WO2003031645A1

WO2003031645A1 - Preincubation assay method using single-stranded nucleic acid oligomer

Info

Publication number: WO2003031645A1
Application number: PCT/JP2002/010029
Authority: WO
Inventors: Kenji Abe
Original assignee: Shionogi & Co., Ltd.
Priority date: 2001-10-01
Filing date: 2002-09-27
Publication date: 2003-04-17
Also published as: JP4199115B2; JPWO2003031645A1

Abstract

An assay method wherein an enzyme-containing solution is incubated in the absence of a test substance and a substrate of the enzyme to thereby reduce false positive ratio in random screening.

Description

Description Pre-incubation assay using single-stranded nucleic acid oligomer

The present invention relates to an Assay method for an activator or an inhibitor of a nucleic acid synthase, and more particularly, to an Assay method for an activator or an inhibitor of a nucleic acid synthase using a single-stranded nucleic acid oligomer. Background art

Nucleic acid synthetase activators or inhibitors may be used to determine the appropriate conditions (eg, buffer conditions, reaction temperature, etc.) for the enzymatic reaction to proceed. A necessary substance (for example, metal ion) is mixed with the test substance, the enzyme reaction is allowed to proceed, and after the reaction is completed, the enzyme activity measured or calculated by some means is measured. It is achieved by comparing with

For example, a nucleic acid synthetase, a labeled substrate, a template, a primer, a metal ion, and a test substance are simultaneously or sequentially mixed to progress the nucleic acid synthase reaction. The labeled substrate is incorporated into the reaction product (Incorporation). After the reaction is completed, the amount of the labeled substrate incorporated into the reaction product is measured, and the calculated enzyme activity is compared with the enzyme activity in the absence of the test substance. However, even if the results of such an assay indicate that the test substance has an activating or inhibitory action on the nucleic acid synthase, the test substance may be based on the structure or properties of the test substance. In some cases, it acts or binds non-specifically to nucleic acid synthases, giving false positives. Therefore, if the result of Atsushi shows that the result is positive, it is necessary to confirm that it is a true nucleic acid synthetase activator or inhibitor by another Atsetsu method. Atsey method requires complicated processes In some cases, however, it is not suitable for primary attestation.

In recent years, high-throughput screening ( _HTS ) has played an important role in the early stages of drug development. HTS enables hundreds to tens of thousands of compounds to be analyzed at once, but how to analyze vast amounts of data is a major issue.

Patent Document 1 describes the possibility of the Atsey method of HCV without adding a primer, but does not disclose preincubation.

On the other hand, Non-Patent Document 1 discloses that the pre-incubation of HCV RNA-dependent RNA synthetase in the presence of a template and a primer increases the enzyme activity. However, the above-mentioned documents merely disclose the properties of the enzyme, and do not disclose at all the activator or inhibitor of the enzyme or the case using a single-stranded nucleic acid oligomer.

In addition, regarding HCV RNA-dependent RNA synthase, when a single-stranded nucleic acid oligomer whose 3 ′ end can serve as its own primer is used as a template, the enzyme reaction proceeds in the absence of a primer. It is disclosed in reference 2. However, no pre-incubation is disclosed.

Patent Document 1: WO 96/37619

Non-Patent Document 1: Journal of General Virology (2000), 81, 759-767

Non-patent document 2: Journal of virology, 2000, 9134-9143 Disclosure of the invention

It is important to obtain useful information from the data obtained as a result of HTS, that is, how to eliminate noise (false positives), and to minimize the noise of activators or inhibitors of nucleic acid synthase. Law development is required.

The present inventors have proposed a preincubation assay, that is, in the absence of a test substance and a substrate for a nucleic acid synthase, a nucleic acid synthase, a single strand in which the 3 ′ terminal portion can function as its own primer. Solution containing the nucleic acid oligomer and metal ions Incubation followed by the selection of an activator or inhibitor of nucleic acid synthase was found to reduce noise from the data obtained from the results of the assay. <5

That is, the present invention

(1) A solution containing a nucleic acid synthase, a single-stranded nucleic acid oligomer capable of functioning as its own primer, and a metal ion in the absence of a test substance and a substrate for the nucleic acid synthase. Inducing the activator or inhibitor of the nucleic acid synthase, comprising a step of incubating the nucleic acid synthase.

About.

More specifically, the following (2) to (17) are provided.

(2) The following steps;

(a) In the absence of a test substance and a substrate for a nucleic acid synthase, a nucleic acid synthase, a single-stranded nucleic acid oligomer capable of functioning as its own primer and a metal ion, and a metal ion Incubating the solution,

(b) mixing the incubated solution with a test substance and the substrate for the nucleic acid synthase, bringing the nucleic acid synthase into contact with the test substance and the substrate for the nucleic acid synthase, Measuring the incorporation of the substrate into the reaction product,

(c) mixing the incubated solution with the nucleic acid synthase substrate, contacting the nucleic acid synthase with the nucleic acid synthase substrate in the absence of a test substance, Measuring incorporation into the reaction product,

(d) comparing the incorporation of the nucleic acid synthase substrate into the reaction product measured in the steps (b) and (C);

Atsey method according to the above (1), wherein

(3) The Atsey method according to (1) or (2), which is an Atsey method for an inhibitor of a viral nucleic acid synthase,

(4) The Atsey method according to the above (3), wherein the viral nucleic acid synthase is a viral RNA-dependent RNA synthase. (5) The method according to (3), wherein the viral RNA-dependent RNA synthase is an HCV RNA-dependent RNA synthase.

(6) The method according to the above (4) or (5), wherein the single-stranded nucleic acid oligomer has a self-complementary nucleic acid sequence at a 3 ′ terminal portion.

(7) The Atsey method according to the above (6), wherein the single-stranded nucleic acid oligomer has a self-complementary nucleic acid sequence at the 3 ′ terminal portion of poly (A).

(8) The Atsey method according to (7), wherein the substrate is ¾-UTP or ³² P-UTP,

(9) The Atsey method according to any one of the above (1) to (8), wherein the metal ion is Mg ²⁺ or Mn ²⁺ .

(10) The method according to any one of (1) to (9), wherein the incubation is performed for 0.5 hours or more.

(11) The method according to any one of the above (1) to (10), wherein the incubation is performed at 20 ° C to 40 ° C.

(12) A method for inhibiting a viral nucleic acid synthase by contacting the virus nucleic acid synthase with the substance obtained by the Atsey method according to (3),

(13) A method for inhibiting virus replication by contacting the virus with a substance obtained by the Atsey method described in (3) above,

(14) The method according to (12) or (13), wherein the virus is a virus belonging to the Flaviviridae family.

(15) The method according to the above (12) or (13), wherein the virus is HCV,

(16) an activator or an inhibitor of nucleic acid synthase obtained by the Atsey method according to any one of (1) to (11) above,

(17) A pharmaceutical composition comprising, as an active ingredient, an activator or an inhibitor of nucleic acid synthase obtained by the Atsey method according to any one of (1) to (11).

Hereinafter, the present invention will be described in detail.

The present invention relates to a technique relating to an activator or an activator of a nucleic acid synthase. You. In the present specification, the assay of the activator or inhibitor of nucleic acid synthase is an assay for evaluating a test substance that is not known to be an activator or inhibitor of nucleic acid synthase, ie, a primary assay. Atsey and atssie to evaluate test substances already known to be activators or inhibitors of nucleic acid synthase, that is, reassessments. The atsee of the present invention can be used for all of these atsees. For example, it can be used to identify substances that activate or inhibit nucleic acid synthase. In particular, it can be used to identify substances that inhibit nucleic acid synthase. For example, identification of substances that inhibit viral nucleic acid synthase, identification of substances that inhibit RNA-dependent RNA synthase, HCV RNA-dependent RNA It can be used to identify substances that inhibit synthase.

The present invention relates to an activator or an inhibitor of a nucleic acid synthase. Atssay is characterized by incubating before starting the enzyme reaction. That is, a solution containing a nucleic acid synthase, a single-stranded nucleic acid oligomer capable of functioning as its own primer and a metal ion in the absence of a test substance and a substrate for the nucleic acid synthase is used. It is characterized by incubating. In this specification, the incubation is also referred to as preincubation. Specifically, at the temperature at which the pre-incubation effect appears in the absence of the test substance and the substrate for the nucleic acid synthase, the time at which the pre-incubation effect appears, the nucleic acid synthase, and the 3 'end become their own primers. This means incubating the resulting solution containing single-stranded nucleic acid oligomer and metal ions.

The temperature of the incubation, that is, the temperature at which the effect of preincubation appears, is specifically about 15 ° C to about 50 ° C, particularly 20 ° C to 40 ° C, Is preferably incubated at 20 to 30 ° C.

The incubation time, that is, the time during which the effect of the pre-incubation appears, is preferably about 15 minutes or more, particularly preferably 0.5 hours or more, more preferably 1 hour or more, and is preferably about 15 minutes to 12 hours, 0 hour or more. It is preferable to incubate for 5 to 3 hours, especially for 1 to 2 hours. In the absence of the test substance and the substrate of the nucleic acid synthase, a solution containing the nucleic acid synthase, a single-stranded nucleic acid oligomer whose 3 ′ terminal portion can function as its own primer, and a metal ion is included. By the basing, the enzyme reaction is likely to proceed.

“In the absence of a test substance and a substrate for a nucleic acid synthase” means that incubation is performed under conditions that do not include a test substance and a substrate for a nucleic acid synthase. For example, what is essential for an enzymatic reaction other than the substrate of the nucleic acid synthase (eg, metal ion) may be incubated with the enzyme.

"Solution containing nucleic acid synthase, single-stranded nucleic acid oligomer whose 3 'end can function as its own primer and metal ion" means Nucleic acid synthase, 3' end functions as its own primer A solution containing at least a single-stranded nucleic acid oligomer and a metal ion that can contain a test substance and a substrate other than a substrate for a nucleic acid synthase. For example, it may contain high molecular compounds, low molecular compounds, metal ions, halogen ions, amino acids, polypeptides, nucleic acids, polynucleotides, and the like.

The present invention relates to a nucleic acid synthase activator activator or an inhibitor, and relates to a nucleic acid elongation synthesis reaction using a single-stranded nucleic acid oligomer whose 3′-terminal part can function as its own primer. Atsushi.

“Single-stranded nucleic acid oligomer whose 3 ′ end can function as its own primer” means a single-stranded template DNA or RNA whose 3 ′ end can function as its own primer. I do. In other words, it means a single-stranded nucleic acid oligomer whose 3 ′ terminal portion hybridizes with its own complementary portion and can form a loop structure. Note that if the 3'-terminal part is a single-stranded nucleic acid oligomer that functions as its own primer in the enzymatic reaction, it forms a loop even if it is present as a single strand without forming a loop in solution. May be. Preferably, it is a single-stranded nucleic acid oligomer which does not form a loop in a solution and exists as a single strand. For example, the complementary bond in the hybridizing part is a hydrogen bond between G and C. And a hydrogen bond between A and U and a hydrogen bond between dA and dT are more preferable than a hydrogen bond between dG and dC. For example, those having an AU repeating sequence at the 3 ′ terminal are preferable.

Examples of the single-stranded nucleic acid oligomer include the following. A single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3'-terminal portion,

poly (dA), poly (dT), poly (dG) ヽ poly (dC) ヽ poly (A), poly (U), poly (G) ヽ poly (C) self-complementary nucleic acid sequence at the 3 ′ end A single-stranded nucleic acid oligomer having

Poly (dA), poly (dT), poly (dG), poly (dC) and other homopolymers (eg, synthetic nucleic acid polymers) at the 3'-end, oligo (dT), oligo (dA) , Oligo (dC), oligo (dG) and other single-stranded DNA oligomers (eg, in the case of DNA-dependent DNA synthase),

Poly (A), poly (U), poly (G), poly (C) and other homopolymers have oligo (U), oligo (A), oligo (C), and oligo ( G) and other single-stranded RNA oligos (eg, in the case of RNA-dependent RNA synthase),

Poly (dA), poly (dT), poly (dG), poly (dC) and other homopolymers (eg, synthetic nucleic acid polymers) have oligo (U), oligo (A), Single-stranded nucleic acid oligomers with oligo (C), oligo (G), etc. (eg, for DNA-dependent RNA synthase),

The oligo (dT), oligo (dA), oligo (dC), and oligo (dG) are added to the 3 'end of homopolymers such as poly (A), poly (U), poly (G), and pol C), respectively. ) And other single-stranded nucleic acid oligomers (for example, in the case of RNA-dependent DNA synthase),

A single-stranded DNA oligomer (for example, DNA) in which a dAdT or dCdG repeating sequence is added to the 3 'end of a homopolymer such as poly (dA), poly (dT), poly (dG), or poly (dC). Dependent DNA synthase),

P. A single-stranded RNA oligomer (such as ly (A), poly (U), poly (G), or poly (C)) with a repeat sequence of AU or CG added to the 3 'end of a homopolymer such as poly (C) , For RNA-dependent RNA synthase), Poly (dA), poly (dT) _s Poly (dG), poly (dC), etc. Single-stranded nucleic acid oligomer (eg, DNA Dependent RNA synthase),

Poly (A), poly (U), poly (G), poly (C) and other homopolymers with a single-stranded nucleic acid oligomer (eg, RNA) Dependent DNA synthase),

A single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3′-terminal portion of a heteropolymer (for example, a naturally occurring nucleic acid polymer, a synthesized nucleic acid polymer, etc.) in which various bases are mixed,

One in which a repeating sequence of dAdT, dCdG, AU, or CG is added to the 3 'end of a heteropolymer (for example, a naturally occurring nucleic acid polymer or a synthesized nucleic acid polymer) in which various bases are mixed. The nucleic acid oligomer of the strand.

Here, the self-complementary nucleic acid sequence means a sequence that forms a loop structure (for example, a hairpin loop) at the 3′-terminal portion and can hybridize with a self-complementary portion.

It should be noted that another nucleic acid may be added to the 5 ′ terminal portion of the homopolymer. For example, when a viral RNA-dependent RNA synthetase is used, a single-stranded nucleic acid oligomer having an AU repeating sequence at the 3 ′ terminal is preferable. In particular, poly (A) having a repeating sequence of AU at the 3 ′ terminal portion is preferable.

The length of the single-stranded nucleic acid oligomer is not particularly limited, and may be appropriately selected depending on the type of the nucleic acid synthase. For example, using a single-stranded nucleic acid oligomer of about 8 bases to about 1000 bases, about 50 bases to about 800 bases, about 100 bases to about 500 bases in length. Can be done.

In a single-stranded nucleic acid oligomer, the number of base pairs that hybridize with a self-complementary portion is 2 or more, preferably 2 to 10, and more preferably 2 to 5. Examples of the metal ion include Mg2 ⁺ , Mn2 ⁺ , Na +, K +. Many nucleic acid synthetases generally require Mg ^{2 +} . The test substance refers to a substance used in Atsushi, and includes not only low molecular weight compounds but also proteins such as polypeptides.

The enzymatic reaction is initiated by adding a nucleic acid synthase substrate to the solution containing the nucleic acid synthase after the preincubation.

In the conventional method (The EMBO Journal vol.15, no.l, ppl2-22, 1996), the enzymatic reaction must be performed at about 22 ° C. However, when the temperature is lower than that, the activity of the nucleic acid synthase becomes dull.

The pre-incubation assay differs from the conventional method in that the nucleic acid synthetase reaction can be performed at about 10 ° C to about 40 ° C. In particular, it can be performed at about 20 ° C to about 40 ° C. Therefore, in an assay using a nucleic acid synthase present in a human or a nucleic acid synthase of a virus that infects a human, it can be performed at the same temperature as the human body temperature (particularly, 37 ° C). Very useful. In addition, by performing the treatment at such a temperature, the enzyme activity increases, and as a result, the detection sensitivity of Atssey increases.

The effect of the test substance on the enzyme reaction of the nucleic acid synthase may be compared by measuring the incorporation of the substrate of the nucleic acid synthase into the reaction product. That is, 1) a solution containing a nucleic acid synthase, a single-stranded nucleic acid oligomer capable of functioning as its own primer, and a metal ion in the absence of a test substance and a substrate for the nucleic acid synthase. 2) Mix the incubated solution with the test substance and the substrate for the nucleic acid synthase, contact the nucleic acid synthase with the substrate for the test substance and the nucleic acid synthase, and react the nucleic acid synthase substrate. Measure the incorporation into the product, and 3) mix the incubated solution with the nucleic acid synthase substrate, and contact the nucleic acid synthase with the nucleic acid synthase substrate in the absence of the test substance. The incorporation of the substrate into the reaction product was measured, and the incorporation of the nucleic acid synthase obtained in steps 4) 2) and 3) into the reaction product was compared. You can check. Even if the (test substance) and the substrate of nuclease are added to the incubated solution, The prepared solution may be added. That is, the nucleic acid synthase substrate may be brought into contact with the nucleic acid synthase substrate.

The phrase “mixing a substrate for a nucleic acid synthase and in the absence of a test substance” means that the test substance is not mixed. It may include something outside. For example, it may contain a reaction solution for dissolving or suspending the substrate of the nucleic acid synthase. That is, any data may be used as long as it is used to create data to be compared with data in the presence of the test substance. A solution containing a nucleic acid synthase that has not been pre-incubated and a substrate for the nucleic acid synthase are mixed, and the nucleic acid synthase when the nucleic acid synthase and the nucleic acid synthase substrate are contacted in the absence of the test substance Incorporation of the substrate into the reaction product can also be used as a comparison. The pre-incubated solution is mixed with the substrate of the nucleic acid synthase, and the substrate of the nucleic acid synthase is brought into contact with the nucleic acid synthase in the absence of the test substance. It is preferable that the incorporation into the reaction product is used as a comparison target. The data used for these comparisons may be performed at the same time as the assay using the test substance, or may be performed separately.

The incorporation of the nucleic acid synthase substrate into the reaction product can be measured by using a labeled nucleic acid synthase substrate. For example, the amount of the labeled nucleic acid synthase substrate incorporated into the reaction product may be measured. As the substrate of the labeled nucleic acid synthetase, a radiolabeled nucleic acid synthase substrate or the like may be used, and in that case, the radioactivity may be measured by a scintillation counter or the like. The labeled substrate of the nucleic acid synthase may be separated from the reaction solution using a commercially available filter (eg, a filter having a acetylaminoethyl group introduced therein). Also, SPA (, Scintillation proximity assay) is suitable.

The low-molecular compound means an organic compound having a molecular weight of 15 to 1000, and its constituent atoms are hydrogen, lithium, boron, carbon, nitrogen, oxygen, fluorine, sodium, magnesium, manganese, Examples include aluminum, sulfur, chlorine, potassium, calcium, iron, barium, bromine, and iodine. Polypeptides include not only naturally-occurring peptides but also structurally modified peptides (for example, peptide isosteres).

Examples of the substrate for the nucleic acid synthase include dATP, dTTP, dGTP, dCTP, ATP, UTP, GTP, and CTP. Preferably those particular labeled, as a substrate for labeled nucleic acid synthesis enzymes, for ^{^{example, 3 H- dATP, 3 H-}} dTTP, 3 H-dGTP, 3 H- dCTP, 3 H-ATP, 3 H- ^{^{UTP, 3 H-GTP, 3}} H- CTP, 32 P-dATPヽ³² P- dTTPヽ³² P-dGTPヽ^{^{32 P- dCTP, 32 P- ATP,}} 32 P- UTP, 32 P-GTP, 32 P- CTP and the like. These substrates may be selected according to the single-stranded nucleic acid oligomer used for the assay.

For example, poly (dA), poly (dT), poly (dG), poly (dC), poly (A) ヽ poly (U) ヽ poly (G ) And poly (C), dTTP, dATP, dCTP, dGTP, UTP, ATP, CTP, and GTP may be used as substrates.

In addition, a single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3 'end of a heteropolymer (for example, a naturally occurring nucleic acid polymer or a synthesized nucleic acid polymer) in which various bases are mixed is used. When a strand nucleic acid oligomer is used, a mixture of dTTP, dATP, dCTP and dGTP, or a mixture of UTP, ATP, CTP and GTP may be used as a substrate. In this case, all the substrates may be labeled, or any of them may be labeled.

For example, when a single-stranded nucleic acid oligomer having a repeat sequence of AU at the 3 ′ end of poly (A) is used, 基質 -UTP or ³² P-UTP may be used as the substrate. it can. '

The term “nucleic acid synthase” means a polynucleotide chain synthase, for example, a DNA synthase (eg, a DNA-dependent DNA synthase, an RNA-dependent DNA synthase), an RNA synthase (eg, a DNA-dependent RNA) Synthase, RNA-dependent RNA synthase, etc.). In the present invention, not only full-length nucleic acid synthases but also deletions, substitutions and additions of nucleic acid synthases having enzyme activity can be used. For example, a recombinant nucleic acid synthase can be used. Also, in the reaction solution Alternatively, a precursor of an enzyme that becomes an enzyme may be used.

As the nucleic acid synthase, viral RNA-dependent RNA synthase is preferable, and HCV RNA-dependent RNA synthase is more preferable.

• The virus is not particularly limited. Examples include HIV (human immunodeficiency virus), HCV (hepatitis C virus), HBV (hepatitis B virus), HTLV (human T-cell leukemia virus), and influenza virus. No. In particular, viruses belonging to the genus Flavivirus are preferred, and HCV is more preferred.

Examples of viral enzymes include HIV reverse transcriptase (RNA-dependent DNA synthase, including those having RNaseH activity), HCV RNA-dependent RNA synthase, HBV DNA synthase (DNA-dependent DNA Those having a synthase activity, an RNA-dependent DNA synthase activity, and RNaseH activity), HTLV reverse transcriptase, and influenza virus RNA-dependent RNA synthase.

The present invention can be used with any nucleic acid synthase. Among the nucleic acid synthases, RNA-dependent RNA synthase (for example, viral RNA-dependent RNA synthase) is preferable, and RNA-dependent RNA synthase of a virus belonging to the Flaviviridae family is particularly preferable. Further, HCV RNA-dependent RNA synthase is preferred.

Viruses having an RNA-dependent RNA synthase include the following: Viruses of the virus family, such as Lamyxoviridae (Paramyxoviridae), Orthomyxoviridae (Orthomyxoviridae), Rhabdoviridae (Rabdoviridae), Arenaviridae (Arenaviridae), and Bunyaviridae Is mentioned. In particular, viruses of the Flaviviridae family are preferred, and HCV is more preferred.

Hepatitis C virus (Hepatitis C virus), yellow fever virus fellow fever virus), tengu iles, JJengue virus), the flavivirus family (Japanese encepnalitis virus), West Nile virus (West Nile fever virus), St. Louis encephalitis virus, St. Louis encephalitis virus, Rocio ゥ inoles (Rocio virus), Murray Valley fl. These viruses have an RNA-dependent RNA synthase. These viruses are also associated with diseases of mammals including humans, and it is useful to find inhibitors of RNA-dependent RNA synthase of these viruses.

-In addition, the nucleic acid synthase includes not only a nucleic acid synthase having a single enzyme activity but also a nucleic acid synthase having a plurality of enzyme activities. For example, examples of the nucleic acid synthetase having a plurality of enzyme activities include HBV DNA synthase (having DNA-dependent DNA synthase activity, RNA-dependent DNA synthase activity, and / or RNaseH activity). Can be

In addition, HIV reverse transcriptase (having RNA-dependent DNA synthase inhibitory activity and RNaseH activity) and the like may be used only in their respective enzymatic activities in the assay of the present invention. A moiety having an enzymatic activity may be used in the present invention.

There are many HCV genotypes, and in the present invention, HCV enzymes having any of these genotypes can be used. Examples of genotypes include genotype la, genotvpe ID, genotvpe lc, genotype 2a, genotype 2b, genotype 2c, genotype 3a, genotype 3b, genotype 4, genotype 5, genotype 6, and the like.

HCV RNA-dependent RNA synthase is encoded in the NS5B (Nonstructural Protein 5B) region of HCV.In the present invention, any enzyme having RNA-dependent RNA synthase activity can be used in the present invention. Can be used. For example, HCV having the above genotype (eg, genotype la, genotvt> e lb, genotype lc, genotype 2a, genotype 2D, genotype 2c, genotype 3a, genotype 3b, genotype 4, genotype 5, genotype 6) Nucleic acid synthase having RNA-dependent RNA synthase activity encoded in the NS5B region can be used.

Activator means a substance that activates an enzymatic reaction. Nucleic acid synthase activity The agent can be used for treating a disease caused by a decrease in the expression level or function of a nucleic acid synthase, and is useful. In addition, when nucleic acid elongation is performed using a nucleic acid synthase, it can be used as a reagent for efficiently performing an enzymatic reaction.

In addition, an inhibitor means a substance that inhibits an enzymatic reaction. In particular, inhibitors of viral nucleic acid synthase can be used for treating diseases caused by viruses and are useful. Inhibitors, e.g., IC ₅ Q value is 1 0 0 M or less, in particular, 1 0 M or less, more, 1 / M the following materials are preferred.

The present invention relates to a pre-incubation assay, and is particularly useful in an assay of an enormous number of compounds, particularly in high-throughput screening (HTS). By implementing the present invention, noise (false positives) can be reduced, and a lead compound can be effectively selected.

That is, the nucleic acid synthase can be activated or inhibited by contacting the nucleic acid synthase with the substance obtained by the Atsey method of the present invention. When the nucleic acid synthetase is a viral nucleic acid synthase, virus replication can be activated or inhibited by contacting the virus with a substance obtained by the Atsey method of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a graph showing the relationship between the preincubation time and the enzyme activity of HCV RNA-dependent RNA synthetase when the enzyme reaction was performed for 3 minutes. In the figure, * indicates the case where an oligo RNA that forms a hairpin loop at the 3 ′ end was used as a template.

Figure 2: Diagram showing the relationship between the presence or absence of preincubation and the enzyme reaction temperature. In the figure, ▲ indicates enzymatic reaction at 37 ° C after pre-incubation, · indicates enzymatic reaction at 25 ° C after pre-incubation, Δ indicates enzymatic reaction at 37 ° C without pre-incubation If the reaction was performed, o is the UM in the HCV RNA-dependent RNA synthase reaction when the enzyme reaction was performed at 25 ° C without pre-incubation. The relationship between the incorporation of P into the reaction product and the enzyme reaction time is shown.

Fig. 3 shows the results of random screening performed by the conventional method (using a template and primers).

Figure 4: Random screening performed by the pre-incubation method (using template and primers). BEST MODE FOR CARRYING OUT THE INVENTION

The “preincubation assay method using a single-stranded nucleic acid oligomer” of the present invention will be described below while comparing with a conventional method. The nucleic acid synthase will be described using HCV RNA-dependent RNA synthase, but is not particularly limited to this enzyme. The present invention can be carried out irrespective of the type of nucleic acid synthase. Particularly preferred are RNA synthase, furthermore, RNA-dependent RNA synthase, and HCV RNA-dependent RNA synthase.

Conventional method

HCV RNA-dependent RNA synthase, oligo RNA as a template (oligo RNA means a single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3 'end), RNA triphosphate as a substrate, Simultaneously mix Mg ion (Mg ²⁺ ), Mn ion (Mn ²⁺ ), and the test substance, start the enzyme reaction, and measure the incorporation of the substrate into the reaction product. By comparing with the value in the absence of the test substance, it is possible to know whether the test substance activates or inhibits HCV RNA-dependent RNA synthase. Pre-incubation atsay method

HCV RNA-dependent RNA synthase, oligo RNA as a template (oligo RNA means a single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3 'end), Mg ion (Mg ²⁺ ), Pre-incubation of Mn ions (Mn ²⁺ ) at about 25 ° C for about 1 to 2 hours, then adding RNA triphosphate as a substrate and a test substance to start the enzyme reaction, This is a method for measuring the incorporation of a substrate into a reaction product. As in the above conventional method, the percentage of inhibition was calculated by comparing with the value in the absence of the test substance. And select inhibitors. The specific procedure may be referred to Example 1 described later.

The “preincubation assay using a single-stranded nucleic acid oligomer” of the present invention is superior to the conventional method in the following points.

① Increase in enzyme activity

The preincubation time was compared with the enzyme activity. The specific procedure is described in Test Example 1. The enzyme activity was determined by measuring the uptake of peridine monophosphate (UMP) into the reaction product. The results are shown in Figure 1. It can be seen that the enzyme activity gradually increases until the pre-incubation time reaches about 90 minutes, and the difference in enzyme activity from the conventional method gradually increases. In addition, it can be seen that about 90 minutes of preincubation shows about eight times the enzymatic activity as compared to the conventional method. This facilitates detection of the measurement results, reduces errors in the inhibition rate, and is useful in screening inhibitors. In addition, when the preincubation time was further increased, it was found that the enzyme activity was almost the same as when preincubation was performed for about 90 minutes.

② Increased thermal stability

The relationship between the presence or absence of preincubation and the enzyme reaction temperature was examined, and the results are shown in FIG. As shown in Fig. 2, in the conventional method, when the enzyme reaction is performed at about 37 ° C, the enzyme is inactivated when the reaction time exceeds 2 hours, and it is not possible to measure an accurate enzyme activity value . In contrast, in the preincubation method, the enzymatic reaction proceeds favorably even at about 37 ° C, and the assay can be performed under conditions closer to the temperature in the living body. Also, it can be seen that the enzyme activity is higher when the enzyme reaction is performed at about 37 ° C than when the enzyme reaction is performed at about 25 ° C. Therefore, in the preincubation assay, it is possible to perform the assay at a temperature close to the biological temperature of about 37 ° C.

③Reduced false positives (noise)

Pre-incubation access method reduces noise in the access results Can be made. This is described below using Tables 1 and 2.

Tables 1 and 2 use 3'-deoxyperidine triphosphate (3'-dUTP) as a competitive inhibitor. 3'-dUTP is a compound capable of inhibiting the enzymatic reaction of HCV RNA-dependent RNA synthase by competing with the substrate in the nucleic acid synthesis reaction and being incorporated into the reaction product to stop elongation of the RNA chain. Compounds A and B are compounds determined to be positive by the conventional method. That is, these compounds inhibit HCV RNA-dependent RNA synthase by about 60% to 80% at a concentration of 20〃Zm 1 in the conventional method without precipitation. However, their inhibition rates fall to about 20% to 30% according to the preincubation assay method. On the other hand, Compound C (0.032 g / ml) and 3'-dUTP (0.08 / M) do not show a decrease in the inhibitory effect in the pre-cubation assay. The structures of Compound A, Compound B and Compound C are shown below.

Compound C

Example

Preparation of HCV RNA-dependent RNA synthase

A HCV RNA-dependent RNA synthase cDNA clone was prepared from HCV-infected plasma, and HCV RNA-dependent RNA synthase was expressed using insect cells or Escherichia coli based on this cDNA clone. HCV RNA-dependent RNA synthase in cell extracts can be analyzed by anion exchange chromatography, heparin affinity chromatography, poly U affinity chromatography, cation exchange chromatography, gel filtration chromatography. Purification was carried out using mouth chromatography. Purification was carried out at a low temperature conditions at about 4 ° C, Echirenjiamin tetraacetate as enzyme solution (I mM), Jichiosurei Torr (1 0 mM), preparative squirrel buffer containing glycerol (2 0%) _(P H7 5) was used. Purified enzymes were stored at 4 ° C or -20 ° C until used in Atsushi.

Example 1

HCV RNA-dependent RNA synthase Preincubation assay

(1) Buffer I containing Tris-hydrochloric acid (100 mM, pH 7.5), magnesium chloride (25 mM), manganese chloride (2.5 mM), dithiothreitol (5 mM), and potassium chloride (125 mM) I * ¹ ) (10.5 1) in polypropylene Oligo RNA Note ²⁾ ice solution (100 µg / ml, 10.5〃1) and water (18.5〃1) were added thereto, and mixed gently.

② Next, HCV RNA-dependent RNA synthase solution (10.5〃1) was added, and preincubation was performed at 25 ° C for 60 minutes.

To ③ dimethyl sulfoxide or 9 6-well microtiter evening first plate water dissolved in the test substance (per well 5.26〃 1) had been dispensed, ② plain-incubated for business purposes has been solution ⁽⁵ 0 1) was added Was.

④ Further, preparative squirrel hydrochloride (100 mM, pH 7.5) and magnesium chloride (25 mM), a salt of manganese (2.5 mM), Jichiosurei Torr (5 mM), Kariumu chloride ^{(l25mM), UTP a 3)} (ΐΛίΜ, including ^4.9 〃Ci ³ mixture of H-UTP) containing buffer II (10.6 1) and water ^{^{(3 9 · 4 Ζ 1)}} ( added admixed 50〃 1), 2 in 5 ° C 6 0 min enzyme reaction Was done.

(5) When the enzyme reaction had elapsed for 60 minutes, an aqueous solution of disodium ethylenediaminetetraacetate (50 mM, 50、1) was added and mixed to stop the enzyme reaction.

⑥ Next, using a cell harvester, transfer the entire amount of the sample to ジェ onto a filter into which a getylaminoethyl group has been introduced (hereinafter abbreviated as DEAE-filter mat), and then use a sodium phosphate buffer ( (0.25Μ, ρΗ7.0), two 10-second rinses and one 10-second rinse in deionized water.

DE Dry the rinsed DEAE-filter mat at 95 ° C for 15 minutes and remove It was sealed in a sample bag together with Chile (10m 1).

放射 Radioactivity was measured with a scintillation counter.

阻害 The inhibition rate was determined by substituting the measured value of radioactivity (unit: cpm) into the following formula. Inhibition rate = 100 — (measured value in the presence of test substance-measured value in the absence of enzyme)

/ (Measurement value in the absence of test substance-Measurement value in the absence of enzyme) X100 Note 1) All ice used for the experimental materials ① to ⑤ was lost in RNAse enzyme by getyl pyrocarbonate. Active treatment. For treatment with getyl pyrocarbonate, dissolve getyl pyrocarbonate (O.lg) in deionized water (100 ml), leave it at 37 ° C for 24 hours, and then autoclave at 120 ° C for 30 minutes. I went to.

AAAUAU A single-stranded oligonucleotide composed of AUAUAU-3 '. Used as template. The GG at the 5 'end is added to the sequence to stop the nucleic acid synthesis reaction. C Note 3) Perysine triphosphate. Used as substrate.

The inhibition rate of HCV RNA-dependent RNA synthase when compound A, compound B, compound C, and 3'-dUTP are used as test substances is shown below. The concentrations of the test substances used are as follows.

Compound A: 20 g / ml Compound B: 20 g / ml

匕合 D: 0.032 g / ml 3'-dUTP: 0.08 M

table 1

Compound No. (concentration) Inhibition rate

Compound A: 20 ig / ml 32%

Compound B: 20 g / ml 22%

Compound C: 0.032 g / ml 36%

3'-dUTP: 0.08〃 M 75%

Reference example 1

HCV RNA-dependent RNA synthase Atsusei Conventional method

① Tris-HCl (100mM, _P H 7.5) and magnesium chloride (25 mM), manganese chloride (2.5 mM), Jichiosurei Torr (5 m M), containing potassium chloride (125 mM) Buffer solution ^u (10.5 1) into a polypropylene container, add an oligo RNA Note ² ) aqueous solution (100 g / ml, 10.5 1) and water (18.5 1) and mix gently. Ice cooled.

② Next, ice-cooled HCV RNA-dependent RNA synthase solution (10.5〃1) was added and mixed.

(3) The ice-cold mixed solution (2) (50 1) was added to a 96-well mic-mouthed Thai plate, into which a test substance (5.26 当り 1 per well) dissolved in dimethyl sulfoxide or water was dispensed. .

④ Next, Tris-hydrochloric acid (100 mM, pH 7.5), magnesium chloride (25 mM), manganese chloride (2.5 mM), dithiothreitol (5 mM), chloride chloride (l25 mM),

UTP Note ³ ) Add a mixed solution (50〃1) of a buffer solution (10.6〃1) containing (4.9 / M, l Ci ³ H-UTP) and water (39.4〃1), mix and add The enzyme reaction was carried out for 60 minutes.

(4) When the enzyme reaction had elapsed for 60 minutes, an aqueous solution of disodium ethylenediaminetetraacetate (50 mM, 50/1) was added and mixed to stop the enzyme reaction.

⑥ Next, using a cell harvester, transfer the entire amount of the sample to フィ onto a filter into which a getylaminoethyl group has been introduced (hereinafter abbreviated as DEAE-filter mat), and then add sodium phosphate buffer ( (0.25 M, pH 7.0), two 10-second rinses and one 10-second rinse in deionized water.

(4) The rinsed DEAE-Fill mat was dried at 95 ° C for 15 minutes, sealed in a sample bag together with liquid scintillator (10 ml).

放射 Radioactivity was measured with a scintillation counter.

阻害 The inhibition rate was determined by substituting the measured value of radioactivity (unit: cpm) into the following formula. Inhibition rate = 100 "(measured value in the presence of test substance-measured value in the absence of enzyme)

/ (Measured value in the absence of test substance-measured value in the absence of enzyme) XI 00 Note 1) All the water used in the experimental materials ① to で is dimethylethyl pyrocarbonate Inactivation treatment is performed. The treatment with getyl pyrocarbonate is performed by dissolving getyl pyrocarbonate (O.lg) in deionized water (100 ml) and then heating at 37 ° C. It was left for 4 hours and then autoclaved at 120 ° C for 30 minutes.

AAA UAU A single-stranded oligonucleotide composed of AUA UAU-3 '. Used as template. The GG at the 5 'end was added to the sequence to stop the nucleic acid synthesis reaction. Note 3) Perysine triphosphate. Used as substrate.

The inhibition rates of HCV RNA-dependent RNA synthase when compound A, compound B, compound (1-1), and 3′-dUTP are used as test substances are shown below. The concentrations of the test substances used are as follows.

Compound A: 20 ^ g / ml Compound B: 20 / g / ml

Compound C: 0.032 g / ml 3'-dUTP: 0.08 M

Table 2

Compound No. (concentration) Inhibition rate

Compound A: 20 Z g / ml 78%

Compound B: 20 g / ml 7 1%

Compound C: 0.032 Z g / ml 36%

3'-dUTP: 0.08〃 M 6 1%

Test example 1

The effect of preincubation, which is a major operational feature of the present method, on enzyme activity was examined. In the description, the RdRp enzyme or HCV RdRp enzyme means HCV RNA-dependent RNA synthase.

Set the pre-incubation time of the mixture of buffer I, oligo RNA, and recombinant RdRp enzyme at ²⁵ ° C to 0, 15, 30, 45, 60, 90, 120 minutes. The reaction was carried out at 25 ° C for 30 minutes (Figure 1). The solvent used was DMSO instead of the test substance.

When the amount of substrate incorporated into the reaction product at this time was compared with the enzyme activity, the enzyme activity increased as the pre-incubation time was longer, and the enzyme activity almost reached a plateau when the pre-cubation time was 90 minutes or more. It became. The increase in enzyme activity was up to 8.5 times higher than without pre-incubation (0 min). From the above results, it was found that preincubation of buffer I, oligo RNA, and recombinant RdRp enzyme before the enzyme reaction induced an increase in enzyme activity.

Test example 2

Next, the effect of preincubation on the thermostability of the enzyme was examined.

The enzymatic reaction temperature was set at 25 ° C and 37 ° C, and the time course of the enzyme activity of HCV RdRp was measured with and without preincubation (Fig. 2). In the conventional method (without preincubation), the enzyme reaction rate at 37 ° C is stopped after 120 minutes. On the other hand, in the preincubation method, although the enzyme reaction rate tends to decrease at 37 ° C after 60 minutes of enzyme reaction, the amount of enzyme reaction increases, and the amount of enzyme reaction increases from 25 minutes to 30 minutes to 180 minutes. The value was 2.6 to 3.0 times higher than that of the enzyme reaction in C.

From the above results, it was found that the preincubation treatment of buffer I, oligo RNA, and recombinant RdRp enzyme enhances the thermal stability of RdRp in the enzyme reaction. When considering the growth of HCV in vivo, the stability of the RdRp enzyme around 37 ° C is essential. From this, the form of RdRp after the pre-incubation treatment may be close to the form of HCV RdRp in vivo.

Test example 3

A major difference between the method of the present invention and the conventional method in screening for an HCV RNA-dependent RNA synthase inhibitor is the difference in inhibitory activity. Compound A and Compound B showed a significant decrease in inhibitory activity by pre-incubation treatment, but Compound C and 3'-dUTP did not show a decrease in inhibitory activity even after pre-incubation treatment. (Table 1, Table 2).

From the above results, it was found that the pre-incubation treatment of buffer I, poly (A), oligo (U), and the recombinant RdRp enzyme caused a large variation in RdRp inhibitory activity depending on the type of compound.

Reference test example In order to compare the conventional method and the preincubation method, instead of a single-stranded nucleic acid oligomer whose 3 'end can function as its own primer, poly (A) as a template and oligo (U) as a primer ) ₁₂ using, a different number of thousands of compounds in chemical structure as a test substance was carried out random screening. First, the compounds were mixed in 10 samples at a time, and the concentration of the test substance was 0.83 Z g / ml. The conventional method (using poly (A) as template and oligo (U) ₁₂ as primer) was used. The enzyme inhibition rate was determined in accordance with the above. The results are shown in Figure 3.

On the other hand, tens of thousands of compounds are used as test substances, and the compounds are mixed in 5 samples each, and the concentration of the test substance as a mixture is determined

The enzyme inhibition rate was determined according to the pre-incubation assay method of the present invention described in Example 1 above (provided that poly (A) was used as the template and oligo (U) ₁₂ was used as the primer). Figure 4 shows the results. Industrial applicability

It was found that the preincubation atsay method can (1) increase enzyme activity, (2) increase thermal stability, and (3) reduce noise.

Claims

The scope of the claims

1. In the absence of the test substance and the substrate for the nucleic acid synthase, a solution containing the nucleic acid synthase, a single-stranded nucleic acid oligomer whose 3 ′ terminal can function as its own primer, and a metal ion is used. A method of using an activator or an inhibitor of nucleate synthase, which comprises a step of cuvating.

2. The following steps:

(a) In the absence of a test substance and a substrate for a nucleic acid synthase, contains a nucleic acid synthase, a single-stranded nucleic acid oligomer capable of functioning as its own primer and a metal ion, and a metal ion. Incubating the solution,

(b) mixing the incubated solution with a test substance and a substrate for the nucleic acid synthase, bringing the nucleic acid synthase into contact with a test substance and a substrate for the nucleic acid synthase, Measuring the incorporation of the enzyme substrate into the reaction product,

(c) mixing the incubated solution with the nucleic acid synthase substrate, contacting the nucleic acid synthase with the nucleic acid synthase substrate in the absence of a test substance, Measuring the incorporation into the reaction product,

(d) comparing the incorporation of the nucleic acid synthase substrate into the reaction product measured in the steps (b) and (c),

2. The Atsey method according to claim 1, comprising:

3. The Atsey method according to claim 1 or 2, which is an Atsey method for an inhibitor of a viral nucleic acid synthase.

4. The method according to claim 3, wherein the viral nucleic acid synthase is a viral RNA-dependent RNA synthase.

5. The method according to claim 3, wherein the viral RNA-dependent RNA synthase is an HCV RNA-dependent RNA synthase.

6. The assay method according to claim 4, wherein the single-stranded nucleic acid oligomer has a self-complementary nucleic acid sequence at the 3′-terminal portion.

7. The Atsey method according to claim 6, wherein the single-stranded nucleic acid oligomer has a self-complementary nucleic acid sequence at the 3 ′ terminal portion of poly (A).

8. The method according to claim 7, wherein the substrate is ³ H-UTP or ³² P-UTP ₍ 9. The method according to claim 1, wherein the metal ion is Mg ²⁺ or Mn ²⁺ . Atsey method according to any one of paragraphs 8 to 9.

10. The method according to any one of claims 1 to 9, wherein the incubation is performed for 0.5 hours or more.

11. The Atsey method according to any one of claims 1 to 10, wherein the incubation is performed at 20 ° C to 40 ° C.

12. A method for inhibiting a viral nucleic acid synthase by contacting the substance obtained by the Atsey method according to claim 3 with a viral nucleic acid synthase.

13. A method for inhibiting the replication of a virus by bringing the virus into contact with the substance obtained by the Atsey method according to claim 3.

14. The method according to claim 12 or 13, wherein the virus is a virus belonging to the Flaviviridae family.

15. The method according to claim 12 or 13, wherein said virus is HCV.

16. An activator or an inhibitor of a nucleic acid synthase obtained by the Atsey method according to any one of claims 1 to 11.

17. A pharmaceutical composition comprising, as an active ingredient, an activator or an inhibitor of a nucleic acid synthase obtained by the Atsey method according to any one of claims 1 to 11.