WO1997019103A1 - Nouveau substrat synthetique pour l'essai d'activite de chromophores ou de fluorochromophores contre la protease ns3 du virus de l'hepatite c - Google Patents

Nouveau substrat synthetique pour l'essai d'activite de chromophores ou de fluorochromophores contre la protease ns3 du virus de l'hepatite c Download PDF

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WO1997019103A1
WO1997019103A1 PCT/JP1996/003398 JP9603398W WO9719103A1 WO 1997019103 A1 WO1997019103 A1 WO 1997019103A1 JP 9603398 W JP9603398 W JP 9603398W WO 9719103 A1 WO9719103 A1 WO 9719103A1
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protease
peptide
substrate
amino acid
amino
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PCT/JP1996/003398
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English (en)
Japanese (ja)
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Yasuaki Shimizu
Kayo Yamaji
Yasuhiko Masuho
Kunitada Shimotohno
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Rational Drug Design Laboratories
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Priority to AU75889/96A priority Critical patent/AU7588996A/en
Publication of WO1997019103A1 publication Critical patent/WO1997019103A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a novel synthetic substrate cleaved by the NS3 protease of hepatitis C virus (hereinafter abbreviated as “HCV”), and the activity of the NS3 protease of HCV using the synthetic substrate.
  • HCV hepatitis C virus
  • Hepatitis C virus is the causative virus of hepatitis C. It is said that hepatitis C has a large number of patients, is likely to become chronic, and has a high probability of progression to liver cirrhosis and liver cancer [HJ Alter et al., N. Engl. J. Med. 321, 1494-1500 (1 989) Natl. Acad. Sci. USA 87, 6547-6549 ( ⁇ 990): K. Shimotohno, Semin. Virol. 4, 305-312 (1993)] It is a clinical problem. Therefore, the drug can be said to be a drug for viral diseases that is still in great demand alongside AIDS drugs. At present, interferon is used for the treatment of hepatitis C, but its efficacy is low and its therapeutic effect is said to be limited.
  • the HCV genome consists of single-stranded RNA (+ strand) consisting of 9400 bases and encodes a single polyprotein consisting of about 3000 amino acids.
  • This precursor protein contains nine types of viral proteins in the j-chain of (NH 2 ) -C-El-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B- (C00H) from the N-terminus [MJ Selby et al., J. Gen. Virol., 74, 1103-1113 (1993): A. Grakoui et al., J. Virol., 67, 1385-1395 (1993): L. To mei et al., J. Virol., 67, 4017-406 (1993)].
  • Protease derived from host cells The polyprotein is processed by two types of proteases (NS3 protease and cprol) that the virus itself encodes, supplying the proteins necessary for the growth of the virus.
  • the N-terminal one-third of non-structural protein 3 has NS3 protease activity, and it has four sites within the non-structural region that encode proteins required for viral replication (each cleavage site (Called NS3 / 4A, NS4A / 4B, NS4B / 5A, and NS5A / 5B) [ ⁇ C. Grakoui et al., J. Virol. 67, 2832-2843 (1993)] .
  • the P1 position is a cysteine
  • the NS3 protease The enzyme has a previously unknown substrate specificity.
  • NS3 protease is necessary for virus growth and has a different substrate specificity from host protease, and thus it is one of the strong targets of anti-HCV drugs. Is considered one. In other words, it is considered that by screening for an NS3 protease inhibitor, it is possible to find a strong candidate for an anti-HCV drug.
  • NS3 protease activity has been confirmed by immunoprecipitation or western blotting, in which in vitro transcription-translation system or intracellular expression system co-expresses protease and substrate, and the substrate is cleaved by immunoprecipitation or Western blot.
  • the two-step method has been known for a long time, and a fluorophore [for example, 7-amino-14-methylcoumarin (7-amino-4-methytri coumarin) / hereinafter abbreviated as "AMC” /
  • AMC fluorophore
  • MCA 4-methhy- coumarine-7-yi-amido
  • AFC 4-trifluoromethylcoumarin-7-ylamide
  • NA in the same case as above, mono-naphthylamide, ⁇ -naphthylamido)] or chromophore [for example, para-nitroaniline) / hereinafter abbreviated as “pNA” / , As well as para-nitroanilid o)], a synthetic substrate is added and cut with the enzyme whose activity is to be measured (primary digestion), and the digest is digested with an excess amount of aminopeptidase (hereinafter abbreviated as “AP”) (2). Secondary digestion) The released fluorophore is a method of measuring the amount of primary digest from the amount of chromophore. Enzyme activities such as renin ⁇ E.
  • the present invention is to develop a fast, simple, high-sensitivity, multi-processable (high througput) NS3 protease atsushi system required for screening for an NS3 protease inhibitor, and particularly to use the atsushi system.
  • the challenge is to provide new synthetic quality.
  • the present inventors set out a two-step method and worked diligently to improve the synthetic substrate and Atsushi system, and completed a novel, rapid, simple, highly sensitive and multi-process NS-3 protease assay system. I let it.
  • Z is an amino acid or peptide
  • X is Leu, Trp or Tyr
  • A is a single bond or peptide
  • Y is a fluorophore, chromophore
  • Z-Cys At least one of the existing peptide bonds is difficult to digest with aminopeptidase In addition, any peptide bonds present in the XA region are eliminated by the aminopeptidase.
  • the chromophore or fluorophore is 7-amino-4-methyl-coumarin, 7-amino-4-trifluoromethylcoumarin (7-amino-4-trif) luoromethyl-coumann), para-nitroaniline or 6-naphthylajamine (5-naphthylajnine), or the synthetic substrate according to (1) above, or
  • amino acid sequence is "Lys-Glu-Asp-Val-Val-Pro-Cys-Ala-Met-Ala-Leu-Y" (SEQ ID NO: 1)
  • the synthetic substrate according to the above (1) is "Lys-Glu-Asp-Val-Val-Pro-Cys-Ala-Met-Ala-Leu-Y" (SEQ ID NO: 1)
  • the present invention provides (7) double digestion of the synthetic substrate according to any one of the above (1) to (6) with hepatitis C virus NS3 protease and aminobeptidase.
  • a method for measuring the activity of hepatitis C virus NS3 protease comprising:
  • the method according to the above (7) further comprising (8) a step of performing double digestion with HC3-derived NS3 protease and aminopeptidase in the presence of the hepatitis C virus NS4A-derived peptide. ) Described method, or
  • NS4A-derived peptide is a peptide containing an amino acid sequence at positions 18 to 40 from the N-terminus of NS4A.
  • a substrate using only the sequence at the ⁇ end of the cleavage point (for example, ⁇ 6 to ⁇ 1), such as “a substrate having a chromophore covalently bound to the C-terminus of a peptide having an amino acid sequence of EDVVPC” is NS 3 It is considered unsuitable as a protease substrate.
  • a shorter sequence at the C-terminus of the cleavage point in the substrate sequence is preferred because it is easier to digest with aminopeptidase and the substrate is easier to synthesize. Therefore, we determined the minimum unit required for cleavage based on the 5A / 5B sequence (GEAGDD IVPCSMSYTWT GAL) used by Nichi K. (N.Kakiuchi) and others for the NS3 protease Atsusy system by HPLC. (See Reference Example 1). So far, mutants of the substrate sequence and NS3 protease have been co-expressed in Escherichia coli or animal cells, and amino acid sequences important for cleavage have been studied. [Y.
  • N3nk KDK IVPC SMS Y
  • NS 3 protease NS 3 protease
  • a two-step method substrate in which pNA was added to the C-terminus of "N3nk” was prepared and subjected to two-step digestion with NS3 protease and aminobeptidase M (hereinafter abbreviated as "APM”).
  • the substrate did not develop well. This was because APM could not sufficiently digest the C-terminal fragment "SMSY-pNA” generated by digesting "N3nk-pNA" with NS3 protease. That is, it was considered that it was an essential requirement for the substrate to efficiently digest the sequence at the C-terminal side of the NS3 protease from the cleavage point with APM, and the present inventors considered that the APM Substrate specificity was studied.
  • the present inventors examined the substrate specificity of APM in the optimal buffer of NS3 protease, and found that Leu, Ala, Met, and Arg were extremely easily digested, but Tyr, Gly, P he is hardly digested, and Ile, Val, Asp, Ser, Pro are hardly digested. (See Reference Example 2).
  • the substrate which has not been digested in the primary digestion may be decomposed by the secondary digestion with aminopeptidase and develop color.
  • the amino group at the N-terminal is replaced with an acetyl group or succinyl group. It is protected with a hydroxyl group, Fmoc or the like.
  • protection of the amino group is usually not preferred because it reduces the solubility of the peptide substrate.
  • the present inventors have found that when an Asp, lye, Ser, Pro, and Va1, which are not easily digested by APM, are included at the N-terminal side of the NS3 protease cleavage point, an N-terminal amino group is added.
  • the synthetic substrate of the present invention is easily cleaved by NS3 protease, and the N-terminal side from the cleavage site of NS3 protease is hardly digested by aminopeptidase and the C-terminal side is easily digested.
  • the most distinctive feature is that a new peptide that is most suitable for NS3 protease activity measurement by the two-step method is newly designed and synthetic quality is provided based on this design.
  • the step method differs from the basic design concept.
  • the above-mentioned features have enabled, for the first time as a measurement method for detecting HCV, multi-processing screening (High Throughput Screening) in which a large amount of compounds can be identified in a short time. It is expected that the use of such a fluorescent substrate will significantly increase the detection sensitivity of NS3 protease.
  • the terms of the present invention will be described in detail.
  • amino acid means a compound having a carboxyl group and amino S in the same molecule, and an imino acid such as proline is also included in the amino acid. Natural and non-natural types are also included (Biochemical Dictionary, Tokyo Kagaku Dojin, 2nd edition, 58-69, 1468-1474 (1992), and Organic Chemistry and Biochemical Nomenclature (2), Nankodo, Revision No. 2nd edition, 59-82 (1989)). In the present invention, the amino acid present at the terminal of the synthetic substrate is also included in the present definition.
  • amino acid residue is a generic term for the above amino acid moieties other than hydrogen atoms and hydroxyl groups that are excluded when forming a peptide bond in a protein or a peptide structural unit (Nikkei Bio 3 ⁇ 4 New Glossary, Nikkei Biotech, 4th ed., 23 (1995) or Biochemical Dictionary, Tokyo Kagaku Dojin, 2nd ed., 61-62 (1992), etc.)
  • Peptide means two or more amino acids linked by peptide bonds.
  • At least one of the peptide bonds present in the Z-Cys region is difficult to be digested with aminopeptidase means that the amino acid or peptide in Z is hardly digested with aminopeptidase. It means that an acid or amino acid residue is present, specifically, 1 le, Va, Asp, Ser, Pro and the like.
  • chromophore or fluorophore is used for measuring the activity of serine protease, thiol protease, aminopeptidase, etc., and any chromophore or fluorophore that achieves the object of the present invention is referred to as "chromophore or fluorophore”.
  • the fluorescent ligase when bound within the substrate of the present invention, the fluorescent ligase has no luminescence, and when released by digestion with aminopeptidase, the fluorescein has a fluorescence or luminescence.
  • pNA, AMC, AFC or / 5NA are examples of the fluorescent ligase.
  • the amino acid sequence to be cleaved by the NS3 protease is not particularly limited, but it is preferable that the amino acid sequence has a length of up to P4, more preferably a length of up to P6 for efficient J-cleavage. Good.
  • the sequence of ⁇ 6 to ⁇ 4 ' may be the NS5A / 5 ⁇ subtype sequence (A. Grakoui et a 1., Journal of Virology, 67, 2832-2843 (1993)) or cut with NS3 protease.
  • P6 is Asp, Asn or Glu
  • P5 is Lys
  • Asp Asp
  • Ser Asn or Gly
  • P4 is I16 or & 1
  • P3 is preferably Val
  • Glu 11 & 1 or 116
  • 2 is preferably syrup 0, hydroxy Pro, ⁇ he, G1u, Va1 or Tyr.
  • P 4 ′ is preferably Trp, Tyr, A1a or Leu.
  • the substrate used in the present invention is preferably a substrate having a cleavage rate of 20% or more by NS 3 -portase under the conditions described in Example (V) below, and more preferably 4%. 0% or more, more preferably 60% or more is used.
  • a hepatitis C virus NS3 protease comprising a step of performing a double digestion of the above-mentioned synthetic substrate with HCV-derived NS3 protease and aminopeptidase.
  • the method for measuring the activity of the enzyme is preferably carried out in the presence of a peptide derived from the hepatitis C virus NS4A.
  • NS4A in the present invention means a fragment, a non-structural protein 4A (NS4A), obtained as a result of digestion of the non-structural protein of the HCV virus with NS3 protease as described above, and a hydrophilic region. And a protein with a total length of 54 amino acids and a hydrophobic region.
  • the NS4A sequence to be added to the Atsushi system of the present invention is not limited to "4A18-40", and any fragment may be used as long as it is a fragment derived from NS4A including the 22nd to 34th positions from the N-terminal. . 4A2 1 _40, 4A 18-37, 4A 18-34, 4 A
  • Examples are 21-34, 4A 22-34.
  • the pH may be ffl in the range of 5.0 to 10.0, preferably 7.0 to 9.0.
  • Sodium chloride may be not added or may be in the range of 20 OmM or less.
  • the DTT concentration may be in the range of 0.05 to 10.
  • OmM preferably 0.5 to 2 mM.
  • the reaction temperature may be ffl in the range of 10 to 50 ° C, preferably 25 to 37 ° C.
  • Synthetic peptide substrates of the present invention are described in “Nobuo Izumiya et al., Principles and Experiments in Peptide Synthesis (1985), Maruzen”, “Novabiochem Peptide Synthesis Manual (1994) , Supervised by Haruaki Yajima, synthesis of peptides (continued drug development 14), Hirokawa Shoten (1991), M, Bodanszky, Peptide Chemistry, A Practical Textbook, Springer-Verlag, Berlin (1988), etc. Can be synthesized.
  • the method for producing a synthetic substrate containing a chromophore or a fluorophore is carried out by a conventional method. For example, "K.
  • Examples of the method for producing the synthetic substrate of the present invention include a liquid phase method or a solid phase method, a peptide synthesis method such as azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, and N method. , N'-dicyclohexylcarbodiimide method, active ester method, carbodiimidazole method, redox method and the like.
  • the synthetic substrate thus obtained is purified or used as it is. Isolation and purification are carried out by a conventional method, such as extraction, distribution, reprecipitation, recrystallization products, or by column chromatography, First c
  • the aminopeptidase used in the present invention is not particularly limited as long as it can digest a C-terminal fragment generated by NS3 protease digestion and release a chromophore or a fluorophore, and preferably APM (leucine aminopeptidase).
  • APM leucine aminopeptidase
  • EC 3.4.1.1.2 is good. More preferably, APM derived from the microsomes of bush kidney is good.
  • Digestion with two proteases can be performed on the same 96-well plate, and the absorbance or fluorescence intensity can be measured as it is. Can be performed quickly.
  • APM may be added after digestion of NS3 protease, or may be added simultaneously with NS3 protease.
  • the enzyme concentration and substrate concentration were 80 ⁇ g / ml and 86 ⁇ M, respectively.
  • the final concentrations of the enzyme and the substrate of 10 to 40 ⁇ g / ml and 1 to 20 ⁇ M are sufficient, which are much higher than those of the conventional Atsushi system. Sensitivity.
  • a pNA substrate it can be used preferably at a concentration of 0.2 to 2%.
  • an MCA substrate it can be used preferably at a concentration of 1 to 100 ⁇ M.
  • NS 3 protease itself has weak substrate cleavage activity, it is desirable to construct an Atsushi system in the presence of NS 4A.
  • the present inventors have previously suggested that the sequence of NS4A required to enhance enzyme activity was located 22 to 34th from the N-terminus of NS4A [Failla et al. al., J. Virol., 68, 3753-3760 (1994): Lin et al., Virol., 68, 8147-8157 (1994): Y. Tanji et al., J.
  • Boc is tertiary butoxycarbonyl
  • tBu is tertiary butyl (tert. Butyl)
  • Clt is chlorotrityl
  • DCC is ⁇ , ⁇ , -dihexylhexyl.
  • Carbodiimide ( ⁇ , ⁇ '-Dicyclohexylcarbodiimide), “DCM” is dichloromethane (dichloromethan), “DIEA” is ⁇ , ⁇ -diisopropyl Ethylamine ( ⁇ , ⁇ -diisopropylethylamine), “DMF” is dimethylformamide, “EDT” is Ethanedithiol, “Fmoc” is 9-Forenylmethoxycarbonyl, “HBTU” is 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethylperonium hexafluorophosphate (2- (1H-benzotriazole-yl)) -1,1,3,3-tetramethyluronium hexafluorophosphate;, “N” is N-Hydroxybenzotriazole, “TFA” is Trifluoroacetic acid, and “TFE” is “Trifluoroethanol” and
  • FIG. 1 shows the time course of substrate digestion by NS3 protease in the presence and absence of 4A 18-40.
  • FIG. 2 is a diagram showing the substrate specificity of APM.
  • FIG. 3 shows a process for synthesizing N307-pNA.
  • FIG. 4 shows the synthesis process of N307-MCA.
  • FIG. 5 is a diagram showing mass spectrometry of N307-pNA.
  • FIG. 6 is a diagram showing mass spectrometry of N307-MCA.
  • FIG. 7 is a graph showing the concentration dependence of NS3 protease in the substrate digestion of N307-pNA.
  • FIG. 8 is a diagram showing temporal changes in certain digestion of N307-MCA.
  • BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
  • the peptides used in this experiment were synthesized and purified using a PS SM-8 type peptide synthesizer manufactured by Shimadzu Corporation according to the manual of the synthesizer.
  • MBP-NS3 MBP binding protein
  • reaction rate increased 15-fold in the presence of 4A18-40 compared to the absence (Fig. 1).
  • all NS3 protease digestions were performed in the presence of 4A18-40.
  • Enzyme reaction solution 50 mM Tris-HCl (pH7.6), 30 mM NaCl, 2 mM DTT) in MBP-NS3 (final concentration 2.2 2.M), 4 A 18 -40 (final concentration 4.4 ⁇ M / enzyme) 2 times the molar concentration) to obtain 47.5 ⁇ 1.
  • MBP-NS3 final concentration 2.2 2.M
  • 4 A 18 -40 final concentration 4.4 ⁇ M / enzyme 2 times the molar concentration
  • Nl DDIVPC- SMSYkdk (SEQ ID NO: 10) 62/5
  • APM was purchased from SIGMA (product number L 0632).
  • a 1 a— pNA, Ala-Al a-Phe-pNA, Arg— pNA, Asp— pNA, G 1 y-pNA, G 1 yPhe-pNA, lie— pNA, Leu— pNA, Met— pNA, Phe— pNA and Va1-pNA were purchased from Bachem, and Tyr-pNA was purchased from NovaMochem.
  • a peptide having the amino acid sequence SMS YTWTG was synthesized using PSSM-8 by a conventional method.
  • the amount of pNA released was determined by reacting the ImM substrate described above with 0.05 U of APM at room temperature in an 83,2 mM DTT buffer (FIG. 2). Asp, I 1 e and Va 1 were shown to be difficult to digest with APM, but Leu, Ala and Arg were easy to cut.
  • NS3 protease was digested under the same conditions as in Reference Example 1 except that the digestion reaction was performed at 37 ° C for 3 hours, and the digestibility was examined.
  • the underlined amino acids are those amino acids that have been modified from their native form to make them more amenable to aminopeptidase cleavage.
  • “N3nk” is a comparative example in which serine (S) that is not cleaved by APM is located at positions Pl and P3.
  • AMA Y can be digested by APM. Digestibility with protease decreased. Therefore, the substrate sequence was further examined, and it was found that the use of the sequence “N307” improved the digestibility with NS 3 protease while maintaining the ease of digestion with APM. In addition, the addition of chromophores or fluorophores further improved the digestibility with NS3 protease.
  • N307-pNA kEDVVPC-AMAL-pNA (SEQ ID NO: 1) 74
  • N307-MCA kEDVVPC-thigh-MCA (SEQ ID NO: 1) 67
  • N307-pNA The synthesis of N307-pNA was performed as follows.
  • the peptide was purified by reversed-phase HPLC (ODS-80 Tm / Tosoh Corporation). At this time, an aqueous solution containing 0.1% TF A was used for solution A, and acetonitrile containing 0.1% TFA was used for solution B, and the separation was performed with a linear gradient of the solution (25 to 60%).
  • the molecular weight of the substrate was confirmed by “Electron spray (ESI) mass spectrometry”. Both ⁇ 307— ⁇ (FIG. 5) and N 307—MCA (FIG. 6) agreed with the target molecular weight.
  • the amino acid composition of the substrate was analyzed by a picotag amino acid analysis method using a Vico-Guyx Station and a gradient system (both manufactured by Bio-Ichiyuzu Co., Ltd.).
  • the value in kazuko indicates the number contained in the synthetic substrate. “Nd” indicates that no data exists.
  • Table 3 The amino acid composition of the substrate was analyzed by a picotag amino acid analysis method using a Vico-Guyx Station and a gradient system (both manufactured by Bio-Ichiyuzu Co., Ltd.).
  • the value in kazuko indicates the number contained in the synthetic substrate. “Nd” indicates that no data exists.
  • N307-pNA and N307-MCA had the amino acid composition predicted from the amino acid sequence.
  • the reaction was performed using a 96-well plate (Maximum immunoplate / Nunc). L to 12 ⁇ g of MBP-NS3 and 4A18-40 (final concentration: 44 ⁇ M) were added to PBS buffer (containing 2 mM DTT) to give 99 ⁇ l. After 30 minutes preheating the temperature at 25 a C, of the present invention N 307- pNA (final concentration 500 ⁇ M) was added for 3 hours at 37 ° C, were substrates digestion. After the completion of the primary reaction, 0.05 U of APM was added, and the reaction was carried out at 55 ° C for 1 hour (secondary reaction). After the completion of the reaction, the absorbance at 405 nm was measured using a "THERMO max" microplate reader (Molecular Devices). NS 3 protease It was confirmed that the substrate digestion progressed in proportion to the enzyme concentration (horizontal axis in Fig. 7) (Fig. 7).
  • the reaction was performed using a 96-well plate (Black Cliniplate Solid, Labsystems, Finland). 4 ⁇ g of MBP-NS 3 and 4A 18-40 (final concentration 22 ⁇ M) were added to a PBS buffer (containing 2 mM DTT) to give 99 ⁇ l. After preheating at 25 ° C for 30 minutes, N307-MCA of the present invention (final concentration: 50 ⁇ M) was added, and substrate digestion was performed at 37 ° C for 3 hours. After the completion of the primary reaction, 0.05 U of APM was added and the reaction was carried out at 37 ° C for 2 hours (secondary reaction).
  • the increase in the light intensity associated with the digestion was measured at 380 nm for the excitation wavelength and 460 nm for the fluorescence wavelength using Fluos Yuichi (Tecan). At a gain of 50, a fluorescence intensity of 1.420 was observed.
  • the reaction was performed using a 96-well plate as in (VI).
  • a PBS buffer including 2mM DTT
  • N307-MCA at a final concentration of 20 ⁇ M
  • NS3 ⁇ S4A fuses the NS3 protease domain (1027-1215) with the NS4A region (1651-1711) containing the C-terminal part of NS3 via the spacer sequence LysLeu. This is a recombinant single-chain active NS3 protease.
  • the protease expression vector was obtained by cutting out the HCV cDNA encoding the above amino acid sequence using the PCR method and ligating it via the HindIII sequence, and then using the NdeI / It was created by inserting it into the BamHI site.
  • the protein was expressed by the method of FW Studier et al. (Methods in Enzymology 185, 60-89, 1990), and the expressed protein was purified with reference to the following literature (FAO Marston, DNA cloning). vollll, pp59-88 IRL Press, 198 7)
  • Figure 8 shows the time course of substrate digestion. The measurement was performed at an excitation wavelength of 360 nm and an emission wavelength of 450 nm using an MTP-32 fluorescent plate reader (Corona).
  • the activity of NS3 protease can be measured quickly, easily and with high selectivity, and the amount of NS3 protease can be measured in a short time. High Throughput Screening has become possible.
  • Leu may be associated with a fluorophore or luminophore.
  • Lys Lys Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr Thr 1 5 10 SEQ ID NO: 8
  • Lys Asp Lys lie Val Pro Cys Ser Met Ser Tyr 1 5 10 SEQ ID NO: 1 6
  • Lys Asp Lys lie Val Pro Cys Ser Met Ser Trp 1 5 10 SEQ ID NO: 1 7
  • Lys Glu Asp lie Val Pro Cys Ser Met Ser Tyr 1 5 10
  • Leu binds to pNA.

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Abstract

L'invention concerne un substrat synthétique contenant une séquence d'acides aminés spécifiques ayant un fluorochromophore ou un chromophore à liaison covalente avec l'extrémité C, et comportant au moins un acide aminé inhibant la digestion d'aminopeptidase du côté de l'extrémité N de la séquence considérée. Grâce à ce substrat, on peut tester efficacement l'activité de protéases NS3. Il est possible d'accroître encore la sensibilité de détection si l'on conduit l'essai en présence de NS4A.
PCT/JP1996/003398 1995-11-22 1996-11-20 Nouveau substrat synthetique pour l'essai d'activite de chromophores ou de fluorochromophores contre la protease ns3 du virus de l'hepatite c WO1997019103A1 (fr)

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AU75889/96A AU7588996A (en) 1995-11-22 1996-11-20 Novel synthetic substrate for activity assay having chromophore or fluorochromophore active against hepatitis virus ns3 protease

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JP7/304881 1995-11-22
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US6251583B1 (en) * 1998-04-27 2001-06-26 Schering Corporation Peptide substrates for HCV NS3 protease assays

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VIROLOGY, Vol. 188, No. 2, 1992, LAUREN C. IACONO-CONNORS and CONNIE S. SCHMALIOHN, "Cloning and Sequence Analysis of the Genes Encoding the Nonstructural Proteins of Langat Virus and Comparative Analysis with Other Flaviviruses", pages 875-80. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6251583B1 (en) * 1998-04-27 2001-06-26 Schering Corporation Peptide substrates for HCV NS3 protease assays

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