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WO2009150845A1 - Procédé de sélection d'un agent antiviral le mieux approprié - Google Patents

Procédé de sélection d'un agent antiviral le mieux approprié Download PDF

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Publication number
WO2009150845A1
WO2009150845A1 PCT/JP2009/002653 JP2009002653W WO2009150845A1 WO 2009150845 A1 WO2009150845 A1 WO 2009150845A1 JP 2009002653 W JP2009002653 W JP 2009002653W WO 2009150845 A1 WO2009150845 A1 WO 2009150845A1
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antiviral agent
patient
target protein
selecting
derived
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PCT/JP2009/002653
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English (en)
Japanese (ja)
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弥重太 遠藤
澤崎達也
山本直樹
梁明秀
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株式会社セルフリーサイエンス
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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a method for selecting an optimal antiviral agent. More specifically, the optimal antiviral agent in each patient is simple, quick and reliable by detecting the inhibitory effect of the antiviral agent on the virus-derived target protein expressed using the cell-free protein synthesis system. Relates to the selection method.
  • This application which is incorporated herein by reference, claims priority from Japanese Patent Application No. 2008-154747.
  • HIV Human immunodeficiency virus
  • UNAIDS / WHO AIDS epidemic update
  • the number of infected people and the number of AIDS patients are small, so the educational effect is not sufficiently improved, and in the developed countries of the world, the increase in the number of infected people is still stopped. Absent.
  • HAART therapy has improved the patient's prognosis, the number of long-term patients has increased, and new complications such as problems with resistance to existing drugs and immune reconstitution syndrome have emerged.
  • HAART therapy a intensive therapy using a combination of two or four reverse transcriptase inhibitors such as azidothymidine (AZT) and protease inhibitors
  • ZHT azidothymidine
  • protease inhibitors such as azidothymidine (AZT) and protease inhibitors
  • the therapeutic effect is limited due to the emergence of resistant viruses and side effects.
  • resistant viruses to administered drugs can easily appear, and multiple drugs based on the same mechanism of action can be selected by careless selection of drugs. It has been reported many times that viruses resistant to drugs have emerged, resulting in difficulty in subsequent treatment.
  • anti-HIV-1 therapy it is important to detect a resistant virus quickly and accurately and select the most effective drug for the virus of each individual patient.
  • Genotyping and Phenotyping drug resistance test methods have been established as criteria for effective drug selection in HIV treatment using anti-HIV-1 drugs (Non-patent Document 1).
  • Genotyping performs base sequence analysis by amplification of the HIV-1 pol gene region. However, it has often been reported that the drug resistance resulting from amino acid mutations in the gene encoding the reverse transcriptase or protease of the viral genome in the specimen does not match the pharmacological drug resistance results of the patient.
  • Phenotyping is a method that reflects the actual drug resistance of viruses. However, Phenotyping is not suitable for processing a large number of samples because it may take several months to obtain a result and requires labor and cost.
  • the anti-HIV-1 drug is mentioned as an example of the antiviral drug. Furthermore, in recent years, drug resistance has become a problem for antiviral agents (neuraminidase inhibitors) against influenza viruses.
  • HCV hepatitis C virus
  • a treatment method is selected depending on the amount and genotype of hepatitis C virus in the patient. It is known that the therapeutic effect of interferon varies depending on the genotype. Therefore, it is necessary to quickly and accurately measure the patient-specific HCV infection status.
  • Patent Document 1 JP 2002-518065 discloses “a method for detecting a nucleotide sequence variation of an HIV protease gene by amplifying a relevant portion of the HIV protease gene in a sample using a characteristic probe”. ing. However, the HIV protease activity based on the nucleotide sequence variation in the sample has not been investigated.
  • Patent Document 2 JP-A-2002-191399 (Patent Document 2) states that “an animal cell capable of expressing a secreted reporter protein by HIV-1 infection is brought into contact with a sample containing HIV-1 in the presence of a test drug. Discloses a method for testing HIV drug resistance, which comprises detecting a reporter protein secreted into the culture supernatant by infection. However, the method for detecting HIV drug resistance described in this publication is clearly different from the method of the present invention because it uses HIV-1-infected cells.
  • JP 2002-508158 discloses "a method for detecting drug resistance of HIV, characterized by identifying a colony containing a drug resistant target protein based on the reporter mechanism of the reporter protein".
  • the method for detecting HIV drug resistance described in this publication is clearly different from the method of the present invention because it uses a bacterial reporter system.
  • Special table 2002-518065 gazette JP 2002-191399 A Special Table 2002-508158 Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents (Jan. 29, 2008: Developed by the DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents)
  • an object of the present invention is to provide an optimal method for selecting an antiviral agent for each patient in a simple, rapid and reliable manner.
  • the present inventors have used a primer that specifically recognizes a sequence not involved in the physiological activity of a virus-derived target protein gene, and has been expressed in a cell-free protein synthesis system.
  • a primer that specifically recognizes a sequence not involved in the physiological activity of a virus-derived target protein gene, and has been expressed in a cell-free protein synthesis system.
  • a method for selecting an antiviral agent suitable for a patient comprising the following steps: 1) A gene amplification solution containing at least one 5 ′ primer and one 3 ′ primer that specifically hybridizes to a sequence not involved in the physiological activity of a target protein gene derived from a virus is brought into contact with a patient-derived sample.
  • Amplifying the target protein gene derived from the virus 2) A step of bringing the target protein gene amplified in 1) into contact with a transcription solution as it is or after purification to construct a translation template for the target protein derived from the virus, 3) A step of expressing the target protein derived from the virus by bringing the translation template of the target protein constructed in 2) into contact with a cell-free protein synthesis system as it is or after purification, 4) The target protein expressed in 3) and one or more antiviral agents are contacted simultaneously or separately to detect the inhibitory effect of the one or more antiviral agents on the target protein derived from the virus.
  • Process, 5) A step of selecting an antiviral agent suitable for the patient based on the detection result of 4), And a method for selecting an antiviral agent suitable for a patient.
  • 2. The method for selecting an antiviral agent suitable for the patient according to item 1 above, wherein the cell-free protein synthesis system is a wheat germ cell-free protein synthesis system. 3.
  • 3. The method for selecting an antiviral agent suitable for the patient according to item 1 or 2, wherein the target protein derived from the virus is a protease, and the antiviral agent is a protease inhibitor. 4).
  • 4. The method for selecting an antiviral agent suitable for the patient according to any one of 1 to 3 above, wherein the step 4) uses ALPHA (amplified luminescence proximity homogeneous assay).
  • the method for selecting an antiviral agent suitable for the patient according to item 4 above, wherein the step 4) includes the following steps: (1) contacting a biotinylated substrate sequence, an acceptor bead capable of directly or indirectly recognizing the biotinylated substrate sequence, a donor bead bound with streptavidin, and the protease and protease inhibitor expressed in the above 3), (2) The inhibitory effect of the protease inhibitor on the protease is detected by a signal change caused by the proximity of the acceptor bead and the donor bead. 6). 6.
  • the kit of claim 9 comprising at least: (1) at least one 5 ′ primer and one 3 ′ primer that specifically hybridize to a sequence other than the sequence portion involved in the physiological activity of the target protein gene derived from the virus, (2) Extract for cell-free protein synthesis derived from eukaryotes. 11.
  • a kit for performing a method of selecting an HIV protease inhibitor suitable for a patient including: (1) primers of SEQ ID NOs: 1 to 5, (2) An extract for cell-free protein synthesis derived from wheat germ.
  • the present invention provides a method for selecting an optimal antiviral agent that is simple, quick and reliable for each patient.
  • Promoter-splitting primer for amplifying HIV protease DNA A: Schematic diagram of HIV protease DNA B: Schematic diagram of biotinylated substrate sequence (“ ⁇ ” in the figure indicates the cleavage site) Detection of HIV protease mutations in vitro using ALPHA Results of examination of optimum base mass Results of examination of optimal amount of protease Results of study on optimal amount of antiviral agent Results of selection of HIV protease inhibitors suitable for patients Results of inhibition of reverse transcriptase activity by non-nucleic acid reverse transcriptase inhibitors Results of inhibition of reverse transcriptase activity by reverse transcriptase inhibitors
  • the method for selecting an antiviral agent suitable for the patient of the present invention mainly has the following characteristics.
  • a virus-derived target protein gene is amplified using a primer that specifically recognizes a sequence not involved in the physiological activity of the virus-derived target protein gene. Thereby, it is possible to detect mutations of all amino acids involved in physiological activity.
  • the target protein gene is synthesized as a transcription template without being introduced into a vector, and is further used as a translation template to express the target protein by a cell-free protein synthesis system. Thereby, since it is not necessary to introduce
  • the inhibitory effect of the antiviral agent on the target protein is preferably detected by a homogeneous assay.
  • an antiviral agent can be selected simply, quickly and easily.
  • the antiviral agent is a nucleic acid reverse transcriptase inhibitor and the target protein is a reverse transcriptase
  • the antiviral agent can be selected regardless of the type of the nucleic acid reverse transcriptase inhibitor. .
  • the “antiviral agent” of the present invention means an agent for treating a disease caused by the presence of a virus in a patient.
  • nucleic acid reverse transcriptase inhibitors for example, nucleic acid reverse transcriptase inhibitors, non-nucleic acid reverse transcriptase inhibitors, HIV-1 infection treatment drugs, protease inhibitors, neuraminidase inhibitors, influenza treatment drugs, HBV treatment drugs, HCV treatment drugs, etc.
  • the “antiviral agent” of the present invention also covers candidate antiviral agents in the research stage or clinical trial stage that are not marketed at this stage.
  • target protein means a virus-derived protein involved in a disease in a patient.
  • virus-derived protein involved in a disease in a patient.
  • HIV protease in HIV-1 infection HIV reverse transcriptase, neuraminidase in influenza virus infection
  • RNA-dependent RNA polymerase (HCV RdRp) in hepatitis C virus infection
  • HBV polymerase (HBV pol) in hepatitis B virus infection, etc.
  • sequence not involved in physiological activity means a sequence other than the base sequence necessary for the action specific to the target protein (for example, protease reaction, reverse transcriptase reaction, polymerase reaction, etc.).
  • sample of the present invention means a biological material obtained directly from a human (or animal) infected with each virus or after culturing.
  • Biological materials include, for example, all types of sputum, bronchial lavage, blood (plasma), skin tissue, biopsy, semen, lymphocyte blood culture, colony, liquid culture, fecal sample, urine, etc. May be. Particularly preferred is blood (plasma).
  • the patient sample means a fraction containing virus RNA extracted from the patient-derived sample (either in solution or in solid state).
  • Contact in each step of the present invention means both of adding the A solution to the B solution or adding the B solution to the A solution.
  • the gene amplification solution may be added to the patient-derived sample, or the patient-derived sample may be added to the gene amplification solution.
  • the “primer” of the present invention is complementary to a nucleic acid chain to be amplified (a gene encoding a target protein derived from a virus).
  • the primer is about 3-100 nucleotides in length, preferably 5-70, more preferably 10-50.
  • the primer of the present invention is characterized in that it specifically hybridizes to a sequence other than the sequence part involved in the physiological activity of the target protein gene. As a result, all amino acid mutations involved in the physiological activity of the target protein can be detected.
  • a primer of the present invention in order to hybridize to a sequence other than the sequence part involved in physiological activity more specifically, a promoter-divided type invented by Sawazaki, one of the inventors of the present application, known per se known nested PCR. It is preferred to use primers (see: WO02 / 018586).
  • the “promoter-splitting primer” means “two types of primers (5 ′ end of the promoter) that satisfy the condition that transcription from DNA constructed using only one type of primer as the 5′-side primer does not occur.
  • a polynucleotide having a sequence complementary to a base sequence including at least a part of a promoter functional site and a polynucleotide having a sequence complementary to a base sequence including at least a part of an RNA polymerase recognition site from the 3 ′ end of the promoter) 3'-side primer "(see: Fig. 1).
  • the individual length and sequence depend on primer usage conditions such as the complexity and temperature of the required gene and ionic strength.
  • the “gene amplification solution” of the present invention means a solution containing RNA of a target protein derived from a virus in a sample as DNA by reverse transcription and further containing an essential component capable of amplifying the DNA.
  • the gene amplification solution is a solution for performing PCR, reverse transcriptase, dNTPs, polymerase, and the primers described in the above paragraph are included.
  • NASBA method Nucleic Acid Sequence Based Amplification method, Nature, 350, 91-92, 1991, Patent No. 2648802 and Patent No.
  • the steps can be greatly reduced, and a large amount of transcription template can be synthesized in a short time with a small number of steps. Become. That is, since a step for preparing a plasmid incorporating a DNA encoding a target protein is not required, the time required for ultracentrifugation for plasmid purification can be shortened.
  • the “transcription solution” of the present invention means a solution containing essential components for using the amplified target protein gene (DNA) as a translation template.
  • it is a solution containing components necessary for a transcription reaction such as RNA polymerase (for example, SP6 RNA polymerase) and a substrate for RNA synthesis (four kinds of ribonucleoside triphosphates).
  • the transcription reaction is carried out by incubating the solution at about 20 ° C. to about 60 ° C., preferably about 30 ° C. to about 42 ° C., for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours. Is called.
  • a transcription solution after transcription containing a target protein translation template can be easily expressed by adding it to the following cell-free protein synthesis system without purification. Thereby, a large amount of target proteins can be prepared easily and rapidly.
  • the “cell-free protein synthesis system” of the present invention preferably means a system in which an extract for cell-free protein synthesis using eukaryotic wheat germ or the like is used, and this is used to express a target protein.
  • Examples of commercially available extracts for protein synthesis include Rabbit Reticulocyte Lysate System (Promega) derived from rabbit reticulocytes and Wheat Germ Expression Premium Kit (WEPRO registered trademark , Cell Free Science Co., Ltd.) derived from wheat germ.
  • the best extract to be applied to the present invention is an extract derived from wheat germ, and an extract from which a metabolite such as glucose that causes inhibition of protein synthesis in the endosperm components and germ tissue cells is further removed.
  • the extract from which the endosperm component has been substantially removed means that the deadenification rate of ribosome is 7% or less, preferably 1% or less. Further preferably, in the cell extract, sugar and phosphorylated saccharide are reduced to 10 mM or less, preferably 6 mM or less (as the glucose concentration in the extract having an absorbance of 200 OD / ml at 260 nm). A method for preparing such an extract is exemplified in WO2005 / 063979 A1.
  • Translation reaction process The amino acid, energy source, various ions, buffer solution, ATP regeneration system, nucleolytic enzyme used as a substrate in the cell extract for protein synthesis to which the translation template of the unpurified or purified target protein obtained as described above is added Add a solution (also called “translation solution”) containing components necessary or suitable for translation reaction, such as inhibitors, reducing agents, polyethylene glycol, folate, antibacterial agents, etc.
  • the translation reaction is performed by incubating for a long time.
  • the substrate amino acids are usually 20 types of L-type amino acids constituting the protein, but analogs and isomers thereof can also be used depending on the purpose.
  • ATP and / or GTP are mentioned as an energy source.
  • Examples of various ions include acetates such as potassium acetate, magnesium acetate, and ammonium acetate, and glutamates.
  • As the buffer Hepes-KOH, Tris-acetic acid or the like is used.
  • the nuclease inhibitor include ribonuclease inhibitors and nuclease inhibitors. Among these, specific examples of ribonuclease inhibitors include human placenta-derived RNase inhibitors (TOYOBO, etc.).
  • Examples of the reducing agent include dithiothreitol.
  • Antibacterial agents include sodium azide, ampicillin and the like. These addition amounts can be appropriately selected within a range that can be usually used in cell-free protein synthesis.
  • the protein synthesis reaction liquid of the following compositions is preferable.
  • a reaction solution [1,000 units / ml ribonuclease inhibitor (RNAsin) (TAKARA) with the following composition and final concentration containing 48% of the wheat germ extract described above (concentration is 200 A 260 nm units / ml).
  • the mode of addition of the translation solution can be appropriately selected according to the translation reaction system to be used.
  • the synthesis system used in the method of the present invention may be any method known per se that can be applied to the cell-free protein synthesis method.
  • the batch method ⁇ Pratt, J. M. et al. , Hames, 179-209, B. D. & Higgins, S. J., eds, IRL Press, Oxford (1984) ⁇ and the multi-layer method (WO02 / 24939).
  • Detecting the inhibitory effect of an antiviral agent on a target protein means that when the target protein gene derived from each patient is mutated, the pharmacological effect of the antiviral agent on the mutated target protein derived therefrom is directly Or it may be indirectly affected, which means confirming it.
  • the target protein is HIV protease and the antiviral agent is an HIV protease inhibitor
  • the target protein is RNA polymerase and the antiviral agent is an RNA polymerase inhibitor
  • the method for detecting the inhibitory effect is to compare the physiological activities of the target protein derived from each patient and the wild-type target protein in the presence of the antiviral agent. Specifically, it is as described below.
  • a patient with a gene amplification solution containing at least one 5 ′ primer and one 3 ′ primer that specifically hybridizes to a sequence other than the sequence part involved in the physiological activity of a protease Directly contacting the sample to amplify the protease gene;
  • the protease gene amplified in (1), as it is or after purification, is brought into contact with a transcription solution to construct a translation template for the protease;
  • a step of expressing the protease by bringing the translation template of the protease constructed in (2) into contact with a cell-free protein synthesis system as it is or after purification, (4) Protease expressed in (3) and one or more protease inhibitors ⁇ Example: RTV (ritonavir), SQV (saquinavir), NFV (nelfinavir), IDV (indina
  • the positions of fluorescence modification at both ends are not close to each other, and the fluorescence intensity increases. That is, a protease having drug resistance does not increase in fluorescence intensity as compared with a protease having no drug resistance.
  • the method for detecting the inhibitory effect of the HCV protease inhibitor on the target protein can be carried out by using a detection kit known per se using the HCV protease expressed in the same manner as described above.
  • the expressed HCV protease and one or more HCV protease inhibitors including candidate HCV protease inhibitors
  • a substrate that is fluorescently modified at both ends eg, EDANA on the N-terminal side and DABCYL on the C-terminal side
  • HCV protease is recognized and cleaved simultaneously or separately
  • the drug resistance of the patient's HCV protease is detected using fluorescence intensity as an index (see: SensoLyte TM 490 HCV Protease Assay Kit, manufacturer: ANA SPEC, Japan distributor: Funakoshi Corporation).
  • a gene comprising at least one 5 ′ primer and one 3 ′ primer that specifically hybridizes to a sequence other than the sequence part involved in the physiological activity of reverse transcriptase (eg, HIV reverse transcriptase) Amplifying the reverse transcriptase gene by contacting the amplification solution directly with the patient sample; (2) a step of bringing the reverse transcriptase gene amplified in (1) into contact with a transcription solution as it is or after purification to construct a translation template for the reverse transcriptase, (3) a step of expressing a reverse transcriptase by bringing the translation template of the reverse transcriptase constructed in (2) into contact with a cell-free protein synthesis system as it is or after purification, (4) The reverse transcriptase expressed in (3) and one or more non-nucleic acid reverse transcriptase inhibitors ⁇ eg, viramune (generic name: nevirap)
  • a gene comprising at least one 5 ′ primer and one 3 ′ primer that specifically hybridizes to a sequence other than the sequence part involved in the physiological activity of reverse transcriptase (eg, HIV reverse transcriptase) Amplifying the reverse transcriptase gene by contacting the amplification solution directly with the patient sample; (2) a step of bringing the reverse transcriptase gene amplified in (1) into contact with a transcription solution as it is or after purification to construct a translation template for the reverse transcriptase, (3) a step of expressing a reverse transcriptase by bringing the translation template of the reverse transcriptase constructed in (2) into contact with a cell-free protein synthesis system as it is or after purification, (4) Reverse transcriptase expressed in (3) and one or more nucleic acid-based reverse transcriptase inhibitors ⁇ eg zidovudine (AZT, ZDV), didanosine
  • reverse transcriptase eg, HIV reverse transcriptase
  • each nucleic acid reverse transcriptase inhibitor used in the present invention preferably uses a triphosphorylated form.
  • nucleic acid-based reverse transcriptase inhibitors become triphosphates, which are active forms, and are incorporated into viral DNA instead of dTTP, dATP, dCTP, dGTP, and the like.
  • DNA chain elongation stops and virus growth is inhibited (see Table 1 below). That is, the template to be used (single-stranded nucleic acid recognized by reverse transcriptase) varies depending on the type of nucleic acid-based reverse transcriptase inhibitor. For this reason, conventionally, templates have been constructed in accordance with the types of various nucleic acid reverse transcriptase inhibitors.
  • a single-stranded nucleic acid (template) that can be commonly used in a method for selecting various nucleic acid reverse transcriptase inhibitors suitable for patients has been newly constructed.
  • the single-stranded nucleic acid is a combination of bases arbitrarily selected from adenine, guanine, cytosine, and uracil.
  • Biotin-dATP and DIG-dCTP are also used in addition to Biotin-dUTP and DIG-dUTP as substrates in order to improve detection sensitivity.
  • a gene amplification solution containing at least one 5 ′ primer and one 3 ′ primer that specifically hybridizes to a sequence other than the sequence part involved in the physiological activity of the neuraminidase gene is directly contacted with a patient sample.
  • Amplifying the neuraminidase gene (2) A step of constructing a neuraminidase translation template by bringing the neuraminidase gene amplified in (1) into contact with a transcription solution as it is or after purification, (3) A step of expressing neuraminidase by contacting the neuraminidase translation template constructed in (2), as it is or after purification, with a cell-free protein synthesis system, (4) Neuraminidase expressed in (3) and one or more neuraminidase inhibitors ⁇ Example: zanamivir (trade name: Relenza), oseltamivir (trade name: Tamiflu) ⁇ and 2 '-(4-methylumbelliferyl) a step of performing enzymatic reaction by contacting ammonium ⁇ -DN-acetylneuraminate simultaneously or separately and detecting drug resistance of neuraminidase in the patient using the enzyme activity as an index;
  • the enzyme reaction can be determined by measuring the fluorescence intensity
  • tritiated UTP tritiated UTP
  • a polymerase without drug resistance has a reduced nucleotide (tritiated UTP) incorporation as compared with a polymerase with drug resistance (see: WO96 / 37619, WO00 / 006529).
  • the detection method includes a homogeneous assay or a heterogeneous assay.
  • the homogeneous assay is more preferable because the washing step can be omitted.
  • the homogeneous assay is selected from any one or more of the following. 1) ALPHA, 2) surface plasmon resonance method, 3) fluorescence correlation analysis method, 4) fluorescence intensity distribution analysis method, 5) FRET, 6) BRET, 7) EFC, 8) FP
  • the heterogeneous assay is selected from any one or more of the following. 1) ELISA, 2) DELFIA, 3) SPA, 4) Flash plate analysis
  • FRET Fluorescence Resonance Energy Transfer
  • ALPHA Analog to Physical Reduction Agent
  • ALPHA Analog to Physical Reduction Agent
  • the method is an analysis method based on the movement of singlet oxygen between a donor bead and an acceptor bead which are brought close to each other. This is because upon excitation at 680 nm, the photosensitizer in the donor bead converts ambient oxygen to singlet state oxygen, which diffuses to a distance of 200 nm.
  • the chemiluminescent group in the acceptor bead transfers energy to the fluorescent acceptor in the bead and subsequently emits about 600 nm light.
  • the acceptor beads are inert substances such as glass, silica gel, and resin, and are carriers for immobilizing the biomolecules.
  • Donor beads are inert substances such as glass, silica gel, and resin, and are carriers for immobilizing streptavidin.
  • SPR Surface plasmon resonance
  • FCS Fluorescence Correlation Spectroscopy
  • FIDA Fluorescence Intensity Distribution Analysis
  • FP Fluorescence Polarization
  • the fluorescent labeling substance binds to a polymer such as an antibody or a receptor
  • the apparent molecular weight increases, so that the molecular motion decreases, and as a result, fluorescence that maintains its polarization (high degree of polarization) is emitted.
  • fluorescence maintaining the polarized light is detected.
  • EFC analysis is based on a processed ⁇ -galactosidase enzyme that consists of two fragments, an enzyme acceptor (EA) and an enzyme donor (ED). When the fragments are separated, ⁇ -galactosidase activity is lost, but when the fragments are combined, they work together (complement) to form an active enzyme.
  • EFC analysis utilizes an ED-analyte conjugate, where the analyte can be recognized by a specific binding protein such as an antibody or receptor. In the absence of a specific binding protein, the ED-analyte conjugate can supplement EA and form active ⁇ -galactosidase, generating a positive luminescent signal.
  • ⁇ BRET Bioluminescent Resonance Energy Transfer
  • the protein fragment complementation method examples include a method using a fluorescent protein monomeric Kusabira-Green (mKG).
  • the fluorescent protein monomeric Kusabira-Green (mKG) is used as follows. A protease expressed in a cell-free protein synthesis system, one or a plurality of protease inhibitors, and a fluorescent protein-modified substrate ⁇ mKG-N terminus-substrate sequence-mKG-C terminus ⁇ are contacted simultaneously or separately to increase the fluorescence intensity. As an index, drug resistance of protease derived from the patient sample is detected (see: CoralHue registered trademark Fluo-chase Kit, Amalgaam product).
  • a biotinylated substrate sequence, acceptor beads capable of directly or indirectly recognizing the biotinylated substrate, donor beads to which streptavidin is bound, expressed HIV protease and HIV protease inhibitor are introduced in vitro.
  • the substrate P2-P7: SEQ ID NO: 12
  • the donor beads and the acceptor beads cannot be brought close to each other, and the signal does not increase.
  • the substrate is not cleaved. Therefore, as shown in the upper diagram of FIG. 3, the donor beads and the acceptor beads are close to each other, and the signal rises.
  • the detection method of a signal is performed by measuring using the fluorescence intensity which an acceptor bead emits, for example.
  • the “method for selecting an antiviral agent suitable for a patient” of the present invention is performed based on the signal detection result. Specifically, the above signal detection is performed using each HIV protease inhibitor, and drug resistance profiling as shown in FIG. 7 is created. Thereby, drug resistance peculiar to each patient can be specified, and one or a plurality of HIV protease inhibitors suitable for each patient can be selected. In FIG. 7, the reaction of only one inhibitor is detected for one sample of each patient. However, naturally, the additive / synergistic effect of a plurality of inhibitors can also be detected for one sample. it can.
  • the “kit for carrying out a method for selecting an antiviral agent suitable for a patient” of the present invention specifically hybridizes at least to a sequence other than a sequence portion involved in the physiological activity of a target protein gene derived from a virus. 5 'primer and one 3' primer, and an extract for cell-free protein synthesis. Furthermore, the “kit for carrying out a method for selecting an HIV protease inhibitor suitable for a patient” includes at least a primer of SEQ ID NOs: 1 to 5 and an extract for synthesis of cell-free protein derived from wheat germ.
  • Patient samples were prepared from blood obtained from patients. Details are as follows. Viral RNA was extracted from blood obtained from an HIV-infected patient, and a buffer was added to prepare a patient sample.
  • the protease gene of the patient sample obtained in Example 1 was amplified. Specifically, a primer specifically recognizing a sequence not involved in protease activity was used. Details are as follows. 5 'primer 1 (S1-plus35-HIVpro: SEQ ID NO: 1), 5' primer 2 (deSP6-E02-S1: SEQ ID NO: 2), 5 'primer 3 (Spu: SEQ ID NO: 3), 3' primer 1 (- 10 ⁇ l gene amplification solution (1 x PCR buffer, 400 nM dNTPs, 0.025 U / ⁇ l thermostable polymerase, reverse) containing 18-HIVpro-sUTR: SEQ ID NO: 4) and 3 ′ primer 2 (sUTR-2nd: SEQ ID NO: 5) was directly introduced into each patient sample obtained in Example 1.
  • S1 is a sequence that can be easily added with various tags to the N-terminus of the target protein when added to the 5 ′ side of the gene for protein synthesis.
  • RT-PCR conditions After 30 minutes at 60 ° C, 30 cycles of (98 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute) were performed.
  • a translation template was prepared from the HIV protease DNA amplified in Example 2. Details are as follows. The solution containing 30 ⁇ l of the amplified HIV protease DNA template obtained in Example 2 was added to the transcription solution ⁇ 30 ⁇ l of 5 ⁇ transcription buffer (400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT). ), 15 ⁇ l of 25 mM 4NTPs, 1.875 ⁇ l of RNAsin (80 Units), 1.875 ⁇ l of SP6 polymerase (80 units), 71.25 ⁇ l water ⁇ , perform a transcription reaction at 37 ° C. for 3 hours, Created.
  • 5 ⁇ transcription buffer 400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT).
  • 15 ⁇ l of 25 mM 4NTPs 1.875 ⁇ l of RNAsin
  • Example 3 (Expression of HIV protease) The translation template obtained in Example 3 was added to a wheat germ cell-free protein synthesis solution (see paragraph “0026”) to express HIV protease from each patient. Details are as follows. The solution containing the translation template (mRNA) obtained in Example 3 was added to a wheat germ cell-free protein synthesis system, and protein synthesis was performed at 26 ° C. for 15 to 20 hours to obtain HIV protease derived from each patient.
  • a biotinylated substrate sequence that was cleaved by the protease activity of HIV protease was generated. Details are as follows.
  • a pEU ⁇ -SP6-E01 (SEQ ID NO: 11) -GST-p2-p7 (SEQ ID NO: 12) -bls ⁇ vector having a nucleotide sequence encoding an amino acid sequence recognized by HIV protease was prepared (see FIG. 2B). ).
  • a transcription template was prepared using PCR using the pEU vector as a template.
  • the transcription template was transferred to a transcription reaction solution [final concentration, 80 mM HEPES-KOH pH 7.8, 16 mM magnesium acetate, 10 mM dithiothreitol, 2 mM spermidine, 2.5 mM 4NTPs (4 types of nucleotide triphosphates), 0.8 U / ⁇ l. RNase inhibitor, 1.6 U / ⁇ l SP6 RNA polymerase] and transcription was performed at 37 ° C. for 3 hours (see Proc Natl Acad Sci USA, 2002, vol 99, p14652-14657: Sawasaki, T et al .). The total amount of the resulting mRNA pellet was added to a wheat germ cell-free protein synthesis solution (see paragraph “0026”), and protein synthesis was performed at 26 ° C. for 15 to 20 hours to obtain a biotinylated substrate sequence.
  • a transcription reaction solution [final concentration, 80 mM HEPES-KOH pH 7.8, 16 mM magnesium a
  • the protein synthesis solution (30 nM: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 ⁇ l) and 3 ⁇ l wild type HIV protease in 15 ⁇ l reaction buffer ⁇ 50 mM Tris-HCl (pH 7.6), 50 mM MgCl 2 , 500 mM CH 3 COOK, 0.1 mM DTT and 1 mg / ml BSA ⁇ were incubated at 23 ° C. for 1 hour to obtain a culture solution.
  • Each culture solution is placed in a 384-well plate ⁇ reaction buffer containing 10 ⁇ l anti-GST antibody, AlphaScreen protein A conjugated Acceptor Bead (PerkinElmer) and streptavidin donor bead (PerkinElmer) in each well: OptiPlate NEW microplate) ⁇ , 23 After 1 hour incubation at 0 ° C., analysis was performed with the Alpha Quest HTS analyzer (the threshold for the positive level of interaction was set to 200 AlphaScreen units). The wild type HIV protease (35-HIVpro (WT) -18) used above was synthesized in the wheat germ cell-free system.
  • WT wild type HIV protease
  • the substrate protein amount was 0.05 to 5.0 ⁇ l, preferably 0.2 to 2.0 ⁇ l. Therefore, in Example 7 below, the substrate protein amount was set to 0.5 ⁇ l.
  • DHFR is amplified using pEU-DHFR (50 ng) as a template and using primers SPu (described above) and AODA2306 (5′-AGCGTCAGACCCCGTAGAAA) to produce a wheat germ cell-free protein synthesis solution (see paragraph “0026” )).
  • Example 9 (Examination of optimal antiviral concentration) The optimal antiviral agent concentration in ALPHA screening in the cell-free protein synthesis system used in Example 9 below was examined. Details are as follows.
  • the protein synthesis solution and 3 ⁇ l wild-type HIV protease were incubated at 23 ° C. for 1 hour in 15 ⁇ l reaction buffer ⁇ 50 mM Tris-HCl (pH 7.6), 50 mM MgCl 2 , 500 mM CH 3 COOK, 0.1 mM DTT and 1 mg / ml BSA ⁇ .
  • Each culture solution is contained in a 384-well plate (each containing a reaction buffer (10 ⁇ l), anti-GST antibody (GE Healthcare), AlphaScreen protein A conjugated Acceptor Bead (PerkinElmer) and streptavidin donor bead (PerkinElmer)) ⁇ I put it in. Furthermore, HIV protease inhibitors (inhibitors A, B, C, D, E, and F) at various concentrations (1, 10, 100, 1000, and 10000 nM) are placed in the reaction buffer and incubated at 23 ° C. for 1 hour. And then analyzed with the Alpha Quest HTS analyzer (the threshold for the positive level of interaction was set to 200 AlphaScreen units).
  • Example 9 the concentration of inhibitor A is 100 nM, the concentration of inhibitor B is 1 ⁇ M, the concentration of inhibitor C is 100 nM, the concentration of inhibitor D is 100 nM, the concentration of inhibitor E is 100 nM, and the concentration of inhibitor F The concentration was set to 1 ⁇ M.
  • DMSO which is not an HIV protease inhibitor, did not detect an inhibitory effect at any concentration.
  • reaction buffer 15 ⁇ l reaction buffer ⁇ 50 mM Tris-HCl (pH 7.6), 50 mM MgCl 2 , 500 mM CH 3 COOK, 0.1 mM DTT and 1 mg / ml BSA containing a biosylated substrate sequence (30 nM: 0.5 ⁇ l) ⁇ At 23 ° C. for 1 hour to obtain a culture solution.
  • Each culture solution is contained in a 384-well plate (with a reaction buffer (10 ⁇ l), anti-GST antibody (GE Healthcare), AlphaScreen protein A conjugated Acceptor Bead (PerkinElmer) and streptavidin donor bead (PerkinElmer)) in each well ⁇ . I put it in.
  • each of post-translational protein synthesis solutions (0001 to 0016) containing HIV protease from 16 HIV-infected patients, and inhibitor A (100 nM), inhibitor B (1 ⁇ M), inhibitor C (100 nM), inhibition Agent D (100 nM), Inhibitor E (100 nM), or Inhibitor F (1 ⁇ M) was placed in a reaction buffer, incubated at 23 ° C. for 1 hour, and then analyzed by Alpha Quest HTS analyzer (positive level of interaction) was set to 200 AlphaScreen units).
  • Reverse transcriptase HIV-RT ⁇ HIV-1 reverse transcriptase (p51, p66) was prepared at a ratio of 1: 1 in the same manner as described in Example 4 ⁇ Reverse transcriptase inhibitor: EFV (0.01nM-1000nM)
  • Reverse transcriptase inhibitor EFV (0.01nM-1000nM
  • Substrate solution DIG-dUTP (75 nM), Biotin-dUTP (75 nM), dTTP (10 ⁇ M) in 50 mM Tris-HCl (pH 7.8)
  • Incubation buffer 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template (Template / primer hybrid): PolyA, oligo (dT) 15 (9A260 / ml)
  • Lysis buffer 50 mM Tris-HCl (pH 7.8), 80 mM potassium chloride, 2.5 mM DTT, 0.75 mM EDTA, 0.5% Triton X-100
  • AlphaScreen TM DIG detection kit No.6760604C (manufactured by PerkinElmer) Beads mix: Streptavidin Donor beads 0.1 ⁇ l, anti-Digoxin / Digoxigenin (DIG) Acceptor Beads 0.1 ⁇ l, 1mg / ml BSA, 1 * PBS
  • the inhibitory effect of various nucleic acid reverse transcriptase inhibitors was confirmed. Details are as follows.
  • the template used is a 6-mer single-stranded nucleic acid (the six nucleic acids are arbitrarily selected from any of adenine, guanine, cytosine, and uracil).
  • the template used as a comparative control is PolyA, oligo (dT) 15 (9A260 / ml).
  • Reverse transcriptase HIV-RT ⁇ HIV-1 reverse transcriptase (p51, p66) was prepared at a ratio of 1: 1 in the same manner as described in Example 4 ⁇
  • Nucleic acid reverse transcriptase inhibitors AZTTP (0,10 nM, 1000 nM), ddCTP (0,10 nM, 1000 nM), 3TCTP (0,10 nM, 1000 nM), Tenofovir.DP (0,10 nM, 1000 nM)
  • Substrate solutions DIG-dUTP (1 ⁇ M), DIG-dCTP (1 ⁇ M), Biotin-dUTP (1 ⁇ M), Biotin-dATP (1 ⁇ M), dTTP (1 ⁇ M), dATP (1 ⁇ M), dCTP (1 ⁇ M), dGTP (1 ⁇ M) in 50 mM Tris-HCl (pH 7.8) Incuba
  • Lysis buffer 50 mM Tris-HCl (pH 7.8), 80 mM potassium chloride, 2.5 mM DTT, 0.75 mM EDTA, 0.5% Triton X-100 Reverse transcriptase assay kit: No.11 468 120 910 (Roche) POD-labeled anti-digoxigenin ⁇ Anti-DIG-POD working dilution (200mU / ml) ⁇ : 50 ⁇ l anti-DIG-POD (10U) and 4.95ml of conjugate dilution buffer ABTS substrate (ABTS substrate solution containing substrate enhancer): 1 mg of the substrate enhancer was dissolved in 1 ml of the substrate solution obtained by dissolving 1 ABTS tablet in 5 ml of the substrate buffer.
  • nucleic acid and non-nucleic acid reverse transcriptase inhibitors suitable for patients can be selected.
  • the present invention provides a simple method for selecting an optimal antiviral agent for each patient.

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Abstract

L'invention concerne un procédé de sélection d'un agent antiviral pour un patient, qui est pratique, rapide, et très fiable. Elle concerne spécifiquement un procédé de sélection de l'agent antiviral qui convient le mieux à un patient. Dans le procédé, une amorce qui peut reconnaître spécifiquement une séquence qui n'est pas impliquée dans l'activité physiologique d'un gène pour une protéine cible dérivée d'un virus est utilisée, et l'effet inhibiteur d'un agent antiviral sur la protéine cible exprimée par un système de synthèse de protéines sans cellule est détecté.
PCT/JP2009/002653 2008-06-12 2009-06-11 Procédé de sélection d'un agent antiviral le mieux approprié WO2009150845A1 (fr)

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CN105759046A (zh) * 2016-04-08 2016-07-13 重庆理工大学 一种新城疫病毒双抗体夹心AlphaLISA检测试剂盒及其检测方法

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WO2013073191A1 (fr) * 2011-11-15 2013-05-23 国立大学法人浜松医科大学 Agent thérapeutique contre accouchement précoce ou avortement utilisant un inhibiteur de l'activateur du plasminogène 1

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Cited By (2)

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WO2013183572A1 (fr) * 2012-06-04 2013-12-12 株式会社セルフリーサイエンス Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase
CN105759046A (zh) * 2016-04-08 2016-07-13 重庆理工大学 一种新城疫病毒双抗体夹心AlphaLISA检测试剂盒及其检测方法

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