WO2009113795A2 - Vector and method for expressing avian influenza virus neuraminidase n1 in e. coli, method of using the vector, and neuraminidase inhibitors - Google Patents

Vector and method for expressing avian influenza virus neuraminidase n1 in e. coli, method of using the vector, and neuraminidase inhibitors Download PDF

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WO2009113795A2
WO2009113795A2 PCT/KR2009/001184 KR2009001184W WO2009113795A2 WO 2009113795 A2 WO2009113795 A2 WO 2009113795A2 KR 2009001184 W KR2009001184 W KR 2009001184W WO 2009113795 A2 WO2009113795 A2 WO 2009113795A2
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nitrophenyl
dihydro
methyl
neuraminidase
amino
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WO2009113795A3 (en
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Doman Kim
Young-Min Kim
Do-Won Kim
Hurng-Chun Lee
Ying-Ta Wu
Vincent Breton
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Industry Foundation Of Chonnam National University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the production of avian influenza (AI) virus neuraminidase N1 in E. coli and inhibitors of the activity of avian influenza virus neuraminidase N1, and more particularly to a vector and method for expressing AI virus neuraminidase N1 in E. coli , a method of using the vector to screen neuraminidase inhibitors, and neuraminidase inhibitors screened by the screening method.
  • AI avian influenza
  • Influenza virus belongs to the Orthomyxoviridae family and causes influenza. Influenza virus is classified into three types: A, B and C. Types A and B often infect humans. Particularly, the serotype of influenza A virus is determined by the difference in the amino acid sequences of the hemagglutinin (H) and neuraminidase (N) protein. There are 15 different hemagglutinin (H) subtypes and 9 different neuraminidase (N) subtypes of influenza A viruses, and a variant of influenza A virus (e.g., H5N1) is determined by the combination of hemagglutinin and neuraminidase subtypes.
  • H hemagglutinin
  • N neuraminidase
  • avian influenza (AI) virus prevailing in Asia is H5N1. It was thought in the past that avian influenza is transmitted between birds and pigs, but it is known that avian influenza also infects humans, because the outbreak of avian influenza virus in Hong Kong in 1997 resulted in 6 deaths among 18 people who came into contact with birds infected with avian influenza. In Hong Kong in 2003, one between 2 people infected with avian influenza died due to H5N1 virus, and in the Netherlands in 2003, one veterinarian among 83 people infected with avian influenza died due to H7N7 virus.
  • AI avian influenza
  • H9N2 virus was also detected in avian influenza which occurred in Hong Kong in 1999. Moreover, a new variant of avian influenza can also emerge according to adaptation to the host's immune system (see Yuen, K.Y. and Wong, S.S.Y., 2005. Hong Kong Med, 11:189-199]).
  • influenza virus The frequent emergence of a new variant of influenza virus lowers the efficacy of vaccines or therapeutic agents, and vaccines cannot protect against new influenza viruses having the ability to infect the human body, such as avian influenza. Thus, the development of various therapeutic agents capable of protecting against influenza viruses is required.
  • Drugs that inhibit the replication of influenza viruses are classified into two categories.
  • the one category includes amantadine, an M2 ion channel inhibitor developed by Dupont in 1964, and it was used before 1980 to treat influenza A virus infections and causes nausea, sleepiness and chronic insomnia (see Long, J.K., et al., 2000. Cleve Clin J. Med., 67:92 ⁇ 95). Since then, rimantadine having reduced side effects compared to amantadine was developed, but it also has a high rate of side effects (see Jefferson et al., 2004. Cochrane Database Syst. Rev., 3:CD001169).
  • the other category includes drugs having neuraminidase inhibitory activity, and zanamivir and oseltamivir have been used to treat influenza viruses (see Dreitlein W.R. et al., 2001. Clin Ther., 23:327-355).
  • Zanamivir is commercially available under the trade name of Relenza from Glaxo Wellcome Inc. and is used as an intranasal powder formulation.
  • Oseltamivir is commercially available under the trade name of Tamiflu from Roche and is used as an oral dosage form in the same manner as amantadine.
  • Relenza can cause difficulty in breathing, and thus can make asthma worse, and Tamiflu causes nausea and vomiting (see McNicholl and McNicholl. 2001, Ann. Pharmacother.
  • Oseltamivir is metabolized to oseltamivir carboxylate in the human body to cause a serious mental disorder (see Izumi Y., et al., 2007, Neuroscience Letters, 426:54-58), and causes environmental pollution and induces spontaneous mutations (see Fick, J., et al., 2007, PLos ONE 2(10):3986; and Singer, A.C., et al., 2007, Env. Health Persp. 115:102-106). Particularly, 8 deaths among patients administered with Tamiflu recently occurred in Japan, and five among them were teenagers.
  • Tamiflu Although teenagers administered with Tamiflu showed abnormal mental states, such as dashing to cars or committing suicide by drowning, the Japanese Ministry of Health, Labor and Welfare prohibited administration of Tamiflu, and the Korean Food and Drug Administration (KFDA) prohibited administration of Tamiflu to teenagers from March 5, 2007.
  • FDA recommended that warning concerning nervous and mental problems (young children or teenagers have hallucinations, wild fancies, offensive personality, suicide ideation, etc. starting immediately from the administration of Tamiflu) be added to Tamiflu.
  • antiviral agents against avian influenza viruses which have reduced toxicity and side effects, need to be developed within a short time. Furthermore, to protect against the frequent emergence of mutants having resistance to inhibitors, the inexpensive and rapid development of inhibitors against avian influenza neuraminidase is urgently required. Also, to protect against the emergence of mutants having drug resistance, various inhibitors are required. For the rapid development of inhibitors, the need to construct recombinant enzymes having mutability exists. For the expression of recombinant neuraminidase, a method of using Baculovirus and insect cells (see Dalakouras, T. et al., 2006.
  • the development of drugs for preventing and treating new diseases and diseases in which mutants frequently occur is performed through in vitro and in vivo experiments for predetermined targets.
  • a general method for developing the drugs is carried out based on a library of specific candidates, and this is a method which can present substances enabling direct treatment of diseases.
  • the time for the development of drugs is most critical.
  • the present inventors have constructed a vector capable of efficiently expressing recombinant neuraminidase N1 in E. coli and succeeded in producing and purifying neuraminidase N1 by culturing E. coli transformed with the vector.
  • the present inventors have used the obtained recombinant neuraminidase to screen compounds showing inhibitory activities superior or equal to those of the existing therapeutic agents, Zanamivir or Oseltamivir.
  • the compounds thus screened can be used as therapeutic agents against H5N1 highly virulent avian influenza virus.
  • an object of the present invention to provide a vector for expressing AI virus neuraminidase N1 in E. coli .
  • Another object of the present invention is to provide E. coli transformed with said vector.
  • Still another object of the present invention is to provide a method of producing AI virus neuraminidase N1 using the transformed E. coli .
  • Still another object of the present invention is to provide a method for screening an inhibitor of AI virus neuraminidase N1, the method comprising the steps of: examining the influence of candidates for AI virus neuraminidase N1 inhibitor on neuraminidase N1 activity in vitro ; and selecting from the candidates a substance inhibiting neuraminidase N1 activity.
  • Yet another object of the present invention is to provide an AI virus neuraminidase inhibitor screened by said method.
  • a first aspect of the present invention relates to a vector for expressing AI virus neuraminidase N1 in E. coli , which comprises an AI virus neuraminidase N1 gene inserted into the multiple cloning site of a pET-23d vector and has a cleavage map of FIG. 2.
  • a second aspect of the present invention relates to E. coli transformed with said expression vector.
  • a third aspect of the present invention relates to a method for producing AI virus neuraminidase N1, which comprises the steps of: culturing the transformed E. coli ; and collecting the culture.
  • a fourth aspect of the present invention relates to a method for screening an AI virus neuraminidase N1 inhibitor, the method comprising the steps of: examining the influence of candidates for AI virus neuraminidase N1 inhibitor on neuraminidase N1 activity in vitro ; and selecting a substance inhibiting neuraminidase N1 activity.
  • a fifth aspect of the present invention relates to a composition for inhibiting AI virus neuraminidase activity, which comprises, as an active ingredient, at least one compound selected from the group consisting of:
  • avian influenza (AI) virus neuraminidase N1 can be expressed in E. coli and rapidly produced at a large amount, and excellent AI neuraminidase N1 inhibitors can be efficiently screened out by measuring and evaluating activities of AI virus neuraminidase inhibitor candidates in vitro using the neuraminidase.
  • AI virus neuraminidase N1 activity were newly screened out in the present invention.
  • FIG. 1 shows a base sequence and a predicted amino acid sequence of the neuraminidase (N1) gene of H5N1 avian influenza virus;
  • FIG. 2 is a cleavage map of a recombinant vector pET-23d-N1 according to the present invention
  • FIG. 3 shows results of Ni-NTA column chromatography of expressed recombinant neuraminidase
  • FIG. 4 shows result of SDS-PAGE of purified recombinant neuraminidase.
  • a recombinant vector pET-23d-N1 comprising an N1 gene was constructed by synthesizing a neuraminidase (N1) gene on the basis of known H5N1 genetic information (see SEQ ID NOS: 1 and 2, and FIG. 1) and inserting the synthesized gene into the multiple cloning site of a pET-23d vector (Novagen, Germany).
  • FIG. 2 is a cleavage map of the constructed pET-23d-N1 vector.
  • Competent E. coli cells for example, E. coli BL21 cells (Novagen, USA) were transformed with the constructed expression vector by, for example, heat shock or electroporation, and the transformed E. coli was cultured.
  • FIG. 3 shows the results of SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) of the purified recombinant neuraminidase.
  • the recombinant neuraminidase produced according to the present invention is easy to purify, because it has six histidine tags attached to the C-terminal thereof.
  • AI virus neuraminidase N1 inhibitors were screened by examining the influence of candidates for AI virus neuraminidase N1 inhibitors on the activity of AI virus neuraminidase N1 in vitro and selecting substances inhibiting the activity of neuraminidase N1.
  • Taiwan Academia Sinica and French CNRS-IN2P3 team completed a work, which would require about 100 years in one computer, within about 6 weeks from April, 2006 using the international supercomputer grid network.
  • a library of virtual compounds 300,000 compounds obtained from ZINC (see Irwin and Shoichet, 2005. J. Chem. Inf.
  • Table 1 shows the name, molecular weight and relative inhibitory activity of each of the screened compounds.
  • relative inhibitory activity is indicated as a percentage relative to the inhibitory activity of purified Tamiflu taken as 100%. Namely, higher percentage values show higher enzyme inhibitory activities.
  • 58 kinds of compounds shown in Table 1 show neuraminidase inhibitory effects superior or equal to that of oseltamivir phosphate, and thus can be advantageously used as agents for preventing or treating highly virulent avian influenza virus infections.
  • the compounds are used as agents for preventing or treating highly virulent avian influenza virus infection, they are preferably administered at a daily dosage of 1 ⁇ g/kg weight to 50 mg/kg weight, but the dosage can be suitably adjusted depending on the age, sex and diet, health condition of a subject, disease severity, administration route, administration time, drug mixing, etc.
  • the compounds of the present invention can be administered according to any conventional method known in the art.
  • the compounds can be administered orally or parenterally, preferably orally.
  • the compounds can be combined with pharmaceutically acceptable carriers, and depending on the purpose of administration, the compounds can be formulated into oral dosage forms such as tablets, hard or soft capsules, granules, chewable tablets, pills, powders, elixirs, suspensions, solutions and syrups, or parenteral dosage forms such as aerosol, sachet, sterile injectable solution, sterile powder, etc.
  • binders such as gum Arabic, corn starch, microcrystalline cellulose or gelatin; excipients such as calcium phosphate or lactose; disintegrating agents such as alginic acid, corn starch or potato starch; lubricants such as magnesium stearate; sweetening agents such as sucrose or saccharin; and flavors such as peppermint, methyl salicylate or fruit flavor can be added.
  • liquid carriers such as polyethylene glycol or fatty oil can be used in addition to the above described components.
  • the injectable solution or suspension for parenteral administration can be administered parenterally, for example, subcutaneously, intravenously, intramuscularly or intraperitoneally.
  • the injectable solution or suspension can be prepared by homogeneously mixing an effective amount of the active ingredient in pharmaceutically acceptable liquid carriers such as water, saline, aqueous dextrose and its related sugar solutions, nonvolatile oil, ethanol, glycerin, polyethylene glycol, propylene glycol, etc.
  • adjuvants such as antibacterial agents, chelating agents, buffers and preservatives can additionally be included.
  • the pharmaceutically acceptable carriers can be any ones, as long as they are pharmaceutically inert, substantially non-toxic and have no adverse effect on the action of the active ingredient.
  • the compounds of the present invention can be used in any forms, including free compounds, pharmaceutically acceptable salts, solvates including hydrates, esters, and stereoisomers, as long as they show the desired neuraminidase inhibitory effect, all of which are intended to fall within the scope of the present invention.
  • the pharmaceutically acceptable salts in the present invention include pharmaceutically acceptable acid addition salts.
  • the pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid or phosphorous acid, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids.
  • inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid or phosphorous acid
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic
  • Example 1 Synthesis of gene and construction of recombinant expression vector
  • a neuraminidase (N1) gene was synthesized on the basis of the genetic information of H5N1[A/Viet Nam/3046/2004(H5N1)] (see SEQ ID NOS: 1 and 2, and FIG. 1).
  • the synthesized gene was introduced into the multiple cloning site of a pET-23d vector (Novagen, Germany), thus constructing a recombinant vector pET-23d-N1 comprising the N1 gene.
  • the pET-23d-N1 has a cleavage map as shown in FIG. 2.
  • the recombinant DNA (5 ⁇ l) obtained in Example 1 was introduced to prepared E. coli BL21 cells (Novagen, USA) and allowed to stand on ice for 1 hour. To insert the DNA, the cells were heat shocked at 42 °C for 90 seconds. Then, 1 ml of LB medium [1%(w/v) tryptone, 0.5%(w/v) yeast extract, and 0.5%(w/v) NaC1] was added to the cells, which were then cultured at 37 °C for 1 hour. It was further cultured in ampicillin (50 ⁇ g/ml)-containing LB medium at 37 °C for 10 hours.
  • the transformed cells obtained in Example 2 were cultured in ampicillin (50 ⁇ g/ml)-containing LB medium at 37 °C for 3-5 hours. Then, lactose or IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) as an activator was added to the cells to a final concentration of 1 mM, and then the cells were cultured at 16 °C for 24 hours. The supernatant and cell lysates obtained by disruption with an ultrasonic homogenizer were analyzed for neuraminidase activity. As shown in FIG. 1, the recombinant neuraminidase consisted of 475 amino acids starting with methionine and was easy to purify, because it had 6 histidine tags attached to the C-terminal thereof.
  • Example 4 In vitro experiment for screening compounds having inhibitory activity against recombinant neuraminidase
  • 4-methylumbelliferyl-neuraminic acid 4-MU-Neu5Ac
  • the crude neuraminidase enzyme obtained in Example 3 was mixed with an inhibitor candidate (0.2 nM to 2 M) and allowed to react with the candidate at 30 °C for 10 minutes to 2 hours, and then the residual activity thereof was measured.
  • the 4-methylumbelliferol was activated at 362 nm, and the emitted fluorescence was measured at 448 nm using Safire 2 (Tecan, Germany).
  • An amount of the enzyme to produce 1 mol of 4-methylumbelliferone per minute from the substrate was regarded as one unit of the enzyme.
  • Tamiflu oseltamivir phosphate
  • Roche Roche, USA
  • Example 5 Isolation and purification of recombinant neuraminidase
  • recombinant neuraminidase was isolated and purified in the following manner.
  • the cell lysate was suspended in a solution containing equilibration buffer [20 mM sodium phosphate (pH 7.2) and 0.5 M NaCl].
  • the suspension was adsorbed onto a nickel-Sepharose column (Amersham Pharmacia Biotech) pre-equilibrated with an equilibration buffer and was eluted by a linear concentration gradient using an equilibration buffer containing 0-0.5 M imidazole. 3 ml of each of the eluted fractions was distributed using a fraction collector.
  • the chromatography results are shown in FIG. 3.
  • each of the fractions was measured in the same manner as in Example 4.
  • the fractions were electrophoresed on 12% polyacrylamide gel at 50 mA.
  • the gel was stained with a staining solution (1 g Coomassie Brilliant Blue R-250, 100 ml acetic acid, 450 ml methanol, and 450 ml distilled water) and destained with 300 ml of a destaining solution (100 ml methanol, 10 ml acetic acid, and 800 ml distilled water) 3-4 times.
  • the size of the protein was determined with reference to protein standards (Bio-Rad (USA)) [lane M: size markers (myosin, 209 kDa; ⁇ - galactosidase, 124 kDa; bovine serum albumin (BSA), 80 kDa; ovalbumin, 49.1 kDa; carbonic anhydrase, 34.8 kDa; and soybean trypsin inhibitor, 28.4 kDa)], and the size of the neuraminidase was shown to be about 50 kDa (see FIG. 4).
  • SM indicates size markers (Bio-Rad)
  • lane 1 indicates the crude enzyme from cell disruption
  • lanes 2-5 indicate fraction samples having activity.
  • SEQ ID No. 1 represents the nucleotide sequence of neuraminidase N1 of Avian Influenza virus
  • SEQ ID No. 2 represents the amino acid sequence of neuraminidase N1 of Avian Influenza virus.

Abstract

Disclosed are a vector and method for expressing neuraminidase Nl of avian influenza (AI) virus in E. coli, a method of using the vector to screen an inhibitor of neuraminidase Nl, and a novel neuraminidase Nl inhibitor screened out by the method.

Description

VECTOR AND METHOD FOR EXPRESSING AVIAN INFLUENZA VIRUS NEURAMINIDASE N1 IN E. COLI, METHOD OF USING THE VECTOR, AND NEURAMINIDASE INHIBITORS
The present invention relates to the production of avian influenza (AI) virus neuraminidase N1 in E. coli and inhibitors of the activity of avian influenza virus neuraminidase N1, and more particularly to a vector and method for expressing AI virus neuraminidase N1 in E. coli, a method of using the vector to screen neuraminidase inhibitors, and neuraminidase inhibitors screened by the screening method.
Influenza virus belongs to the Orthomyxoviridae family and causes influenza. Influenza virus is classified into three types: A, B and C. Types A and B often infect humans. Particularly, the serotype of influenza A virus is determined by the difference in the amino acid sequences of the hemagglutinin (H) and neuraminidase (N) protein. There are 15 different hemagglutinin (H) subtypes and 9 different neuraminidase (N) subtypes of influenza A viruses, and a variant of influenza A virus (e.g., H5N1) is determined by the combination of hemagglutinin and neuraminidase subtypes.
Thus, there are 135 different combinations of hemagglutinin and neuraminidase subtypes. Among them, the serotype of avian influenza (AI) virus prevailing in Asia is H5N1. It was thought in the past that avian influenza is transmitted between birds and pigs, but it is known that avian influenza also infects humans, because the outbreak of avian influenza virus in Hong Kong in 1997 resulted in 6 deaths among 18 people who came into contact with birds infected with avian influenza. In Hong Kong in 2003, one between 2 people infected with avian influenza died due to H5N1 virus, and in the Netherlands in 2003, one veterinarian among 83 people infected with avian influenza died due to H7N7 virus. H9N2 virus was also detected in avian influenza which occurred in Hong Kong in 1999. Moreover, a new variant of avian influenza can also emerge according to adaptation to the host's immune system (see Yuen, K.Y. and Wong, S.S.Y., 2005. Hong Kong Med, 11:189-199]).
However, it is known that all 15 hemagglutinin subtypes and 9 neuraminidase subtypes do not infect humans, but rather 3 hemagglutinin subtypes (H1, H2 and H3) and 2 neuraminidase subtypes (N1 and N2) have the ability to infect humans. In addition, H5N1 found in poultry such as chickens or ducks can also infect humans (see Kigon, B.L., 2005. Semin Pediatr. Infect Dis 16:325-335).
The frequent emergence of a new variant of influenza virus lowers the efficacy of vaccines or therapeutic agents, and vaccines cannot protect against new influenza viruses having the ability to infect the human body, such as avian influenza. Thus, the development of various therapeutic agents capable of protecting against influenza viruses is required.
Drugs that inhibit the replication of influenza viruses are classified into two categories. The one category includes amantadine, an M2 ion channel inhibitor developed by Dupont in 1964, and it was used before 1980 to treat influenza A virus infections and causes nausea, sleepiness and chronic insomnia (see Long, J.K., et al., 2000. Cleve Clin J. Med., 67:92~95). Since then, rimantadine having reduced side effects compared to amantadine was developed, but it also has a high rate of side effects (see Jefferson et al., 2004. Cochrane Database Syst. Rev., 3:CD001169). The other category includes drugs having neuraminidase inhibitory activity, and zanamivir and oseltamivir have been used to treat influenza viruses (see Dreitlein W.R. et al., 2001. Clin Ther., 23:327-355). Zanamivir is commercially available under the trade name of Relenza from Glaxo Wellcome Inc. and is used as an intranasal powder formulation. Oseltamivir is commercially available under the trade name of Tamiflu from Roche and is used as an oral dosage form in the same manner as amantadine. Relenza can cause difficulty in breathing, and thus can make asthma worse, and Tamiflu causes nausea and vomiting (see McNicholl and McNicholl. 2001, Ann. Pharmacother. 35(1):57-70). Oseltamivir is metabolized to oseltamivir carboxylate in the human body to cause a serious mental disorder (see Izumi Y., et al., 2007, Neuroscience Letters, 426:54-58), and causes environmental pollution and induces spontaneous mutations (see Fick, J., et al., 2007, PLos ONE 2(10):3986; and Singer, A.C., et al., 2007, Env. Health Persp. 115:102-106). Particularly, 8 deaths among patients administered with Tamiflu recently occurred in Japan, and five among them were teenagers. Because teenagers administered with Tamiflu showed abnormal mental states, such as dashing to cars or committing suicide by drowning, the Japanese Ministry of Health, Labor and Welfare prohibited administration of Tamiflu, and the Korean Food and Drug Administration (KFDA) prohibited administration of Tamiflu to teenagers from March 5, 2007. FDA recommended that warning concerning nervous and mental problems (young children or teenagers have hallucinations, wild fancies, offensive personality, suicide ideation, etc. starting immediately from the administration of Tamiflu) be added to Tamiflu.
Accordingly, antiviral agents against avian influenza viruses, which have reduced toxicity and side effects, need to be developed within a short time. Furthermore, to protect against the frequent emergence of mutants having resistance to inhibitors, the inexpensive and rapid development of inhibitors against avian influenza neuraminidase is urgently required. Also, to protect against the emergence of mutants having drug resistance, various inhibitors are required. For the rapid development of inhibitors, the need to construct recombinant enzymes having mutability exists. For the expression of recombinant neuraminidase, a method of using Baculovirus and insect cells (see Dalakouras, T. et al., 2006. 1136, 48-56, Journal of Chromatography A) and a method of using Madin-Darby canine kidney (MDCK ) cells have been mainly used (see Govorkova, E.A., et al., 2001, 45(10) 2723-2732, Antimicrobial Agents and Chemotheraphy, and Yen, H.L., et al., 2006, 80(17) 8787-8795, J. of Virology). The rapid production of recombinant neuraminidase is necessary for the rapid development of inhibitors protecting against the frequent emergence of mutants, but an example of success in expressing recombinant neuraminidase of avian influenza virus using E. coli has not yet been reported.
The development of drugs for preventing and treating new diseases and diseases in which mutants frequently occur is performed through in vitro and in vivo experiments for predetermined targets. A general method for developing the drugs is carried out based on a library of specific candidates, and this is a method which can present substances enabling direct treatment of diseases. However, in cases such as avian influenza, drug-resistant mutants frequently emerge, and thus, for the treatment of the mutants, the time for the development of drugs is most critical.
Nevertheless, the development of new therapeutic agents using current methods requires a long time reaching 10-12 years and incurs much cost.
Furthermore, this method is not considered to be effective against the emergence of mutants having resistance to existing drugs.
Recently, to develop compounds having new activities from a library of a wide range of candidates, docking studies using virtual screening have been conducted. Most recently, an in silico virtual screening method using an International Grid Network(EGEE program-Enabling Grid for E-sciencE), which is being attempted by European research teams, is receiving a great deal of attention and being newly conducted. However, the activities of drug compounds presented by this method have not yet been confirmed by in vitro methods, because active proteins having neuraminidase activity, which are target proteins for avian influenza virus, could not be obtained.
In order to develop inhibitors of H5N1 AI virus, the present inventors have constructed a vector capable of efficiently expressing recombinant neuraminidase N1 in E. coli and succeeded in producing and purifying neuraminidase N1 by culturing E. coli transformed with the vector.
Furthermore, the present inventors have used the obtained recombinant neuraminidase to screen compounds showing inhibitory activities superior or equal to those of the existing therapeutic agents, Zanamivir or Oseltamivir.
The compounds thus screened can be used as therapeutic agents against H5N1 highly virulent avian influenza virus.
It is, therefore, an object of the present invention to provide a vector for expressing AI virus neuraminidase N1 in E. coli.
Another object of the present invention is to provide E. coli transformed with said vector.
Still another object of the present invention is to provide a method of producing AI virus neuraminidase N1 using the transformed E. coli.
Still another object of the present invention is to provide a method for screening an inhibitor of AI virus neuraminidase N1, the method comprising the steps of: examining the influence of candidates for AI virus neuraminidase N1 inhibitor on neuraminidase N1 activity in vitro; and selecting from the candidates a substance inhibiting neuraminidase N1 activity.
Yet another object of the present invention is to provide an AI virus neuraminidase inhibitor screened by said method.
A first aspect of the present invention relates to a vector for expressing AI virus neuraminidase N1 in E. coli, which comprises an AI virus neuraminidase N1 gene inserted into the multiple cloning site of a pET-23d vector and has a cleavage map of FIG. 2.
A second aspect of the present invention relates to E. coli transformed with said expression vector.
A third aspect of the present invention relates to a method for producing AI virus neuraminidase N1, which comprises the steps of: culturing the transformed E. coli; and collecting the culture.
A fourth aspect of the present invention relates to a method for screening an AI virus neuraminidase N1 inhibitor, the method comprising the steps of: examining the influence of candidates for AI virus neuraminidase N1 inhibitor on neuraminidase N1 activity in vitro; and selecting a substance inhibiting neuraminidase N1 activity.
A fifth aspect of the present invention relates to a composition for inhibiting AI virus neuraminidase activity, which comprises, as an active ingredient, at least one compound selected from the group consisting of:
(1)1-(3-bromophenyl)-4-{[5-(2-chloro-5-nitrophenyl)-2-furyl]methylene}-3,5-pyrazolidinedione
(2)6-amino-4-(4-chloro-3-nitrophenyl)-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
(3)3-{2-[(4-chloro-2-methylphenoxy)acetyl]carbonohydrazonoyl}phenyl 4-nitrobenzoate
(4)8-nitro-3-(phenoxymethyl)[1,2,4]triazolo[3,4-b][1,3,4]benzothiadiazepine
(5)N-[3-(5,7-dichloro-1,3-benzoxazol-2-yl)phenyl]-2-nitrobenzamide
(6)3-(4-methoxyphenyl)-N'-[3-(2-methoxyphenyl)-2-propen-1-ylidene]-1H-pyrazole-5-carbohydrazide
(7)N-1-(2-aminoethyl)-N-3-benzyl-4-nitro-1,3-benzenediamine hydrochloride
(8)N'-(2-bromo-3-phenyl-2-propen-1-ylidene)-4-methyl-3-phenyl-1H-pyrazole-5-carbohydrazide
(9)4-[3-(4-chlorobenzoyl)-4-hydroxy-5-oxo-2-phenyl-2,5-dihydro-1H-pyrrol-1-yl]butanoic acid
(10)3-hydroxy-5-(3-nitrophenyl)-1-(1,3,4-thiadiazol-2-yl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one
(11)ethyl6-[(benzylthio)methyl]-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydro-5-pyrimidinecarboxylate
(12)2-amino-6-methyl-5-(3-nitro-1H-1,2,4-triazol-1-yl)-4-phenyl-4H-pyran-3-carbonitrile
(13)N-(4-{[(3-methoxy-2-pyrazinyl)amino]sulfonyl}phenyl)-3-(5-nitro-2-thienyl)acrylamide
(14)N'-[(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene]-2,3-dihydro-1,4-benzodioxine-2-carbohydrazide
(15)N-[3-(5,7-dimethyl-1,3-benzoxazol-2-yl)phenyl]-3-(3-nitrophenyl)acrylamide
(16)6-amino-7-(1H-benzimidazol-2-yl)-5-[3-(diethylamino)propyl]-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile
(17)2-[5-(4-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid
(18)4-{[3-(1-carboxy-2-phenylethyl)-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid
(19)1-[3-(diethylamino)propyl]-3-hydroxy-4-(5-methyl-2-furoyl)-5-(4-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(20)methyl 4-(4-hydroxy-3-nitrophenyl)-2-methyl-5-oxo-7-(2-thienyl)-1,4,5,6,7,8-hexahydro-3-quinolinecarboxylate
(21)sodium 5-acetyl-3-[2-(acetylamino)ethyl]-1H-indole-2-carboxylate
(22)4,6-dimethyl-1-[(4-nitrobenzylidene)amino]-2-oxo-1,2-dihydro-3-pyridinecarboxamide
(23)1-[2-(diethylamino)ethyl]-4-(4-fluorobenzoyl)-3-hydroxy-5-(4-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(24)2-{3-nitro-5-[2-(1-pyrrolidinylcarbonyl)phenoxy]-1H-1,2,4-triazol-1-yl}acetamide
(25)N'-(3,4-dihydro-1(2H)-naphthalenylidene)-3-nitrobenzohydrazide
(26)2-(1H-benzimidazol-2-ylthio)-N'-(2-nitrobenzylidene)acetohydrazide
(27)2-[(4-methylphenyl)amino]-N'-{[5-(3-nitrophenyl)-2-furyl]methylene}acetohydrazide (non-preferred name)
(28)2-[allyl(1-methyl-3-phenylpropyl)amino]-1-(3-nitrophenyl)ethanol hydrochloride
(29)2-(1-naphthyl)-N'-[(5-nitro-2-furyl)methylene]acetohydrazide
(30)3-(4-fluorophenyl)-2-(2-methylphenyl)-5-(3-nitrophenyl)dihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione
(31)4,6-dimethyl-1-[(4-nitrobenzylidene)amino]-2-oxo-1,2-dihydro-3-pyridinecarboxamide
(32)4-(4-chlorobenzoyl)-3-hydroxy-1-methyl-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(33)2-isopropyl-5-methylbenzo-1,4-quinone1-[O-(3-nitrobenzoyl)oxime]
(34)3-(4-ethoxyphenyl)-N'-[(2-methyl-1H-indol-3-yl)methylene]-1H-pyrazole-5-carbohydrazide
(35)2-[4-(3-chlorophenyl)-1-piperazinyl]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazol-4(5H)-one
(36)4-[3-benzoyl-4-hydroxy-2-(4-nitrophenyl)-5-oxo-2,5-dihydro-1H-pyrrol-1-yl]butanoic acid
(37)4-(4-chlorobenzoyl)-3-hydroxy-1-[3-(4-morpholinyl)propyl]-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(38)4-(4-chlorobenzoyl)-1-(4,5-dimethyl-1,3-thiazol-2-yl)-3-hydroxy-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(39)2-(1-naphthyl)-N'-[(6-nitro-1,3-benzodioxol-5-yl)methylene]acetohydrazide
(40)N-{[(4-chlorobenzyl)amino][(4,6-dimethyl-2-pyrimidinyl)amino]methylene}-4-methylbenzenesulfonamide
(41)N-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-{[5-(4-methylphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazol-3-yl]thio}acetamide
(42)9,9-dimethyl-12-(4-methyl-3-nitrophenyl)-8,9,10,12-tetrahydrobenzo[a]acridin-11(7H)-one
(43)5-[2-(1-naphthylmethoxy)-5-nitrobenzylidene]-2-thioxo-1,3-thiazolidin-4-one
(44)1-(4-nitrophenyl)-1H-pyrrole-2-carbaldehyde thiosemicarbazone
(45)1-[3-(dimethylamino)propyl]-3-hydroxy-4-(4-methoxybenzoyl)-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(46)2-[(4-chloro-3-nitrobenzoyl)amino]-N-(2-furylmethyl)-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide
(47)1-[2-(dimethylamino)ethyl]-3-hydroxy-4-(4-methylbenzoyl)-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
(48)3-(3-methyl-4-{[5-(4-methyl-2-nitrophenyl)-2-furyl]methylene}-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid
(49)6-amino-7-(1H-benzimidazol-2-yl)-5-[3-(diethylamino)propyl]-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile
(50)4-[(3-nitrobenzylidene)amino]-5-(phenoxymethyl)-4H-1,2,4-triazole-3-thiol
(51)N'-(2-bromo-3-phenyl-2-propen-1-ylidene)-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide
(52)Ethyl 7-methyl-2-(3-nitrobenzylidene)-3-oxo-5-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
(53)3-(4-chloro-3-nitrophenyl)-N-[3-(5,7-dimethyl-1,3-benzoxazol-2-yl)phenyl]acrylamide
(54)4-benzyl-3-(2-furyl)-5-[(4-nitrobenzyl)thio]-4H-1,2,4-triazole
(55)ethyl7-methyl-2-[(5-methyl-2-furyl)methylene]-5-(3-nitrophenyl)-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
(56)Butyl 3-{[3-(4-nitro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoyl]amino}benzoate
(57)2-(4-nitrophenyl)-2-oxoethyl 2-(benzoylamino)benzoate
(58)2-phenylethyl 7-(4-methoxyphenyl)-2-methyl-4-(6-nitro-1,3-benzodioxol-5-yl)-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinecarboxylate,
or a pharmaceutically acceptable salt, hydrate or ester thereof
According to the present invention, avian influenza (AI) virus neuraminidase N1 can be expressed in E. coli and rapidly produced at a large amount, and excellent AI neuraminidase N1 inhibitors can be efficiently screened out by measuring and evaluating activities of AI virus neuraminidase inhibitor candidates in vitro using the neuraminidase. As a result, 58 kinds of compounds which effectively inhibit AI virus neuraminidase N1 activity were newly screened out in the present invention.
FIG. 1 shows a base sequence and a predicted amino acid sequence of the neuraminidase (N1) gene of H5N1 avian influenza virus;
FIG. 2 is a cleavage map of a recombinant vector pET-23d-N1 according to the present invention;
FIG. 3 shows results of Ni-NTA column chromatography of expressed recombinant neuraminidase; and
FIG. 4 shows result of SDS-PAGE of purified recombinant neuraminidase.
Hereinafter, the present invention will be described in detail.
In the present invention, a recombinant vector pET-23d-N1 comprising an N1 gene was constructed by synthesizing a neuraminidase (N1) gene on the basis of known H5N1 genetic information (see SEQ ID NOS: 1 and 2, and FIG. 1) and inserting the synthesized gene into the multiple cloning site of a pET-23d vector (Novagen, Germany). FIG. 2 is a cleavage map of the constructed pET-23d-N1 vector. Competent E. coli cells, for example, E. coli BL21 cells (Novagen, USA) were transformed with the constructed expression vector by, for example, heat shock or electroporation, and the transformed E. coli was cultured. A recombinant neuraminidase protein was purified from the obtained culture by, for example, Ni-NTA (Nickel-NitriloTriacetic Acid) column chromatography (FIG. 3). FIG. 4 shows the results of SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) of the purified recombinant neuraminidase. The recombinant neuraminidase produced according to the present invention is easy to purify, because it has six histidine tags attached to the C-terminal thereof.
Furthermore, in the present invention, AI virus neuraminidase N1 inhibitors were screened by examining the influence of candidates for AI virus neuraminidase N1 inhibitors on the activity of AI virus neuraminidase N1 in vitro and selecting substances inhibiting the activity of neuraminidase N1. Taiwan Academia Sinica and French CNRS-IN2P3 team completed a work, which would require about 100 years in one computer, within about 6 weeks from April, 2006 using the international supercomputer grid network. Also, from a library of virtual compounds (300,000 compounds) obtained from ZINC (see Irwin and Shoichet, 2005. J. Chem. Inf. Model., 45(1):177-182), compounds which could readily bind to the active site of H5N1 neuraminidase were calculated in a virtual space (see WISDOM, Kasam, v., et al., J. Chem. Inf. Model. 2007. 47(5):1818-1828, and Lee, H.C. et al., 2006, 5(4):288-295, IEEE Trans Nanobioscience). The process of obtaining the compounds using the international grid is as follows:
(1) preparing a data base for compounds (300,000 compounds) for examining the binding of neuraminidase, and preparing models of the three-dimensional structures of proteins, and determining binding sites associated with activity;
(2) virtually binding each compound to the binding site of a target protein, calculating binding energy, and then secondarily analyzing compounds (the highest ranking 5%) showing good binding in consideration of molecular dynamics; and
(3) presenting compounds (falling within the highest ranking 5%) showing good binding ability.
In the present invention, 308 compounds among the presented compounds were purchased from Cambridge (USA), the effectiveness of virtual screening results was analyzed using the purchased compounds, and compounds having inhibitory activities superior or equal to those of the existing inhibitors were screened from the purchased compounds. The neuraminidase inhibitory activities of the compounds screened in virtual screening were determined by adding each compound (0.2 nM to 2 M) to a substrate, a recombinant enzyme and an enzymatic reaction solution and measuring a decrease in activity compared to a reference sample to which the compound was not added. As the control, Tamiflu (oseltamivir phosphate) which is currently used as a drug was used after purification.
Table 1 below shows the name, molecular weight and relative inhibitory activity of each of the screened compounds. In Table 1, relative inhibitory activity is indicated as a percentage relative to the inhibitory activity of purified Tamiflu taken as 100%. Namely, higher percentage values show higher enzyme inhibitory activities.
Figure PCTKR2009001184-appb-I000001
Figure PCTKR2009001184-appb-I000002
Figure PCTKR2009001184-appb-I000003
Figure PCTKR2009001184-appb-I000004
58 kinds of compounds shown in Table 1 show neuraminidase inhibitory effects superior or equal to that of oseltamivir phosphate, and thus can be advantageously used as agents for preventing or treating highly virulent avian influenza virus infections. When the compounds are used as agents for preventing or treating highly virulent avian influenza virus infection, they are preferably administered at a daily dosage of 1 ㎍/㎏ weight to 50 ㎎/㎏ weight, but the dosage can be suitably adjusted depending on the age, sex and diet, health condition of a subject, disease severity, administration route, administration time, drug mixing, etc.
The compounds of the present invention can be administered according to any conventional method known in the art. For example, the compounds can be administered orally or parenterally, preferably orally. The compounds can be combined with pharmaceutically acceptable carriers, and depending on the purpose of administration, the compounds can be formulated into oral dosage forms such as tablets, hard or soft capsules, granules, chewable tablets, pills, powders, elixirs, suspensions, solutions and syrups, or parenteral dosage forms such as aerosol, sachet, sterile injectable solution, sterile powder, etc. For the purpose of oral administration, when the active ingredient of the present invention is formulated in the form of elixirs, suspensions, solutions, syrups, etc., binders such as gum Arabic, corn starch, microcrystalline cellulose or gelatin; excipients such as calcium phosphate or lactose; disintegrating agents such as alginic acid, corn starch or potato starch; lubricants such as magnesium stearate; sweetening agents such as sucrose or saccharin; and flavors such as peppermint, methyl salicylate or fruit flavor can be added. In the case in which the unit dosage form is a capsule, liquid carriers such as polyethylene glycol or fatty oil can be used in addition to the above described components. Also, the injectable solution or suspension for parenteral administration can be administered parenterally, for example, subcutaneously, intravenously, intramuscularly or intraperitoneally. In general, the injectable solution or suspension can be prepared by homogeneously mixing an effective amount of the active ingredient in pharmaceutically acceptable liquid carriers such as water, saline, aqueous dextrose and its related sugar solutions, nonvolatile oil, ethanol, glycerin, polyethylene glycol, propylene glycol, etc. In addition, adjuvants such as antibacterial agents, chelating agents, buffers and preservatives can additionally be included. The pharmaceutically acceptable carriers can be any ones, as long as they are pharmaceutically inert, substantially non-toxic and have no adverse effect on the action of the active ingredient.
The compounds of the present invention can be used in any forms, including free compounds, pharmaceutically acceptable salts, solvates including hydrates, esters, and stereoisomers, as long as they show the desired neuraminidase inhibitory effect, all of which are intended to fall within the scope of the present invention. The pharmaceutically acceptable salts in the present invention include pharmaceutically acceptable acid addition salts. The pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid or phosphorous acid, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Specific examples thereof include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.
[Examples]
Hereinafter, the present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited in any way by these examples.
Example 1: Synthesis of gene and construction of recombinant expression vector
A neuraminidase (N1) gene was synthesized on the basis of the genetic information of H5N1[A/Viet Nam/3046/2004(H5N1)] (see SEQ ID NOS: 1 and 2, and FIG. 1). To express an active protein, the synthesized gene was introduced into the multiple cloning site of a pET-23d vector (Novagen, Germany), thus constructing a recombinant vector pET-23d-N1 comprising the N1 gene. The pET-23d-N1 has a cleavage map as shown in FIG. 2.
Example 2: Transformation of E. coli
The recombinant DNA (5 ㎕) obtained in Example 1 was introduced to prepared E. coli BL21 cells (Novagen, USA) and allowed to stand on ice for 1 hour. To insert the DNA, the cells were heat shocked at 42 ℃ for 90 seconds. Then, 1 ml of LB medium [1%(w/v) tryptone, 0.5%(w/v) yeast extract, and 0.5%(w/v) NaC1] was added to the cells, which were then cultured at 37 ℃ for 1 hour. It was further cultured in ampicillin (50 ㎍/㎖)-containing LB medium at 37 ℃ for 10 hours.
Example 3: Production of recombinant neuraminidase
The transformed cells obtained in Example 2 were cultured in ampicillin (50 ㎍/㎖)-containing LB medium at 37 ℃ for 3-5 hours. Then, lactose or IPTG (isopropyl β-D-1-thiogalactopyranoside) as an activator was added to the cells to a final concentration of 1 mM, and then the cells were cultured at 16 ℃ for 24 hours. The supernatant and cell lysates obtained by disruption with an ultrasonic homogenizer were analyzed for neuraminidase activity. As shown in FIG. 1, the recombinant neuraminidase consisted of 475 amino acids starting with methionine and was easy to purify, because it had 6 histidine tags attached to the C-terminal thereof.
Example 4: In vitro experiment for screening compounds having inhibitory activity against recombinant neuraminidase
To examine the activity of the recombinant neuraminidase, 4-methylumbelliferyl-neuraminic acid (4-MU-Neu5Ac) as a substrate was dissolved in 40 mM sodium phosphate buffer (pH 6-7.5) at a concentration of 40 mM. The crude neuraminidase enzyme obtained in Example 3 was mixed with an inhibitor candidate (0.2 nM to 2 M) and allowed to react with the candidate at 30 ℃ for 10 minutes to 2 hours, and then the residual activity thereof was measured. After the enzymatic reaction, the 4-methylumbelliferol was activated at 362 nm, and the emitted fluorescence was measured at 448 nm using Safire 2 (Tecan, Germany). An amount of the enzyme to produce 1 mol of 4-methylumbelliferone per minute from the substrate was regarded as one unit of the enzyme.
To compare inhibitory activities against the recombinant neuraminidase, Tamiflu (oseltamivir phosphate; Roche, USA) which is currently used as a neuraminidase inhibitor, was purchased, purified by Sephadex LH-20 column chromatography and HPLC (TSK amide 80 (4.6 x 300 ㎜); 85%(v/v) acetonitrile; flow rate: 0.8 ㎖/min; and detector: UV 226 ㎚), and then diluted in a reaction solution for examining activity.
As a result, as shown in Table 1, 58 kinds of compounds showing inhibitory activities superior or equal to that of Tamiflu were screened.
Example 5: Isolation and purification of recombinant neuraminidase
From the cell lysate obtained in Example 3, recombinant neuraminidase was isolated and purified in the following manner. The cell lysate was suspended in a solution containing equilibration buffer [20 mM sodium phosphate (pH 7.2) and 0.5 M NaCl]. The suspension was adsorbed onto a nickel-Sepharose column (Amersham Pharmacia Biotech) pre-equilibrated with an equilibration buffer and was eluted by a linear concentration gradient using an equilibration buffer containing 0-0.5 M imidazole. 3 ml of each of the eluted fractions was distributed using a fraction collector. The chromatography results are shown in FIG. 3.
The activity of each of the fractions was measured in the same manner as in Example 4. To confirm the presence of neuraminidase enzyme in the fractions showing activity, the fractions were electrophoresed on 12% polyacrylamide gel at 50 mA. After completion of the electrophoresis, the gel was stained with a staining solution (1 g Coomassie Brilliant Blue R-250, 100 ㎖ acetic acid, 450 ㎖ methanol, and 450 ㎖ distilled water) and destained with 300 ml of a destaining solution (100 ㎖ methanol, 10 ㎖ acetic acid, and 800 ㎖ distilled water) 3-4 times. The size of the protein was determined with reference to protein standards (Bio-Rad (USA)) [lane M: size markers (myosin, 209 kDa; β- galactosidase, 124 kDa; bovine serum albumin (BSA), 80 kDa; ovalbumin, 49.1 kDa; carbonic anhydrase, 34.8 kDa; and soybean trypsin inhibitor, 28.4 kDa)], and the size of the neuraminidase was shown to be about 50 kDa (see FIG. 4). In FIG. 4, SM indicates size markers (Bio-Rad), lane 1 indicates the crude enzyme from cell disruption, and lanes 2-5 indicate fraction samples having activity.
SEQ ID No. 1 represents the nucleotide sequence of neuraminidase N1 of Avian Influenza virus; and
SEQ ID No. 2 represents the amino acid sequence of neuraminidase N1 of Avian Influenza virus.

Claims (5)

  1. A vector for expressing avian influenza (AI) virus neuraminidase N1 in E. coli, which comprises an AI virus neuraminidase N1 gene inserted into the multiple cloning site of a pET-23d vector, and has a cleavage map of FIG. 2.
  2. E. coli transformed with the expression vector of Claim 1.
  3. A method for producing avian influenza virus neuraminidase N1, which comprises the steps of: culturing the transformed E. coli of Claim 2; and collecting the culture.
  4. A method for screening inhibitor of an avian influenza virus neuraminidase N1, the method comprising the steps of: examining the influence of candidates for avian influenza virus neuraminidase N1 inhibitor on the activity of neuraminidase N1 in vitro; and selecting from the candidates a substance inhibiting the activity of neuraminidase N1.
  5. A composition for inhibiting avian influenza virus neuraminidase, which comprises, as an active ingredient, at least one compound selected from the group consisting of:
    (1)1-(3-bromophenyl)-4-{[5-(2-chloro-5-nitrophenyl)-2-furyl]methylene}-3,5-pyrazolidinedione
    (2)6-amino-4-(4-chloro-3-nitrophenyl)-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
    (3)3-{2-[(4-chloro-2-methylphenoxy)acetyl]carbonohydrazonoyl}phenyl 4-nitrobenzoate
    (4)8-nitro-3-(phenoxymethyl)[1,2,4]triazolo[3,4-b][1,3,4]benzothiadiazepine
    (5)N-[3-(5,7-dichloro-1,3-benzoxazol-2-yl)phenyl]-2-nitrobenzamide
    (6)3-(4-methoxyphenyl)-N'-[3-(2-methoxyphenyl)-2-propen-1-ylidene]-1H-pyrazole-5-carbohydrazide
    (7)N-1-(2-aminoethyl)-N-3-benzyl-4-nitro-1,3-benzenediamine hydrochloride
    (8)N'-(2-bromo-3-phenyl-2-propen-1-ylidene)-4-methyl-3-phenyl-1H-pyrazole-5-carbohydrazide
    (9)4-[3-(4-chlorobenzoyl)-4-hydroxy-5-oxo-2-phenyl-2,5-dihydro-1H-pyrrol-1-yl]butanoic acid
    (10)3-hydroxy-5-(3-nitrophenyl)-1-(1,3,4-thiadiazol-2-yl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one
    (11)ethyl6-[(benzylthio)methyl]-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydro-5-pyrimidinecarboxylate
    (12)2-amino-6-methyl-5-(3-nitro-1H-1,2,4-triazol-1-yl)-4-phenyl-4H-pyran-3-carbonitrile
    (13)N-(4-{[(3-methoxy-2-pyrazinyl)amino]sulfonyl}phenyl)-3-(5-nitro-2-thienyl)acrylamide
    (14)N'-[(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene]-2,3-dihydro-1,4-benzodioxine-2-carbohydrazide
    (15)N-[3-(5,7-dimethyl-1,3-benzoxazol-2-yl)phenyl]-3-(3-nitrophenyl)acrylamide
    (16)6-amino-7-(1H-benzimidazol-2-yl)-5-[3-(diethylamino)propyl]-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile
    (17)2-[5-(4-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid
    (18)4-{[3-(1-carboxy-2-phenylethyl)-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid
    (19)1-[3-(diethylamino)propyl]-3-hydroxy-4-(5-methyl-2-furoyl)-5-(4-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (20)methyl 4-(4-hydroxy-3-nitrophenyl)-2-methyl-5-oxo-7-(2-thienyl)-1,4,5,6,7,8-hexahydro-3-quinolinecarboxylate
    (21)sodium 5-acetyl-3-[2-(acetylamino)ethyl]-1H-indole-2-carboxylate
    (22)4,6-dimethyl-1-[(4-nitrobenzylidene)amino]-2-oxo-1,2-dihydro-3-pyridinecarboxamide
    (23)1-[2-(diethylamino)ethyl]-4-(4-fluorobenzoyl)-3-hydroxy-5-(4-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (24)2-{3-nitro-5-[2-(1-pyrrolidinylcarbonyl)phenoxy]-1H-1,2,4-triazol-1-yl}acetamide
    (25)N'-(3,4-dihydro-1(2H)-naphthalenylidene)-3-nitrobenzohydrazide
    (26)2-(1H-benzimidazol-2-ylthio)-N'-(2-nitrobenzylidene)acetohydrazide
    (27)2-[(4-methylphenyl)amino]-N'-{[5-(3-nitrophenyl)-2-furyl]methylene}acetohydrazide (non-preferred name)
    (28)2-[allyl(1-methyl-3-phenylpropyl)amino]-1-(3-nitrophenyl)ethanol hydrochloride
    (29)2-(1-naphthyl)-N'-[(5-nitro-2-furyl)methylene]acetohydrazide
    (30)3-(4-fluorophenyl)-2-(2-methylphenyl)-5-(3-nitrophenyl)dihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione
    (31)4,6-dimethyl-1-[(4-nitrobenzylidene)amino]-2-oxo-1,2-dihydro-3-pyridinecarboxamide
    (32)4-(4-chlorobenzoyl)-3-hydroxy-1-methyl-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (33)2-isopropyl-5-methylbenzo-1,4-quinone1-[O-(3-nitrobenzoyl)oxime]
    (34)3-(4-ethoxyphenyl)-N'-[(2-methyl-1H-indol-3-yl)methylene]-1H-pyrazole-5-carbohydrazide
    (35)2-[4-(3-chlorophenyl)-1-piperazinyl]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazol-4(5H)-one
    (36)4-[3-benzoyl-4-hydroxy-2-(4-nitrophenyl)-5-oxo-2,5-dihydro-1H-pyrrol-1-yl]butanoic acid
    (37)4-(4-chlorobenzoyl)-3-hydroxy-1-[3-(4-morpholinyl)propyl]-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (38)4-(4-chlorobenzoyl)-1-(4,5-dimethyl-1,3-thiazol-2-yl)-3-hydroxy-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (39)2-(1-naphthyl)-N'-[(6-nitro-1,3-benzodioxol-5-yl)methylene]acetohydrazide
    (40)N-{[(4-chlorobenzyl)amino][(4,6-dimethyl-2-pyrimidinyl)amino]methylene}-4-methylbenzenesulfonamide
    (41)N-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-{[5-(4-methylphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazol-3-yl]thio}acetamide
    (42)9,9-dimethyl-12-(4-methyl-3-nitrophenyl)-8,9,10,12-tetrahydrobenzo[a]acridin-11(7H)-one
    (43)5-[2-(1-naphthylmethoxy)-5-nitrobenzylidene]-2-thioxo-1,3-thiazolidin-4-one
    (44)1-(4-nitrophenyl)-1H-pyrrole-2-carbaldehyde thiosemicarbazone
    (45)1-[3-(dimethylamino)propyl]-3-hydroxy-4-(4-methoxybenzoyl)-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (46)2-[(4-chloro-3-nitrobenzoyl)amino]-N-(2-furylmethyl)-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide
    (47)1-[2-(dimethylamino)ethyl]-3-hydroxy-4-(4-methylbenzoyl)-5-(3-nitrophenyl)-1,5-dihydro-2H-pyrrol-2-one
    (48)3-(3-methyl-4-{[5-(4-methyl-2-nitrophenyl)-2-furyl]methylene}-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid
    (49)6-amino-7-(1H-benzimidazol-2-yl)-5-[3-(diethylamino)propyl]-5H-pyrrolo[2,3-b]pyrazine-2,3-dicarbonitrile
    (50)4-[(3-nitrobenzylidene)amino]-5-(phenoxymethyl)-4H-1,2,4-triazole-3-thiol
    (51)N'-(2-bromo-3-phenyl-2-propen-1-ylidene)-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide
    (52)Ethyl 7-methyl-2-(3-nitrobenzylidene)-3-oxo-5-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
    (53)3-(4-chloro-3-nitrophenyl)-N-[3-(5,7-dimethyl-1,3-benzoxazol-2-yl)phenyl]acrylamide
    (54)4-benzyl-3-(2-furyl)-5-[(4-nitrobenzyl)thio]-4H-1,2,4-triazole
    (55)ethyl7-methyl-2-[(5-methyl-2-furyl)methylene]-5-(3-nitrophenyl)-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
    (56)Butyl 3-{[3-(4-nitro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propanoyl]amino}benzoate
    (57)2-(4-nitrophenyl)-2-oxoethyl 2-(benzoylamino)benzoate
    (58)2-phenylethyl 7-(4-methoxyphenyl)-2-methyl-4-(6-nitro-1,3-benzodioxol-5-yl)-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinecarboxylate,
    or a pharmaceutically acceptable salt, hydrate or ester thereof.
PCT/KR2009/001184 2008-03-10 2009-03-10 Vector and method for expressing avian influenza virus neuraminidase n1 in e. coli, method of using the vector, and neuraminidase inhibitors WO2009113795A2 (en)

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CN103951602A (en) * 2014-03-19 2014-07-30 河南理工大学 Synthesis method for pyrrole thiosemicarbazone copper or nickel coordination compound having anti-tumor activity
CN105646495A (en) * 2016-01-06 2016-06-08 中山大学 Pyrrolo[2,3-b]pyrazine derivatives, and preparation method and application thereof
WO2017059401A3 (en) * 2015-10-01 2018-01-25 Duke University Androgen receptor ligands
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Cited By (6)

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WO2010035727A1 (en) * 2008-09-25 2010-04-01 塩野義製薬株式会社 Novel pyrrolinone derivative and medicinal composition containing same
EP4130273A1 (en) * 2009-12-28 2023-02-08 Sanofi Vaccine Technologies, S.A.S. Production of heterologous polypeptides in microalgae, microalgal extracellular bodies, compositions, and methods of making and uses thereof
CN103951602A (en) * 2014-03-19 2014-07-30 河南理工大学 Synthesis method for pyrrole thiosemicarbazone copper or nickel coordination compound having anti-tumor activity
CN103951602B (en) * 2014-03-19 2016-06-15 河南理工大学 There is the synthetic method of pyrroles's thiosemicarbazones copper of anti-tumor activity, nickel complex
WO2017059401A3 (en) * 2015-10-01 2018-01-25 Duke University Androgen receptor ligands
CN105646495A (en) * 2016-01-06 2016-06-08 中山大学 Pyrrolo[2,3-b]pyrazine derivatives, and preparation method and application thereof

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