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WO2003031645A1 - Procede de dosage de preincubation utilisant un oligomere d'acide nucleique monocatenaire - Google Patents

Procede de dosage de preincubation utilisant un oligomere d'acide nucleique monocatenaire Download PDF

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Publication number
WO2003031645A1
WO2003031645A1 PCT/JP2002/010029 JP0210029W WO03031645A1 WO 2003031645 A1 WO2003031645 A1 WO 2003031645A1 JP 0210029 W JP0210029 W JP 0210029W WO 03031645 A1 WO03031645 A1 WO 03031645A1
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Prior art keywords
nucleic acid
synthase
acid synthase
substrate
rna
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PCT/JP2002/010029
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English (en)
Japanese (ja)
Inventor
Kenji Abe
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Shionogi & Co., Ltd.
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Priority to JP2003534615A priority Critical patent/JP4199115B2/ja
Publication of WO2003031645A1 publication Critical patent/WO2003031645A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • 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.
  • 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
  • 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
  • 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).
  • 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.
  • the test substance may be based on the structure or properties of the test substance.
  • Atsushi 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.
  • HTS high-throughput screening
  • Patent Document 1 describes the possibility of the Atsey method of HCV without adding a primer, but does not disclose preincubation.
  • 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.
  • 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.
  • 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
  • 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
  • the viral nucleic acid synthase is a viral RNA-dependent RNA synthase.
  • the viral RNA-dependent RNA synthase is an HCV RNA-dependent RNA synthase.
  • 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).
  • the present invention relates to a technique relating to an activator or an activator of a nucleic acid synthase.
  • 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.
  • nucleic acid synthase 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.
  • the nucleic acid synthase 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 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 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.
  • 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.
  • what is essential for an enzymatic reaction other than the substrate of the nucleic acid synthase eg, metal ion
  • 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
  • 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.
  • the complementary bond in the hybridizing part is a hydrogen bond between G and C.
  • 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.
  • those having an AU repeating sequence at the 3 ′ terminal are preferable.
  • Examples of the single-stranded nucleic acid oligomer include the following.
  • poly (dA), poly (dT), poly (dG) ⁇ poly (dC) ⁇ poly (A), poly (U), poly (G) ⁇ poly (C) self-complementary nucleic acid sequence at the 3 ′ end A single-stranded nucleic acid oligomer having
  • 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 have oligo (U), oligo (A), Single-stranded nucleic acid oligomers with oligo (C), oligo (G), etc. (eg, for DNA-dependent RNA synthase),
  • 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.
  • homopolymers such as poly (A), poly (U), poly (G), and pol C), respectively.
  • other single-stranded nucleic acid oligomers for example, in the case of RNA-dependent DNA synthase
  • a single-stranded DNA oligomer for example, DNA
  • 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).
  • a homopolymer such as poly (dA), poly (dT), poly (dG), or poly (dC).
  • 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) ,
  • RNA-dependent RNA synthase Poly (dA), poly (dT) s Poly (dG), poly (dC), etc.
  • Single-stranded nucleic acid oligomer eg, DNA Dependent RNA 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,
  • 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.
  • a heteropolymer for example, a naturally occurring nucleic acid polymer or a synthesized nucleic acid polymer
  • the nucleic acid oligomer of the strand for example, a naturally occurring nucleic acid polymer or a synthesized nucleic acid polymer
  • 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.
  • nucleic acid may be added to the 5 ′ terminal portion of the homopolymer.
  • a single-stranded nucleic acid oligomer having an AU repeating sequence at the 3 ′ terminal is preferable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount of the labeled nucleic acid synthase substrate incorporated into the reaction product may be measured.
  • 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).
  • SPA Scintillation proximity assay
  • 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).
  • the substrate for the nucleic acid synthase examples include dATP, dTTP, dGTP, dCTP, ATP, UTP, GTP, and CTP.
  • 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 ⁇ 32 P- dTTP ⁇ 32 P-dGTP ⁇ 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.
  • poly (dA), poly (dT), poly (dG), poly (dC), poly (A) ⁇ poly (U) ⁇ poly (G ) And poly (C), dTTP, dATP, dCTP, dGTP, UTP, ATP, CTP, and GTP may be used as substrates.
  • 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
  • a heteropolymer for example, a naturally occurring nucleic acid polymer or a synthesized nucleic acid polymer
  • a strand nucleic acid oligomer 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.
  • ⁇ -UTP or 32 P-UTP may be used as the substrate. it can. '
  • 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.).
  • a DNA synthase eg, a DNA-dependent DNA synthase, an RNA-dependent DNA synthase
  • RNA synthase eg, a DNA-dependent RNA Synthase
  • RNA-dependent RNA synthase eg. RNA-dependent RNA synthase, etc.
  • not only full-length nucleic acid synthases but also deletions, substitutions and additions of nucleic acid synthases having enzyme activity can be used.
  • a recombinant nucleic acid synthase can be used.
  • 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.
  • 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.
  • 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 HTLV reverse transcriptase
  • influenza virus RNA-dependent RNA synthase influenza virus RNA-dependent RNA synthase.
  • RNA-dependent RNA synthase for example, viral RNA-dependent RNA synthase
  • RNA-dependent RNA synthase of a virus belonging to the Flaviviridae family is particularly preferable.
  • 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.
  • 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
  • St. Louis encephalitis virus St. Louis encephalitis virus
  • Rocio ⁇ 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.
  • 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.
  • 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).
  • HIV reverse transcriptase having RNA-dependent DNA synthase inhibitory activity and RNaseH activity
  • HIV reverse transcriptase having RNA-dependent DNA synthase inhibitory activity and RNaseH activity
  • a moiety having an enzymatic activity may be used in the present invention.
  • HCV enzymes having any of these genotypes can be used.
  • 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.
  • any enzyme having RNA-dependent RNA synthase activity can be used in the present invention.
  • 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.
  • nucleic acid elongation is performed using a nucleic acid synthase, it can be used as a reagent for efficiently performing an enzymatic reaction.
  • an inhibitor means a substance that inhibits an enzymatic reaction.
  • 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).
  • HTS high-throughput screening
  • 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.
  • virus replication can be activated or inhibited by contacting the virus with a substance obtained by the Atsey method of the present invention.
  • 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.
  • * 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.
  • ⁇ indicates enzymatic reaction at 37 ° C after pre-incubation
  • ⁇ indicates enzymatic reaction at 25 ° C after pre-incubation
  • ⁇ 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).
  • 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.
  • 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.
  • Mg ion Mg 2+
  • Mn ion Mn 2+
  • oligo RNA means a single-stranded nucleic acid oligomer having a self-complementary nucleic acid sequence at the 3 'end
  • Mg ion Mg 2+
  • Mn 2+ Pre-incubation of Mn ions 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HCV RNA-dependent RNA synthase solution (10.5 ⁇ 1) was added, and preincubation was performed at 25 ° C for 60 minutes.
  • 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.
  • 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 ⁇ ⁇ 1 per well) dissolved in dimethyl sulfoxide or water was dispensed. .
  • Tris-hydrochloric acid 100 mM, pH 7.5
  • magnesium chloride 25 mM
  • manganese chloride 2.5 mM
  • dithiothreitol 5 mM
  • chloride chloride l25 mM
  • 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.
  • the RdRp enzyme or HCV RdRp enzyme means HCV RNA-dependent RNA synthase.
  • 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).
  • the enzyme reaction rate at 37 ° C is stopped after 120 minutes.
  • 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.
  • 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).
  • the preincubation atsay method can (1) increase enzyme activity, (2) increase thermal stability, and (3) reduce noise.

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Abstract

L'invention concerne un procédé de dosage qui consiste à laisser incuber une solution contenant des enzymes en l'absence d'une substance d'essai et d'un substrat de l'enzyme afin de réduire les probabilités de rapport faussement positif dans le criblage aléatoire.
PCT/JP2002/010029 2001-10-01 2002-09-27 Procede de dosage de preincubation utilisant un oligomere d'acide nucleique monocatenaire WO2003031645A1 (fr)

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JP2003534615A JP4199115B2 (ja) 2001-10-01 2002-09-27 一本鎖の核酸オリゴマーを使用したプレインキュベーションアッセイ法

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JP2001-305558 2001-10-01
JP2001305558 2001-10-01

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WO2003031645A1 true WO2003031645A1 (fr) 2003-04-17

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077371A1 (fr) * 2000-04-07 2001-10-18 Shionogi & Co., Ltd. Procede de dosage de preincubation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077371A1 (fr) * 2000-04-07 2001-10-18 Shionogi & Co., Ltd. Procede de dosage de preincubation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TOMEI L. ET AL.: "Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking of the C-terminal hydrophobic sequence", JOURNAL OF GENERAL VIROLOGY, vol. 81, March 2000 (2000-03-01), pages 759 - 767, XP002907465 *
ZHONG W. ET AL.: "Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase", JOURNAL OF VIROLOGY, vol. 74, no. 19, October 2000 (2000-10-01), pages 9134 - 9143, XP002957697 *

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JPWO2003031645A1 (ja) 2005-01-27

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