubated and reacting said
polymerase with said substrate of said polymerase with a virus
RNA-dependent RNA polymerase activity measured by mixing only a
substrate RNA of said polymerase with said solution incubated and
reacting said polymerase with said substrate of said
polymerase.
(14) The method for an assay according to the above (12) or (13)
which comprises incubating said solution for 0.5 hours or more.
(15) The method for an assay according to any one of the above (12)
to (14) which comprises incubating said solution at 20 to
40.degree. C.
(16) The method for an assay according to any one of the above (12)
to (15) wherein said template RNA is poly(A), said primer RNA is
oligo(U), said metal ion is Mg.sup.2+ and said substrate RNA is
.sup.3H-UTP or .sup.32P-UTP.
(17) A method for inhibiting a virus RNA-dependent RNA polymerase
which comprises contacting said virus RNA-dependent RNA polymerase
with a compound which is obtained by the method for an assay
according to any one of the above (12) to (16).
(18) A method for inhibiting replication of a virus which comprises
contacting said virus with a compound obtained by the method for an
assay according to any one of the above (12) to (16).
(19) The method according to any one of the above (12) to (18)
wherein said virus belongs to Flavivirus genus.
(20) The method according to any one of the above (12) to (1.8)
wherein said virus is an HCV.
(21) A compound of the formula: A-C(.dbd.O)--CH.dbd.C(OH)--COOH
wherein A is an organic residue, which is obtained by the method
for an assay according to any one of the above (12) to (16), (19)
and (20).
(22) A pharmaceutical composition for treating hepatitis C which
comprises as an active ingredient a compound obtained by the method
for an assay according to any one of the above (12) to (16), (19)
and (20).
[0015] (23) A pharmaceutical composition for treating hepatitis C
which comprises as an active ingredient a compound of the formula:
A-C(.dbd.O)--CH.dbd.C(OH)--COOH wherein A is an organic residue,
which is obtained by the method for an assay according to any one
of the above (12) to (16), (19) and (20).
[0016] The present invention is explained in detail below.
[0017] The present invention relates to a method for an assay of an
activator or an inhibitor of an enzyme. In the present
specification, an assay of an activator or an inhibitor to an
enzyme includes the first assay which evaluates test substances
that are unknown to be an activator or an inhibitor of an enzyme
and the revaluation assay which evaluates test substances that are
known to be an activator or an inhibitor of an enzyme. The assay of
the present invention can be used for all kinds of assay. For
example, the present assay can be used for an identification of an
activator or an inhibitor to a polymerase, an inhibitor to a virus
polymerase, an inhibitor to RNA-dependent RNA polymerase or an
inhibitor to HCV RNA-dependent RNA polymerase.
[0018] The present invention is used for an assay of an activator
or an inhibitor to an enzyme and characterized in that an
incubation is carried out before an enzyme reaction, in detail,
that an incubation of a solution containing said enzyme in the
absence of a test substance and a substrate of said enzyme. In the
present specification, the above incubation is also expressed as a
pre-incubation. In detail, the present invention includes
incubating a solution containing an enzyme in the absence of a test
substance and a substrate of the enzyme at a temperature where an
effect of a pre-incubation can be exhibited for an incubation time
where an effect of a pre-incubation can be exhibited.
[0019] A temperature where an effect of a pre-incubation can be
exhibited is, for example, approximately 15.degree. C. to
50.degree. C., preferably 20.degree. C. to 40.degree. C., and more
preferably 20.degree. C. to 30.degree. C.
[0020] An incubation time where an effect of a pre-incubation can
be exhibited is approximately 15 minutes or more, preferably 0.5
hour or more, more preferably 1 hour or more. For example,
preferred is approximately 15 minutes to 12 hours, 0.5 to 3 hours,
0.5 to 2 hours, especially 1 hour.
[0021] An incubation of a solution containing an enzyme in the
absence of a test substance and a substrate of the enzyme seems to
form a condition where an enzyme reaction can easily be
preformed.
[0022] The term "the absence of a test substance and a substrate of
an enzyme" includes a condition where a test substance and a
substrate of an enzyme are not included. A solution containing said
enzyme may be incubated together with anything essential to an
enzyme reaction except a substrate of an enzyme.
[0023] The term "a solution containing an enzyme" includes a
solution containing at least an enzyme, preferably a solution
containing an enzyme and anything essential to an enzyme reaction
except a substrate of an enzyme.
[0024] Anything essential to an enzyme reaction except a substrate
of an enzyme includes, depending on the enzyme to be used, for
example, high molecular weight compounds, low molecular weight
compounds, metal ions, halogenous ions, amino acids, polypeptides,
nucleic acids, polynucleotides or the like.
[0025] The assay of the present invention is useful for an assay of
an activator or an inhibitor of a polymerase, specially an assay
relating to a nucleic acid elongation reaction in the presence of a
primer. When an enzyme is a polymerase, a solution preferably
contains a polymerase, a template, a primer and a metal ion.
[0026] A template includes a template DNA or a template RNA, for
example, a homopolymer such as a synthesized homopolymer, poly(dA),
poly(dT), poly(dG), poly(dC), poly(A), poly(U), poly(G), poly(C) or
the like or a heteropolymer such as a natural type of nucleic acid
polymer, a synthesized heteropolymer or the like. A length of a
template is not limited and can be selected depending on a kind of
an enzyme. For example, a template of approximately 8 to 1000
bases, 50 to 800 bases, 100 to 500 bases can be used. A
commercially available template or the like may be used.
[0027] A primer includes a primer DNA or a primer RNA. A primer
complementary to a template can be used. When a template is a
homopolymer, a primer such as oligo(dA), oligo(dT), oligo(dG),
oligo(dC), oligo(A), oligo(U), oligo(G), oligo(C) or the like can
be used. When a template is homopolymer, a primer partially
complementary to a template can be used. A length of a primer
depends on a kind of an enzyme. A primer of 12 to 18 bases can
generally be used.
[0028] A metal ion includes Mg.sup.2+, Mn.sup.2+, Na.sup.+, K.sup.+
or the like. Mg.sup.2+ is generally essential to a polymerase.
[0029] A test substance includes a substance used for an assay, for
example, a protein such as a polypeptide as well as a low molecular
weight compound.
[0030] An enzyme reaction after a pre-incubation starts by adding a
substrate of the enzyme to a solution containing the enzyme. The
prior method described in The EMBO Journal vol. 15, no. 1, pp
12-22, 1996 requires an enzyme reaction temperature of
approximately 22.degree. C. An enzyme loses an activity of itself
over such a temperature and does not work below the temperature.
The pre-incubation assay method is different from the prior method
and an enzyme reaction can be carried out at approximately 10 to
40.degree. C., preferably 20 to 40.degree. C. When a human enzyme,
an enzyme of a virus infecting human being or the like is used in
the assay of the present invention, a enzyme reaction can be
carried out at the same temperature as a human temperature,
specially 37.degree. C. Therefore, the present invention is very
useful. Further such temperature heightens an activity of an enzyme
activity. As a result, the detectability of an assay increases.
[0031] An activating effect or inhibiting effect of a test
substance to an enzyme can be identified by comparing an enzyme
activity, which is measured by mixing a test substance and a
substrate of the enzyme with a solution incubated and reacting the
enzyme with the substrate of the enzyme, with an enzyme activity,
which is measured by mixing only the substrate of the enzyme with a
solution incubated and reacting the enzyme with the substrate of
the enzyme. To a solution incubated may be added a substrate of an
enzyme and an optional test substance. To a substrate of an enzyme
and an optional test substance may be added a solution incubated.
The addition order does not matter as far as a substrate of an
enzyme contacts with an enzyme.
[0032] The term "mixing only a substrate of said enzyme" includes
mixing no test substance. As far as no test substance is included,
a solution may include anything other than a substrate of an
enzyme. For example, a reaction solution for dissolving or
suspending a substrate of an enzyme may be included. Anything may
be mixed as far as data for being compared with data in the
presence of a test substance can be prepared. An enzyme activity
measured by mixing only a substrate of a enzyme with a
non-incubated solution and reacting the enzyme with the substrate
of the enzyme can be used as a reference. Preferred is an enzyme
activity measured by mixing only a substrate of a enzyme with a
solution incubated and reacting the enzyme with the substrate of
the enzyme.
[0033] An enzyme activity can be measured by using a labeled
substrate of the enzyme. When a polymerase is used, an amount of
the labeled substrate of the enzyme incorporated to the reaction
product can be measured. An isotope-labeled substrate of an enzyme
can be used as a labeled substrate of the enzyme and a
radioactivity can be measured by a scintillation counter or the
like. A labeled substrate of an enzyme or the like can be separate
from a reaction solution by using a commercially available filter
such as a filter modified by a dimethylaminoethyl group or the
like.
[0034] A low molecular weight compound includes an organic compound
of molecular weight 15 to 1000. Atoms constituting such compound
includes hydrogen, lithium, boron, carbon, nitrogen, oxygen,
fluorine, sodium, magnesium, aluminum, sulfur, chlorine, potassium,
calcium, iron, barium, bromine, iodine or the like.
[0035] A polypeptide includes not only a natural peptide but also a
modified peptide such as peptide isoster or the like.
[0036] A substrate of an enzyme includes a substance on which an
enzyme acts, for example, a high molecular weight compound, a low
molecular weight compound, an amino acid, a polypeptide, a nucleic
acid, a polynucleotide or the like. An example of a substrate of a
polymerase includes dATP, dTTP, dGTP, dCTP, ATP, UTP, GTP, CTP or
the like. An example of a labeled substrate of an enzyme includes
.sup.3H-UTP, .sup.32P-UTP or the like.
[0037] An enzyme includes a protein catalyzing an enzyme reaction,
for example, any kind of oxygenase, any kind of phosphotransferase
(e.g., phosphokinase, protein kinase or the like), any kind of
protease, any kind of polymerase, any kind of synthetase or the
like. In the present invention, not only a full length enzyme but
also a deletion mutant, a substitution mutant or an addition mutant
having an enzyme activity can be used. For example, a recombinant
enzyme can be used.
[0038] A polymerase includes a polynucleotide chain synthetase, for
example, DNA polymerase (e.g., DNA-dependent DNA polymerase,
RNA-dependent DNA polymerase) or RNA polymerase (e.g.,
DNA-dependent RNA polymerase, RNA-dependent RNA polymerase).
[0039] A protease includes a proteolytic enzyme, an enzyme
catalyzing hydrolysis of a peptide bond. An example of a protease
includes a serine protease, a cysteine protease, a metalloprotease
or an aspartic protease. A protease includes an endopeptidase and
an exopeptidase.
[0040] A virus is not limited to the specific virus. An example of
a virus includes HIV (human immunodeficiency virus), HCV (hepatitis
C virus), HBV (hepatitis B virus), HTLV (human T cell leukemia
virus), influenza virus or the like. Preferred is a virus which
belongs to Flavivirus genus, especially HCV.
[0041] An enzyme of HIV includes a reverse transcriptase (a
RNA-dependent DNA polymerase, a RNA-dependent DNA polymerase having
a RNaseH activity), an integrase, a protease and the like. An
enzyme of HCV includes a RNA-dependent RNA polymerase, protease
(serine protease, metalloprotease), an helicase and the like. An
enzyme of HBV includes a DNA polymerase (an enzyme having a
DNA-dependent DNA polymerase activity, a RNA-dependent DNA
polymerase activity and/or a RNaseH activity) and the like. An
enzyme of HTLV includes a reverse transcriptase, an integrase, a
protease and the like. An enzyme of influenza virus includes a
RNA-dependent RNA polymerase, an endonuclease, a sialidase and the
like.
[0042] Any enzyme can be used in the present invention. A protein
having an enzyme activity can be used. Preferred is an enzyme
relating to a long length chain of nucleic acids (e.g., an
integrase, a helicase, a polymerase or the like), especially a
polymerase. Preferred among polymerases is a RNA-dependent RNA
polymerase (e.g., a virus RNA-dependent RNA polymerase), especially
a virus RNA-dependent RNA polymerase of a flavivirus, more
especially a HCV RNA-dependent RNA polymerase.
[0043] An example of a virus having a RNA-dependent RNA polymerase
includes a virus which belongs to Picornaviridae, Caliciviridae,
Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae,
Orthomyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae or the
like. Preferred is a virus which belongs to Flaviviridae,
especially a virus which belongs to Flaviviridae family Flavivirus
genus.
[0044] An example of a virus which belongs to Flavivirus genus
includes a hepatitis C virus, a yellow fever virus, a dengue virus,
a Japanese/encephalitis virus, a West Nile fever virus, a St. Louis
encephalitis virus, a Rocio encephalitis virus, a Murray Valley
encephalitis virus, a Venezuelan equine encephalitis virus or the
like. These viruses have a RNA-dependent RNA polymerase. Since
these viruses relate to diseases of a mammal including human being,
it is important to find inhibitors of these virus RNA-dependent RNA
polymerase.
[0045] An enzyme includes not only an enzyme having one enzyme
activity, but also an enzyme having two or more enzyme activities.
For example, a example of an enzyme having two or more enzyme
activities includes a HBV DNA polymerase (an enzyme having a
DNA-dependent DNA polymerase activity, a RNA-dependent DNA
polymerase activity and/or a RNaseH activity) or the like.
[0046] When a HIV reverse transcriptase (an enzyme having a
RNA-dependent DNA polymerase activity and a RNaseH activity) is
used, not only a part having each enzyme activity but also a part
having both enzyme activities can be used for the present
invention.
[0047] There are many genotypes of HCV. In the present invention,
an enzyme of any genotype of HCV can be used. An example of
genotype of HCV includes genotype 1a, genotype 1b, genotype 2a,
genotype 2b, genotype 3, genotype 4 or the like.
[0048] HCV RNA-dependent RNA polymerase is encoded in the NS5B
(nonstructural protein 5B) region of HCV. In the present invention,
any enzyme having a RNA-dependent RNA polymerase activity can be
used. For example, an enzyme having a RNA-dependent RNA polymerase
activity encoded in NS5B of the above genotype of HCV (e.g.,
genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3,
genotype 4).
[0049] An activator includes a substance activating an enzyme
reaction.
[0050] An inhibitor includes a substance inhibiting an enzyme
reaction. Preferred is an inhibitor having IC.sub.50 of 100 .mu.M
or less, specially 10 .mu.M or less, more specially 1 .mu.M or
less.
[0051] The present invention relates to a pre-incubation assay
method and is useful for an assay of an enormous number of
compounds, especially a high throughput assay (HTS). The present
invention enables pseudo-positive data to be reduced and lead
compounds can efficiently be selected.
[0052] An enzyme can be activated or inhibited by contacting a
substance obtained by the assay of the present invention with an
enzyme. A replication of a virus can be activated or inhibited by
contacting a substance obtained by the assay of the present
invention with a virus.
BRIEF DESCRIPTION OF DRAWINGS
[0053] FIG. 1 shows an enzyme activity of HCV RNA-dependent RNA
polymerase measured by the prior method.
[0054] FIG. 2 shows an enzyme activity of HCV RNA-dependent RNA
polymerase measured by the pre-incubation assay method.
[0055] FIG. 3 shows a relationship between pre-incubation time and
an enzyme activity of HCV RNA-dependent RNA polymerase when an
enzyme reaction time is 30 minutes. In the figure, .box-solid. is
used when a solvent dissolving a test substance is DMSO and
.tangle-solidup. is used when the solvent is water.
[0056] FIG. 4 shows a relationship between the presence or the
absence of a pre-incubation and an enzyme reaction temperature. In
the figure, each symbol represents a relationship between an
incorporation of UMP by HCV RNA-dependent RNA polymerase and an
enzyme reaction time. .DELTA. is used when an enzyme reaction after
a pre-incubation is carried out at 37.degree. C., .quadrature. is
used when an enzyme reaction after a pre-incubation is carried out
at 25.degree. C., .circle-solid. is used when an enzyme reaction is
carried out at 25.degree. C. without a pre-incubation and
.box-solid. is used when an enzyme reaction is carried out at
37.degree. C. without a pre-incubation.
[0057] FIG. 5 shows a relationship between a pre-incubation time
and each inhibitory activity of 3'-dUTP, Compound A, Compound B and
Compound C to HCV RNA-dependent RNA polymerase. In the figure,
.largecircle. is used when a test substance is 3'-dUTP, .box-solid.
is used when a test substance is Compound A, .tangle-solidup. is
used when a test substance is Compound B and .circle-solid. is used
when a test substance is Compound C.
[0058] FIG. 6 shows a relationship between an inhibitory activity
of 3'-dUTP to HCV RNA-dependent RNA polymerase and a pre-incubation
temperature. In the figure, .circle-solid. is used when a
pre-incubation temperature is 25.degree. C., .box-solid. is used
when a pre-incubation temperature is 20.degree. C. and
.tangle-solidup. is used when a pre-incubation temperature is
2.degree. C.
[0059] FIG. 7 shows a relationship between an inhibitory activity
of Compound I-1 to HCV RNA-dependent RNA polymerase and a
pre-incubation temperature. In the figure, .circle-solid. is used
when a pre-incubation temperature is 25.degree. C., .box-solid. is
used when a pre-incubation temperature is 20.degree. C. and
.tangle-solidup. is used when a pre-incubation temperature is
2.degree. C.
[0060] FIG. 8 shows a relationship between an inhibitory activity
of Compound C to HCV RNA-dependent RNA polymerase and a
pre-incubation temperature. In the figure, .circle-solid. is used
when a pre-incubation temperature is 25.degree. C., .box-solid. is
used when a pre-incubation temperature is 20.degree. C. and
.tangle-solidup. is used when a pre-incubation temperature is
2.degree. C.
[0061] FIG. 9 shows a result of random screening carried out
through the prior method.
[0062] FIG. 10 shows a result of random screening carried out
through the pre-incubation method.
BEST MODE FOR CARRYING OUT THE INVENTION
[0063] A pre-incubation assay method of the present invention is
explained below, compared with a prior method. Though it is
explained by using HCV RNA-dependent RNA polymerase, one of
polymerase as an example of an enzyme, an enzyme is not limited to
such polymerase. Any kind of an enzyme can be used in the present
invention. Preferred is a polymerase, specially a RNA polymerase,
especially RNA-dependent RNA polymerase, more especially HCV
RNA-dependent RNA polymerase.
Prior Method
[0064] A HCV RNA-dependent RNA polymerase, template RNA, primer
RNA, substrate RNA, Mg ion (Mg.sup.2+) and a test substance are
mixed at the same time to start an enzyme reaction and measure an
enzyme activity. It can be known whether a test substance activates
or inhibits an activity of a HCV RNA-dependent RNA polymerase by
comparing the above value of an enzyme activity with that measured
in the absence of a test substance. IC.sub.50 value can be used,
which is a concentration of a test substance able to inhibit 50% of
an enzyme activity. For example, one of prior methods can be
carried out in accordance with the later-described Reference 1.
Pre-Incubation Assay Method
[0065] A HCV RNA-dependent RNA polymerase, template RNA, primer RNA
and Mg ion (Mg.sup.2+) are pre-incubated at approximately
25.degree. C. for approximately 1 hour. After that, substrate RNA
and a test substance are added to start an enzyme reaction and
measure an enzyme activity. IC.sub.50 value can be calculated by
comparing with an enzyme activity measured in the absence of a test
substance as well as the above prior method. An inhibitor can be
selected by comparing IC.sub.50. An example of a procedure of this
method is shown in the later described Example 1.
[0066] A pre-incubation assay method of the present invention is
superior to a prior method in the following points.
1) Higher Enzyme Activity
[0067] FIGS. 1 and 2 shows charts of enzyme activities at each
concentration of Mg.sup.2+ and Mn.sup.2+ in a prior method and a
pre-incubation assay method. When a concentration of Mg.sup.2+ is 3
mM and a concentration of Mn.sup.2+ is 0.1 mM, an enzyme activity
is 29,095 ccpm in a pre-incubation assay method whereas that is
6,288 ccpm in a prior method. An enzyme activity is 4 to 5 times as
high as that in a prior method. Therefore, assay results can easily
be detected and experimental errors decrease. It is useful for
screening inhibitors.
[0068] A relationship between a pre-incubation time and an enzyme
activity was examined. An example of a procedure is described in
Examination 1. An enzyme activity is represented by an amount of an
incorporation of uridine monophosphate (UMP) to a reaction product.
A result is shown in FIG. 3. An enzyme activity gradually gets
higher until a pre-incubation time is approximately 60 minutes. The
difference of enzyme activities between a prior method and a
pre-incubation assay method gradually gets larger. An enzyme
activity measured after pre-incubating for approximately 60 minutes
is approximately 4 times as high as that measured by a prior
method. When a pre-incubation time is more than 60 minutes, an
enzyme activity is the same as that measured after pre-incubating
for approximately 60 minutes. This results shows that a preferable
pre-incubation time is approximately 60 minutes.
2) Higher Thermostability
[0069] A relationship between the presence or absence of a
pre-incubation and an enzyme reaction temperature is examined and
shown in FIG. 4. In a prior method, when an enzyme reaction
temperature is approximately 37.degree. C., a longer reaction time
deactivates an enzyme. Therefore, an accurate enzyme activity can
not be measured and an enzyme reaction must be carried out at
approximately 25.degree. C. in a prior method. On the other hand,
an enzyme reaction in a pre-incubation method proceeds well at
approximately 37.degree. C. and an assay can be carried out at a
temperature more close to that of a mammal. An enzyme activity at
approximately 37.degree. C. is higher than that at approximately
25.degree. C. Therefore, a pre-incubation method enables an assay
to be carried at approximately 37.degree. C., which is close to a
temperature of a mammal.
3) Reducing Pseudo-Positive Data
[0070] A pre-incubation method enable to pseudo-positive data to
decrease. It is explained by using FIG. 5.
[0071] In FIG. 5, 3'-deoxyuridine triphosphate (3'-dUTP) is used as
a competitive inhibitor. 3'-dUTP is incorporated as a substrate,
stops the elongation of a RNA chain and can inhibit an activity of
a HCV RNA-dependent RNA polymerase. Compound A, B and C show
positive data in a prior method. These compounds (3'-dUTP (0.7
.mu.M), Compound A (100 .mu.g/ml), Compound B (50 .mu./ml) or
Compound C (25 .mu./ml)) inhibit an activity of a HCV RNA-dependent
RNA polymerase at approximately 50 to 70% of an inhibition in a
prior method which is not accompanied with a pre-incubation. The
structures of Compound A, B and C are shown below. ##STR1##
[0072] When these four compounds are assayed in a pre-incubation
assay method, an enzyme inhibition rate of Compound A, B and C are
under 15%, whereas an enzyme inhibition rate of a positive control
compound (3'-dUTP) does not change. Compound A, B and C are judged
to be negative compounds.
[0073] Compounds which show positive data in a prior method in
spite of no inhibition activity to a HCV RNA-dependent RNA
polymerase are judged to be negative compounds in a pre-incubation
assay.
[0074] This is important in a search for an inhibitor to an enzyme
which can not be examined by the other method. Specially, it is
important in a search for an inhibitor to a HCV RNA-dependent RNA
polymerase because there is no HCV infection experiment. It can be
judged by a pre-incubation of the present invention whether it is a
real inhibitor or not.
[0075] It is specially important in a random screening wherein a
large amount of compounds are examined at the same time. FIG. 9
shows a result of a random screening in a prior method. FIG. 10
shows a result of a random screening in a pre-incubation method. A
bar chart shows numbers of samples at each inhibition rate (left
vertical axis: a number of samples, horizontal axis: an inhibition
rate). Right vertical axis shows percentages of positive compounds
to the total number of all assayed compounds. FIG. 9 shows a
percentage of compounds wherein their inhibition rates are over 75%
is approximately 3.8% to the total number of all test substances in
a prior method. FIG. 10 shows a percentage of compounds wherein
their inhibition rates are over 40% is approximately 3.2% to the
total number of all test substances in a prior method. Therefore,
it is easy to select lead compounds in a pre-incubation assay
because a number of pseudo-positive data decreases whereas it is
difficult to select lead compounds in a prior method because a
number of compounds showing positive date is large.
[0076] Lead compounds can effectively be found through a
pre-incubation assay method of the present invention. Results
obtained by using compounds obtained through the above screening
are shown as examples. FIG. 6 shows a result obtained by using a
positive control compound (3'-dUTP). FIG. 7 shows a result obtained
by using Compound I-1 which was effectively found in a
pre-incubation assay method. FIG. 8 shows a result obtained by
using Compound C. The structure of Compound I-1 is shown below.
##STR2##
[0077] Compound I-1 can be selected as an effective lead compound
because it shows a positive result in a pre-incubation method as
well as a positive control compound (3'-dUTP).
[0078] On the other hand, Compound C showing a positive result in a
prior method can be excluded from candidates of lead compounds
because it shows a negative result in a pre-incubation method.
[0079] FIG. 8 shows the effect of the pre-incubation is not found
at a pre-incubation temperature of 2.degree. C. Preferred as a
pre-incubation temperature is 20 to 30.degree. C.
[0080] Compound I-1 was selected as a lead compound as shown above
and structurally close compounds (compounds of the formula:
A-C(.dbd.O)--CH.dbd.C(OH)--COOH wherein A is an organic residue)
were prepared. Inhibition activities of them to HCV RNA-dependent
RNA polymerase were examined in accordance with a manner described
in the following Example 1. The results are shown in the following
Table 1. A compound of the formula: A-C(.dbd.O)--CH.dbd.C(OH)--COOH
can form a structure of the formula:
A-CH(OH).dbd.CH--C(.dbd.O)--COOH or of the formula:
A-C(.dbd.O)--CH.sub.2--C(.dbd.O)--COOH, wherein A is an organic
residue. A compound of any structure is within the scope of the
present invention. TABLE-US-00001 TABLE 1 Inhibition activities of
compounds of the formula: A--C(.dbd.O)--CH.dbd.C(OH)--COOH to HCV
RNA-dependent RNA polymerase Inhibition rate at a concentration
Comp. of 100 .mu.g/ml IC.sub.50 No. Group of the formula: - A (%)
(.mu.g/ml) I-1 3-hydroxyphenyl >99 0.4 I-2 4-hydroxyphenyl 99
0.9 I-3 4-methylphenyl 99 1.0 I-4 4-aminophenyl >99 1.4 I-5
phenyl 98 2.3 I-6 5-meyhylthiophen-2-yl 97 3.1 I-7 naphthalen-2-yl
95 6.9 I-8 indol-2-yl 95 6.9 I-9 4-chlorophenyl 94 7.1 I-10
thiophen-3-yl 94 7.2 I-11 4-fluorophenyl 94 7.4 I-12
3-methoxyphenyl 94 8.4 I-13 thiophen-2-yl 92 8.7 I-14 3-bromophenyl
93 8.9 I-15 1-methylpyrrol-2-yl 94 9.8 I-16 pyrrol-2-yl 92 10 I-17
4-hydroxy-3-methoxyphenyl 92 11 I-18 2,4-dichlorophenyl 90 13
[0081] An activator or an inhibitor to an enzyme can effectively be
found in the present assay method. A compound of the formula:
A-C(.dbd.O)--CH.dbd.C(OH)--COOH wherein A is an organic residue has
an inhibition activity to a HCV RNA-dependent RNA polymerase and is
useful as an agent for treating hepatitis C. The above result shows
an organic residue in A is not limited to the specific residue and
the structure of the formula: --C(.dbd.O)--CH.dbd.C(OH)--COOH is
important for exhibiting a inhibition activity to a HCV
RNA-dependent RNA polymerase.
[0082] A HCV RNA-dependent RNA polymerase can be inhibited by
contacting a compound obtained through an assay of the present
invention and a HCV RNA-dependent RNA polymerase. A replication of
HCV can be inhibited by contacting a compound obtained through an
assay of the present invention and HCV.
EXAMPLE
Preparation of HCV RNA-Dependent RNA Polymerase
[0083] A cDNA encoding HCV RNA-dependent RNA polymerase was made by
use of HCV positive human plasma and HCV RNA-dependent RNA
polymerase was expressed using the insect cells or the E. coli. HCV
RNA-dependent RNA polymerase in the cell extract was purified by
use of an anion exchange chromatography, a heparin affinity
chromatography, a polyU affinity chromatography, a cation exchange
chromatography and gel filtration chromatography. The purification
was performed at 4.degree. C. and Tris buffer (pH7.5) containing
ethylenediaminetetraacetic acid (1 mM), dithiothreitol (10 mM) and
glycerol (20%) was used. The enzyme purified was stored at
4.degree. C. until use for the assays.
Example 1
the Assay Method with Pre-Incubation Using HCV RNA-Dependent RNA
Polymerase
[0084] 1) The buffer 1 .sup.1) (10.5 .mu.l) containing Tris-HCl
(100 mM, pH 7.5), MgCl.sub.2 (25 mM), dithiothreitol (5 mM), KCl
(125 mM) and ethylenediaminetetraacetic acid (5 mM) was transferred
to a polypropylene tube and then poly(A) .sup.2) solution (100
.mu.g/ml, 10.5 .mu.l), oligo(U).sub.12 .sup.3) solution (1
.mu.g/ml, 10.5 .mu.l) and water (8 .mu.l) were added into the tube
and mixed.
2) Next, HCV RNA-dependent RNA polymerase (10.5 .mu.l) was added
into the tube and pre-incubated at 25.degree. C. for 60 min.
3) The enzyme solution pre-incubated (50 .mu.l) was added to a test
compound (5.26 .mu.l/well) dissolved with dimethyl sulfoxide or
water in a well of a 96-wells microtiter plate.
[0085] 4) Additionally, the buffer II (10.6 .mu.l) containing
Tris-HCl (100 mM, pH 7.5), MgCl.sub.2 (25 mM), dithiothreitol (5
mM), KCl (125 mM), ethylenediaminetetraacetic acid (5 mM) and UTP
4) (2.1 .mu.M, 1 .mu.Ci .sup.3H-UTP), was mixed with water (39.4
.mu.l). The mixture (50 .mu.l) was added into the well and mix, and
then the enzyme reaction was performed at 25.degree. C. for 30
min.
5) After enzyme reaction for 30 min, the enzyme reaction was
stopped by adding disodium ethylenediaminetetraacetic acid (50 mM,
50 .mu.l).
[0086] 6) By use of the cell-harvester, all volume of the enzyme
reaction sample was transferred onto a filtermat bound with
diethylaminoethyl base (referred to as DEAE-filtermat) and washed
twice for 10 sec with sodium phosphate buffer (0.25 M, pH 7.0) and
once for 10 sec with de-ionized water.
7) The DEAE-filtermat was dried at 95.degree. C. for 15 min and
transferred into a sample bag with liquid scintillater (10 ml), and
then a sample bag was sealed.
8) The radio activity was measured by a scintillation counter.
[0087] 9) The inhibitory rate was calculated from the radioactivity
measured (ccpm) as follows. Inhibitory .times. .times. rate = 100 -
( ccpm .times. .times. with .times. .times. test .times. .times.
compound - ccpm .times. .times. without .times. .times. enzyme ) /
( ccpm .times. .times. without .times. .times. test .times. .times.
compound - ccpm .times. .times. without .times. .times. enzyme )
.times. 100 ##EQU1##
[0088] The concentration of compound at 50% inhibition (IC.sub.50
value) was calculated from the compound concentration used and the
inhibitory rate calculated.
[0089] When use of a compound at .alpha. .mu.g/ml results in A %
inhibition and use of a compound at .beta. .mu.g/ml results in B %
inhibition on condition of A.gtoreq.50>B the IC50 value is
obtained by the calculation as follows.
IC.sub.50(.mu.g/ml)=10.sup.[(50-B)/(A-B).times.(log.sup..alpha..sup.-log.-
sup..beta..sup.)+log.sup..beta..sup.]
[0090] .sup.1) All of water used as an experimental material from
1) to 5) was treated with diethyl pyrocarbonate to inactivate
ribonuclease. As the treatment of diethyl pyrocarbonate, diethyl
pyrocarbonate (0.1 g) was dissolved in de-ionized water (100 ml),
incubated at 37.degree. C. for 24 hr and then autoclaved at
120.degree. C. for 30 min.
[0091] .sup.2) Poly(A) is a single strand polynucleotide consisted
of adenylic acid only and used as template.
[0092] .sup.3) Oligo(U).sub.12 is a single strand oligonucleotide
consisted of 12 molecules of uridylic acid and used as primer.
[0093] .sup.4) UTP is uridine triphosphate and used as
substrate.
[0094] When compound I-1,3'-dUTP, compound A, compound B and
compound C were used as test compounds, the inhibitory rates of the
compounds for HCV RNA-dependent RNA polymerase were shown in Table
2. Concentrations of the compounds were as follows. TABLE-US-00002
Compound I-1: 0.5 .mu.g/ml 3'-dUTP: 0.7 .mu.M Compound A: 100
.mu.g/ml Compound B: 50 .mu.g/ml Compound C: 25 .mu.g/ml
[0095] TABLE-US-00003 TABLE 2 Compound No. (concentration)
Inhibitory rate I-1 (0.5 .mu.g/ml) 62% 3'-dUTP (0.7 .mu.M) 79% A
(100 .mu.g/ml) 19% B (50 .mu.g/ml) 1% C (25 .mu.g/ml) 2%
Reference 1
[0096] The prior assay method using HCV RNA-dependent RNA
polymerase
[0097] 1) The buffer 1 .sup.1) (10.5 .mu.l) containing Tris-HCl
(100 mM, pH 7.5), MgCl.sub.2 (25 mM), dithiothreitol (5 mM), KCl
(125 mM) and ethylenediaminetetraacetic acid (5 mM) was transferred
to a polypropylene tube and then poly(A) .sup.2) solution (100
.mu.g/ml, 10.5 .mu.l), oligo(U).sub.12 .sup.3) solution (1
.mu.g/ml, 10.5 .mu.l) and water (8 .mu.l) were added into the tube.
After mixing, the tube was cooled on ice.
2) Next, HCV RNA-dependent RNA polymerase (10.5 .mu.l) cooled on
ice was added into the tube and mixed.
3) The mixture cooled (50 .mu.l) was added to a test compound (5.26
.mu.l/well) dissolved with dimethyl sulfoxide or water in a well of
a 96-wells microtiter plate.
[0098] 4) Additionally, the buffer II (10.6 .mu.l) containing
Tris-HCl (100 mM, pH 7.5), MgCl.sub.2 (25 mM), dithiothreitol (5
mM), KCl (125 mM), ethylenediaminetetraacetic acid (5 mM) and UTP
.sup.4) (2.1 .mu.M, 1 .mu.Ci .sup.3H-UTP), was mixed with water
(39.4 .mu.l). The mixture (50 .mu.l) was added into the well and
mix, and then the enzyme reaction was performed at 25.degree. C.
for 30 min.
5) After enzyme reaction for 30 min, the enzyme reaction was
stopped by adding disodium ethylenediaminetetraacetic acid (50 mM,
50 .mu.l).
[0099] 6) By use of the cell-harvester, all volume of the enzyme
reaction sample which was described in 5) was transferred onto a
DEAE-filtermat and washed twice for 10 sec with sodium phosphate
buffer (0.25 M, pH 7.0) and once for 10 sec with de-ionized
water.
7) The DEAE-filtermat was dried at 95.degree. C. for 15 min and
transferred into a sample bag with liquid scintillater (10 ml), and
then a sample bag was sealed.
8) The radio activity was measured by a scintillation counter.
[0100] 9) The inhibitory rate was calculated from the radioactivity
measured (ccpm) as follows. Inhibitory .times. .times. rate = 100 -
( ccpm .times. .times. with .times. .times. test .times. .times.
compound - ccpm .times. .times. without .times. .times. enzyme ) /
( ccpm .times. .times. without .times. .times. test .times. .times.
compound - ccpm .times. .times. without .times. .times. enzyme )
.times. 100 ##EQU2##
[0101] When compound I-1,3'-dUTP, compound A, compound B and
compound C were used as test compounds, the inhibitory rates of the
compounds for HCV RNA-dependent RNA polymerase were shown in Table
3. Concentrations of the compounds were as follows. TABLE-US-00004
Compound I-1: 0.5 .mu.g/ml 3'-dUTP: 0.7 .mu.M Compound A: 100
.mu.g/ml Compound B: 50 .mu.g/ml Compound C: 25 .mu.g/ml
[0102] TABLE-US-00005 TABLE 3 Compound No. (concentration)
Inhibitory rate I-1 (0.5 .mu.g/ml) 62% 3'-dUTP (0.7 .mu.M) 68% A
(100 .mu.g/ml) 50% B (50 .mu.g/ml) 63% C (25 .mu.g/ml) 67%
Examination 1
[0103] Effects of the pre-incubation that is a special feature of
the assay method as the present invention were evaluated. In this
specification, RdRp or HCV RdRp means HCV RNA-dependent RNA
polymerase.
[0104] The mixture of the buffer 1, poly(A), oligo(U).sub.12 and
recombinant RdRp was pre-incubated for 0 min, 15 min, 30 min, 45
min, 60 min, 90 min, 120 min, 150 min and 180 min respectively,
before the enzyme reaction at 25.degree. C. for 30 min (FIG. 3).
The solvent, DMSO or water was used instead of test compound
solution.
[0105] Enzyme activity was estimated by determining 3H-UTP as
substrate incorporated into the product. The enzyme activity
increased with an increase in the pre-incubation time and leveled
off after approximately 60 min. The increase of enzyme activity was
appeared in both cases of DMSO and water as the solvent of
compound. When DMSO and water was used, the enzyme with
pre-incubation showed 2.7 times and 4.2 times respectively in the
best case as active as that without pre-incubation.
[0106] From the above-mentioned findings, it was confirmed that
increase of enzyme activity was brought about by pre-incubating the
buffer I, poly(A), oligo(U) and recombinant RdRp before the enzyme
reaction.
Examination 2
[0107] Next, effects of pre-incubation on thermostability of RdRp
were determined.
[0108] Time course of HCV RdRp activity at 25.degree. C. or
37.degree. C., with or without pre-incubation was shown in FIG. 4.
In the prior assay method without pre-incubation, the velocity of
enzyme reaction at 37.degree. C. was getting lower after the enzyme
reaction for 20 min and the amount of the product in enzyme
reaction for 60 min at 37.degree. C. was about 50% less than that
at 25.degree. C. On the other hand, in the pre-incubation assay
method, the velocity of enzyme reaction at 37.degree. C. tended to
decrease after the enzyme reaction for 20 min but the amount of the
product in enzyme reaction increased and the amount of the product
in enzyme reaction for 60 min at 37.degree. C. was a higher value
at about 125% as compared with that at 25.degree. C.
[0109] These data showed that the pre-incubation of the buffer 1,
poly(A), oligo(U) and recombinant RdRp resulted in increase of
thermostability of RdRp during the enzyme reaction.
Examination 3
[0110] In the screening for HCV RNA-dependent RNA polymerase
inhibitor, there is a dissociation of inhibitory effect as a
different point between an assay method of the present invention
and a prior assay method. Treatment of pre-incubation resulted in
great decrease of inhibitory effect of many compounds. Therefore,
the influence of pre-incubation on the inhibitory value of enzyme
inhibitor was determined. Using several compounds which evaluated
RdRp inhibition in the prior assay method, the time course of HCV
RdRp inhibitory effect was determined for 0 min, 15 min, 30 min, 45
min, 60 min, 90 min, 120 min, 150 min and 180 min as a
pre-incubation time (FIG. 5).
[0111] There was not a large influence by pre-incubation using
3'-dUTP as an analog of substrate UTP. However, when the other
three compounds, compound A, compound B and compound C were used,
the inhibitory effect decreased with increasing pre-incubation time
and was stable in about 60 min of pre-incubation time.
[0112] These results showed that the treatment of pre-incubation of
the buffer I, poly(A), oligo(U) and recombinant RdRp lead to great
change of RdRp inhibitory effect by kinds of compounds.
Examination 4
[0113] Influence of pre-incubation on inhibitory value of enzyme
inhibitor was determined in Examination 3. The temperature of
pre-incubation in Examination 3 was changed and the time course of
inhibitory effect was determined at variant temperature of
pre-incubation.
[0114] Using several compounds, 3'-dUTP, compound I-1 and compound
C, which evaluated RdRp inhibition in the prior assay method, the
time course of HCV RdRp inhibitory effect was determined at
2.degree. C., 20.degree. C. and 25.degree. C. as a pre-incubation
temperature for 0 min, 20 min, 40 min, 60 min, 80 min, and 100 min
as a pre-incubation time (FIG. 6, FIG. 7, FIG. 8). There was not
influence by pre-incubation at any temperatures using 3'-dUTP as an
analog of substrate UTP and compound I-1. However, when compound C
was used, the inhibitory effect decreased greatly with increasing
pre-incubation time at 20.degree. C. and 25.degree. C. but did not
appear clear influence by pre-incubation at 2.degree. C.
[0115] These results showed that the treatment of pre-incubation
which lead to great change of RdRp inhibitory effect had variant
influence by change of temperature.
Examination 5
[0116] To compare a pre-incubation assay method with the prior
assay method, random screening assay was performed using several
ten thousands compounds with different chemical structures each to
each as test substances.
[0117] First, the compounds were mixed ten by ten. The
concentration of each test compound mixed was adjusted to 0.83
.mu.g/ml. The inhibitory rate was obtained according to the prior
assay method described in Reference 1. The result is shown in FIG.
9. On the other hand, the compounds were mixed five by five. The
concentration of each test compound mixed was adjusted to 49
.mu.g/ml. The inhibitory rate was obtained according to a
pre-incubation assay method of the present invention described in
Example 1. The result is shown in FIG. 10.
Example 2
[0118] The assay method with pre-incubation using HCV RNA-dependent
RNA polymerase without radioisotope-labeled substrate.
[0119] In Example 1, .sup.3H-UTP, that is UTP labeled with tritium
as radioisotope was used as substrate in the assay with
pre-incubation using HCV RNA-dependent RNA polymerase. Herein,
digoxigenin-labeled UTP (DIG-UTP) instead of .sup.3H-UTP was used
as substrate in the assay with pre-incubation using HCV
RNA-dependent RNA polymerase.
1) Streptavidin (1 mg/ml) was dissolved in the phosphate buffer
saline (PBS) containing NaCl (0.137 M), KCl (2.68 mM), potassium
dihydrogenphosphate (1.47 mM) and sodium phosphate (8.06 mM).
2) The streptavidin solution was diluted 25-fold with the carbonate
buffer containing sodium carbonate (0.9 M) and sodium
hydrogencarbonate (0.1 M).
3) The 25-fold streptavidin solution (50 .mu.l/well) was
transferred to a well of 96 wells-microtiter palate with flat
bottom for immunoassay and then the plate was kept at 4.degree. C.
over night.
4) After the 96 wells-microtiter plate was coated with
streptavidin, the well was washed twice by adding, mixing and
draining PBS (200 .mu.l/well).
5) The skim milk dissolved with PBS (1%, 300 .mu.l) was transferred
into each well and was kept at room temperature for 30 min.
6) After the plate was washed once with PBS (200 .mu.l/well), the
well was filled with PBS (300 .mu.l/well) and then the plate was
stored at 4.degree. C.
7) After the PBS was drained from the streptavidin-coated plate
stored under the condition described in 6), the well was washed
once with PBS (200 .mu.l/well) and was filled with de-ionized water
(300 .mu.l/well).
[0120] 8) The buffer I (10 .mu.l) containing Tris-HCl (100 mM, pH
7.5), MgCl.sub.2 (25 mM), dithiothreitol (5 mM), KCl (125 mM) and
ethylenediaminetetraacetic acid (5 mM) was transferred to a
polypropylene tube and then poly(A) solution (6.25 .mu.g/ml, 10
.mu.l), biotin-labeled oligo(U).sub.12 solution (3 .mu.g/ml, 10
.mu.l), water (10 .mu.l) and HCV RNA-dependent RNA polymerase
solution (10 .mu.l) were added into the tube and mixed calmly.
9) De-ionized water was drained from the plate described in 7), and
the solution (50 .mu.l/well) at once after the mixing described in
8) was transferred to each well. After mixing by a plate mixer, the
solution was pre-incubated at 25.degree. C. for 1 hr.
[0121] 10) On the other hand, the buffer I (10 .mu.l) was
transferred into another tube, and UTP (1 .mu.M, 10 .mu.l), DIG-UTP
(300 .mu.M, 1 .mu.l), water (24 .mu.l) and a test compound
dissolved with dimethyl sulfoxide or water (5 .mu.l) were added to
the tube and mixed calmly. Concentrations of the compounds used
were as follows. TABLE-US-00006 Compound (I-1): 0.5 .mu.g/ml
3'-dUTP: 0.7 .mu.M Compound A: 100 .mu.g/ml Compound B: 50 .mu.g/ml
Compound C: 25 .mu.g/ml
11) The mixed solution (50 .mu.l/well) described in 10) was
transferred into each well after the pre-incubation described in
9), mixed by a plate mixer and then incubated at 25.degree. C. for
1 hr. 12) When the enzyme reaction was in 1 hr, the enzyme reaction
was stopped by adding ethylenediaminetetraacetic acid (0.1 M, 50
.mu.l/well). 13) The plate was washed twice with PBS (200
.mu.l/well) after end of the enzyme reaction. 14) Next, alkaline
phosphatase-labeled anti-DIG antibody (3 U/ml, 100 .mu.l/well)
diluted 250-fold with PBS containing skim milk (0.5%) was
transferred each well. After mixing by a plate mixer,
immuno-reaction was performed at 25.degree. C. for 1 hr. 15) Each
well was washed twice with PBS (200 .mu.l/well) containing skim
milk (0.05%) and then washed twice with PBS (200 .mu.l/well). 16)
Substrate solution (150 .mu.l/well) for alkaline phosphatase was
prepared by mixing of Tris-HCl (100 mM, pH 9.5), NaCl (100 mM),
MgCl.sub.2 (5 mM) and p-nitrophenyl phosphate (6 mM) just before
use. The substrate solution was transferred into each well of the
plate, mixed by a plate mixer and then incubated at 25.degree. C.
for 1 hr until the color appears in the solution. 17) The plate was
set on a plate reader and the absorbance was measured at 405 nm.
18) The inhibitory rate was calculated from the optical density
(unit; OD) measured as follows. Inhibitory .times. .times. rate =
100 - ( OD .times. .times. with .times. .times. test .times.
.times. compound - OD .times. .times. without .times. .times.
enzyme ) / ( OD .times. .times. without .times. .times. test
.times. .times. compound - OD .times. .times. without .times.
.times. enzyme ) .times. 100 ##EQU3##
[0122] Table 4 summarized the comparison between inhibitory rates
in the assay with the pre-incubation and those in the assay without
the pre-incubation. TABLE-US-00007 TABLE 4 Inhibitory rate
Treatment of pre- incubation Compound No. (concentration) + - I-1
(0.5 .mu.g/ml) 52% 59% 3'-dUTP (0.7 .mu.M) 50% 44% A (100 .mu.g/ml)
24% 87% B (50 .mu.g/ml) 20% 28% C (25 .mu.g/ml) 15% 87%
INDUSTRIAL APPLICABILITY
[0123] The pre-incubation assay method can heighten 1) an activity
to an enzyme and 2) thermostability and reduce 3) pseudo-positive
data.
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