WO1997019103A1 - Novel synthetic substrate for activity assay having chromophore or fluorochromophore active against hepatitis c virus ns3 protease - Google Patents

Abstract

A synthetic substrate which contains a specific amino acid sequence, has a fluorochromophore or chromophore covalently bonded to the C-terminus, and carries at least one amino acid inhibiting the aminopeptidase digestion on the N-terminal side of the above sequence. The use of this substrate makes it possible to efficiently assay the activity of an NS3 protease. By effecting the assay in the presence of NS4A, the detection sensitivity can be further elevated.

Description

 Details ΐ

Novel synthetic substrate with chromophore or fluorophore for hepatitis C virus NS3 protease for measuring activity

 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. Related to the measurement method. Body

 Hepatitis C virus (HCV) 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 (NS3) 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) [Α C. Grakoui et al., J. Virol. 67, 2832-2843 (1993)] . As a result, in the region from NS3 to NS5B, five proteins, NS3 (p70), awake (p4), NS4B (p27), NS5A (p58 / 56) and NS5 Mp66) bind (Hijikata K. et al. Natl. Acad. Sci. USA, 90, 10773 (1993)). It is known that NS3 protease alone has weak activity, and NS4A, another nonstructural protein, becomes a cofactor (cofactor) and enhances the protease cleavage activity. [C. Failla et al., J. Virol., 68, 3753-3760 (1994)]. Of the four t-sequences that are cleaved by NS3 protease, of the three t-sequences cleaved by trans (NS4A / 4B, NS4B / 5A, NS5A / 5B), the P1 position is a cysteine, and the NS3 protease The enzyme has a previously unknown substrate specificity. As described above, 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.

By the way, to screen for NS3 protease inhibitors, it goes without saying that a system for measuring the activity of HCV NS3 protease is required, but a quick and simple HCV NS3 protease using synthetic substrates is required. At present, the activity measurement system has not been established yet. In particular, synthetic substrates for measuring enzyme activity must satisfy the following four points: high sensitivity and high specificity to enzymes, good solubility in water or biological test solutions, and easy detection of digests. Although it is said that is important, there is no knowledge about a synthetic substrate for NS3 protease that satisfies these conditions. To date, 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. [Y. Komoda et al., J. Virol. 68, 7351-7357 (1994): P. Bouffard et al., Virology, 209, 52-59 (1995): B. Hahm et al., J. Virol., 69, 2534-2539 (1995): R. Bartenschlager et al., J. Virol., 68, 5045-5055 (1994): CL Failla et al., J, Virol., 68, 3753-3760 (1994): C. Lin et al., J. Virol., 68, 5063-5073 (1994)]. Since these methods require immunoprecipitation and electrophoresis, they cannot be said to be simple Atsusei methods for screening for PI1 harmful agents, and it is difficult to determine the expression levels of enzymes and substrates. Was not suitable for biological analysis.

 In addition, an Atsuy system of NS3 protease using a synthetic peptide substrate has also been reported [N. Kakiuchi et al., BBR, 210, 1059-1065 (1995)]. This system uses a substrate with a dansyl group introduced at the N-terminus of 20 amino acids, which mimics the sequence between NS5A and 5B. Because of the time and effort involved, this method is not a suitable method for random screening of inhibitors that require a large number of samples.

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" / When covalently bound to a peptide or amino acid, it is abbreviated as “MCA” (4-methhy- coumarine-7-yi-amido); 7-amino-4-trifluoromethyl-coumarin (7-amino-4-trifluoromethyl-coumarin) / Abbreviated as "AFC" / In the same case as above, 4-trifluoromethylcoumarin-7-ylamide / Hereinafter abbreviated as “? NA” / in the same case as above, mono-naphthylamide, β-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 ゃ E. coli leader beptidase have been measured using these substrates [A. Reinhaiz and M. Roth, European J. Biochem., 7, 334-339 (1969): K. Murakami et al., Anal. Biochem., 110, 232-239 (1981): D. Kuo et al., Biochemistry, 33, 8347-8354 (1994)]. However, the traditional two-step method basically utilized a substrate containing the native sequence containing the cleavage site of the enzyme whose activity was to be measured. Also, there was no report that a two-step method group was applied to the activity measurement of NS3 protease. Disclosure of the invention

 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.

That is, the present invention

 (1) a synthetic substrate represented by the following formula (I),

 (N terminal) Z— Cys— Al a— Me t— Al a— X— A— Y (C terminal)

 (I)

(However, the formula, 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, and 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. )

 Preferably, (2) 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

 (3) The amino acid or amino acid residue which is difficult to digest with aminopeptidase in the Z-Cys region is represented by Asp, Ser, Pro, 116 or & 1. ) Or the synthetic substrate according to (2),

 More preferably, (4) the 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),

 (5) The synthetic substrate according to the above (4), wherein the chromophore bound to the C-terminal side is para-nitroaline.

 (6) The synthetic substrate according to the above (4), wherein the chromophore bound to the C-terminal side is 7-amino-4-methylcoumarin.

 Further, 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:

 Preferably, 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

(9) The method according to (8), wherein the NS4A-derived peptide is a peptide containing an amino acid sequence at positions 18 to 40 from the N-terminus of NS4A. Hereinafter, the present invention will be described in detail.

 The cleavage site on the HCV polyprotein has already been reported (A. Grakoui et al., J. Virol., 67, 2832-2843 (1993)). Comparison of the three cleavage sites that are cleaved by trans and the subtypes of each cleavage site revealed that the ¾-charged amino acid at position P6 (83 or 01u), Cys at position P1, and P1, position 1. The conservation of hydrophobic amino acids (Tyr, Trp, Ala or Leu) at the Ser or A1a, P4, positions has been pointed out. To the N-terminus and P1, Ρ2, Ρ3 · · · to the C-terminus and Pl ', P2,, P3, ···). That is, unlike serine proteases such as chymotrypsin, trypsin, and elasase, the sequence at the C-terminal side of the cleavage point seems to be essential for group recognition. Therefore, a substrate using only the sequence at the Ν end of the cleavage point (for example, の 6 to 切断 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.

When designing a two-step 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. Komoda et al., J. Vi. rol., 68, 7351-7357 (1994): Y. Tanji et al., Gene, 145, 215-219 (1994): A. Alexander et al., J. Virol., 68, 7525-7533 (1994). ], It has been reported that the basic sequence from the P4 position to the P1, position is important for cleavage. However, when the synthetic peptide is used as a substrate, the present inventors have found that the C-terminal side from the cleavage point to P 1 ′ is not sufficient, and has a sequence (SMS) from P 3 to Ser. If the sequence up to Tyr at position P4 (SMSY) is present, it will be more easily digested. I came out. In addition, the N-terminal side from the cleavage point is digested if it has the sequence up to Ile at P4 (IVPC), but the sequence up to Gy at P7 or Asp at P6. (GDD I VC C or DD I VC C) was found to be more easily digested. Therefore, a substrate having a peptide sequence from P6 to P3 'is preferable as a substrate for NS3 protease. Those having a peptide sequence from 6 to 4 'would be more preferred. Furthermore, in consideration of the water solubility of the substrate, the sequence of KDK IVPC SMS Y (hereinafter referred to as “N3nk”) in which both G 1 y at P 7 and Asp of P 5 have been replaced with Lys is taken as NS 3 protease It was found to be particularly suitable as the basic sequence of the substrate (see Reference Example 1).

 By the way, 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.

Until now, the substrate specificity of APM has been studied only for some limited amino acids using amino acid pNA substrates, MCA proteins and dipeptide substrates [It. K. Kania, et al., J. Biol. Chem., 252, 4929-4934 (1977): M. Nakanis hi, et al., J. Biochem., 106, 818-825 (1989): S. Ishiura, et al., J. Biochem. ., 102, 1023-1031 (1987)], and there were many unknowns about the substrate specificity of APM. In the present activity measurement system, it is preferable that NS3 protease and APM react in the same buffer for simplicity. Therefore, 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).

 Therefore, in consideration of the amino acid sequence that is easily digested by APM, the modifications of P1 ′ to Ρ4 ′ were performed to simultaneously satisfy the substrate specificities of both NS3 protease and APM. As a result, a substrate cleaved by NS3 protease and whose C-terminal fragment was digested by APM was found (see Reference Example 3).

In general, in the two-step method, the substrate which has not been digested in the primary digestion may be decomposed by the secondary digestion with aminopeptidase and develop color.Therefore, 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. However, 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. Even without protection, it was confirmed that the undigested substrate did not develop color in APM during the time when the C-terminal cleavage fragment of NS3 protease was secondarily digested by APM, and water solubility was ensured. . Then, it was confirmed that the use of a chromogenic and fluorescent synthetic group in which pNA or AMC was added to the C-terminus of the substrate makes it possible to measure NS3 protease activity easily and with high sensitivity (see Example) The present invention has been completed. That is, 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. Specifically, alanine (A la), arginine (A rg), asparagine (A sn), aspartic acid (A sn), cysteine (Cys), glutamine (G 1 u), glutamic acid (G in), Glycine (G1y), histidine (His), isoleucine (I1e), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro) , Serine (Ser), threonine (T hr), tryptophan (T rp), tyrosine (T yr), quinoline (V a1), β-alanine (i3 A la), 2-aminobutyric acid (A bu ), A-Aminoisobutyric acid (A ib), α-aminosuberic acid (A su), 4-clofenerulanalanine, citrulline (C it), and —Hexa hexylalanine (Cha) , 3, 4 1, 2, 3 — young 4-fluoropheniralanine, homoserine (hSer), hydroxyaline (Hyp), β-hydroxyvalin, 4-nitrophen-alanine, norleucine (Nle), norvaline (Nva), orditin O rn), Be-Silamine (P en), Phenylglycine (P hg), Pyroglutamine, Sarcosine (S ar), β- (2-Chenyl) alanine (T hi), Pipecolic acid (P ip), Naphthylalanine, buco pargyglycine (P ra) and the like.

"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 ¾ New Glossary, Nikkei Biotech, 4th ed., 23 (1995) or Biochemical Dictionary, Tokyo Kagaku Dojin, 2nd ed., 61-62 (1992), etc.) _c "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.

 "Every peptide bond present in the region of X-A is digested by aminopeptidase", which means that not only the bond between XA and A is cut by aminopeptidase, but also It means that the amino acid residues except for the amino acid residues which are not easily digested by the above aminopeptidase are present in the peptide therein.

 The term "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". Specifically, 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. And more specifically, pNA, AMC, AFC or / 5NA. In the present invention, 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 Ρ6 to Ρ4 'may be the NS5A / 5Β subtype sequence (A. Grakoui et a 1., Journal of Virology, 67, 2832-2843 (1993)) or cut with NS3 protease. The sequence confirmed by the present inventors to be cleaved, more specifically, P6 is Asp, Asn or Glu, P5 is Lys, Asp, Ser, Asn or Gly, P4 is I16 or & 1 , P3 is preferably Val, Glu, 11 & 1 or 116, and 2 is preferably syrup 0, hydroxy Pro, Ρhe, G1u, Va1 or Tyr. P 4 ′ is preferably Trp, Tyr, A1a or Leu.

Examples of preferred substrates include, for example, “Ly s—G 1 u—Asp—Va 1—Va 1Pro—Cys—Al a—Me t—A la—Leu—pNA ”or“: Lys—G lu—As p—Val—Val—Pro—Cys—A la—Me t— Al a — Le eu — AMC ”(SEQ ID NO: 1). In general, 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.

 Further, the present invention, specifically, 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 number after 4A indicates the amino acid number counted from the N-terminus of NS4A at the N-terminus and C-terminus of each NS4A fragment.) 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. Murakami et al., Anal.Biochem., 110, 232-239 (1981)", "A. Reinharz & M. Roth, Eu ropean J. Biochem., 7, 334-339 (1969)", "0.Kuo et al., Biochemistry, 33, 8347-8354", "Konig W. & R. Geiger, Chem. Ber., 103, 788-798 (1970)",

Methods such as "T. Morita et al., J. Biochemistry, 82, 1495-1498 (1977)" and "M. Zierman et al., Anal.Biochei., 78, 41-51 (1977)" This is done with reference to. 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. In synthesizing peptides by the solid-phase method, excellent automatic peptide synthesizers, such as Applied Biosystems Inc. peptide dynamic synthesizer 430A, are commercially available, and according to the standard operation program of such an apparatus. Just do it. The method for producing the synthetic substrate of the present invention is not limited only to the application of a commercially available device at present.

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). 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. According to the above-mentioned Atsushi method by Kakiuchi et al. (N. Kakiuchi et al., BBRC., 210, 1059-1065 (1995)), the enzyme concentration and substrate concentration were 80 終 g / ml and 86〃M, respectively. However, in the case of the MCA substrate of the present invention, the final concentrations of the enzyme and the substrate of 10 to 40 μg / ml and 1 to 20 μM are sufficient, which are much higher than those of the conventional Atsushi system. Sensitivity. In the case of a pNA substrate, it can be used preferably at a concentration of 0.2 to 2%. In the case of an MCA substrate, it can be used preferably at a concentration of 1 to 100〃M.

 Since 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. Virol., 69, 4017-4026 (1995)], an 18th to 40th peptide containing this sequence (LTTGSVVIVGR II LSGRPAVVPD; hereinafter abbreviated as "4A 18-40") was synthesized and added to the Atsushi system. It was found that when 4A18-40 "was added in an equimolar amount or more to the enzyme, the substrate cleavage activity of NS3 protease was significantly enhanced (see Comparative Example 1). For example, when “4A18-40” was added twice as much as the amount of the enzyme, the reaction rate increased about 15 times. The knowledge of the present inventors has made it possible to measure protease activity with high sensitivity even with a small amount of enzyme.

 In addition, the abbreviation of the compound in this specification has the following meaning.

"Boc" is tertiary butoxycarbonyl, "tBu" is tertiary butyl (tert. Butyl), "Clt" is chlorotrityl, and "DCC" is Ν, Ν, -dihexylhexyl. Carbodiimide (Ν, Ν'-Dicyclohexylcarbodiimide), “DCM” is dichloromethane (dichloromethan), “DIEA” is は, Ν-diisopropyl Ethylamine (Ν, Ν-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 “Trt” represent trityl, respectively.

 In the present specification, the one-letter and three-letter notations of amino acids conform to the rules of the IUPAC C Biochemical Nomenclature Committee (CBN). For example, "Biochemical Dictionary (second edition)" Chemical Dojin, 1990, p. 1468 ". BRIEF DESCRIPTION OF THE FIGURES

 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. Unless otherwise specified, 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.

 [Comparative Example 1] Enhancement effect of NS3 protease by adding 4A18-40 The method of expressing NS3 protease is known, but in the following examples, Kakiuchi et al.

Kakiuchi et al., BBR, 210, 1059-1065 (1995)) used a NS3 region expressed as a fusion protein with MBP (Maltose binding protein) (hereinafter referred to as “MBP-NS3”). Call). Add MBP-NS3 (final concentration 2.2〃M) to the enzyme reaction mixture (50 mM Tris-HC 1 (ρΗ7.6), 30 mM NaC 2 mM DTT), and add 4A 18-40 (final concentration 4.4 enzyme concentration). The total amount was 47.5 1 in the presence or absence. After preheating at 25 ° C for 30 minutes, 2 Omer peptide group imitating NS 5A / 5B sequence: HI (GEAGDD IVPCSMSYTWTGAL) 2. Add 5〃1 (final concentration 100〃M) and digest at 37 C The reaction was performed. The reaction was stopped by adding 1〃1 of 5M acetic acid every hour. After the reaction was stopped, the reaction solution was separated by reversed-phase HPLC, and the digestibility of the substrate was determined from the decrease rate relative to the peak area of the substrate when the enzyme was not digested.

 As a result, the reaction rate increased 15-fold in the presence of 4A18-40 compared to the absence (Fig. 1). In the following examples, all NS3 protease digestions were performed in the presence of 4A18-40.

[Reference Example 1] Identification of minimum unit of substrate necessary for NS3 protease digestion of HCV To design a suitable synthetic substrate for two-step method used for measuring NS3 protease activity, substrate sequence We examined how much can be shortened. Based on the sequence between NS5A / 5B, one of the recognition sequences of the NS3 protease of HCV, the peptides shown in Table 1 below were synthesized. Lysine and aspartic acid shown in lowercase letters on the N-terminal side and C-terminal side are artificially added to increase water solubility. 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. After preheating at 25 ° C for 30 minutes, add the following peptide substrates to each of 2.5 (1 (final concentration 100 M), 314 at 25 ° (3 hours at 25 ° C, otherwise The digestion reaction was performed for 6 hours at C. The reaction was stopped by adding 1〃1 of 5 M acetic acid .. After the reaction was stopped, the reaction solution was reverse-phase HPLC (ODS 80 Tm / TOSO CORPORATION / 2. (15 x 30 cm), and the digestibility of the substrate was determined from the reduction ratio of the substrate to the beak area of the enzyme before digestion.

Table 1 Peptide sequence Digestibility (%) dNS4 + / dNS4-

PS GEAGDDIVPC--SMSYTWTGAL (SEQ ID NO: 2) 85/19

Dl EAGDDIVPC- -SMSYTWTGA (SEQ ID NO: 3) 92/14

D2 AGDDIVPC- -S SYTWTG (SEQ ID NO: 4) 75/8

D3 GDDIVPC- -SMSYTWT (SEQ ID NO: 5) 59 / nd

D4 GDDIVPC- -SMSYTW (SEQ ID NO: 6) 44/4

CO kkGDDIVPC- -SMSYT (SEQ ID NO: 7) 88 / nd

CI kkGDD! VPC- -SMSY (SEQ ID NO: 8) 89/7

C2G GDDIVPC- -SMS (SEQ ID NO: 9) 22/2

Nl DDIVPC- SMSYkdk (SEQ ID NO: 10) 62/5

N3 IVPC- -SMSYkdk (SEQ ID NO: 11) 49/9

N3d VPC- -SMSYkd (SEQ ID NO: 12) 2/0

N3k IVPC- -SMSYk (SEQ ID NO: 13) 6/2

N3Y IVPC- -SMSY (SEQ ID NO: 14) 3/0

N3nk kDklVPC- -SMSY (SEQ ID NO: 15) 85/8

314 kDklVPC- -SMSW (SEQ ID NO: 16) 22/2 dNS4 +: in the presence of 4A18-40

dNS4-: In the absence of 4A18-40

nd: not tested [Reference Example 2] Substrate specificity of APM

 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.

 100 one? The amount of pNA released (absorption at 405 nm) 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.

 In addition, it was determined in reverse phase how APM can digest the C-terminal fragment SMSYTWTG or SMSY kdk generated by digesting the substrate indicated by `` D2 '' or `` N3 '' in Table 1 with NS3 protease. Analysis by HPLC revealed that both were hardly digested from the first residue to the third residue.

 [Reference Example 3] Peptide substrate satisfying both substrate specificity of NS3 protease and APM

It was confirmed that the peptides shown in Table 2 below satisfied the substrate specificity of both NS3 protease and APM under the same conditions as in Reference Example 1 except that the digestion reaction was performed at 37 ° C for 3 hours. That is, 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. In "N301", which replaces Ser in "N3nk" with Ala, 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.

Beptide sequence Digestibility (%)

N3nk kDklVPC-SMSY (SEQ ID NO: 15) 40

 N301 kDklVPC-AMAY (SEQ ID NO: 17) 19

 ED kEDIVPC-SMSY (SEQ ID NO: 18) 60

 N303 kDklVPC-AMAL (SEQ ID NO: 19) 21

 N307 kEDVVPC-AMAL (SEQ ID NO: 1) 41

 N307-pNA kEDVVPC-AMAL-pNA (SEQ ID NO: 1) 74

 N307-MCA kEDVVPC-thigh-MCA (SEQ ID NO: 1) 67

[Example] Production of chromogenic and fluorescent synthetic substrate for measuring NS3 protease activity

 (I) Synthesis of KEDVVPCAMAL-A (N307-pNA) (shown in Figure 3)

Fmo c-Ly s (Bo c)-Glu (t Bu) -Asp (t B u) — Val-Val—Pro (fragment 1) is obtained by using Fro—2 _ C 1 t -Cys (trt) -Ala-Met-Ala (fragment 2) was synthesized using A1a-2-C1t resin by a conventional method as follows. 1. Swell the resin with DMF.

 2. Dissolve the desired f-moc amino acid (20 ol) in 20 ml of DMF.

 3. Add 0.45M HBTU / HOBT dissolved in 40ml DMF and 7ml DIEA to the amino acid solution and stir for 5 minutes.

 4. Add the product from step 3 to the resin and stir for 1 hour.

 5. Wash the resin with DMF.

 6. Add 100 ml of DMF containing 20% piperidine to the resin and stir for 20 minutes.

 7. Repeat the above procedure with the f-moc amino acid to be added next.

The protection peptide was cut out by treating with a solution of acetic acid: TFE: DCM = 1: 2: 7 for 1 hour. After distilling off the solvent, the peptide was precipitated by ether.

Synthesis of Cys (trt) —Ala—Met—Ala—Leu—pNA (SEQ ID NO: 20) was performed as follows.

 1. Dissolve fragment 2 (3.85 g), Leu-pNAHC1 (1.63 g), HOBT (0.65 g) and DIEA (0.774 ml) in 50 ml of DMF.

2. Add lg of DCC dissolved in DMF at 0 ° C.

 3. After stirring overnight at room temperature, remove the urea with a filter.

 4. After distilling off the solvent, suspend the residue in 300 ml of ethyl acetate.

5. 0.5N HCK 5 NaHC0 after it it washed with ₃ and brine, then recover the machine solvent layer is distilled off.

 6. Add 100 ml of DMF solution containing 20% piperidine to the residue and treat for 20 minutes. 7. After distilling off the solvent, precipitate the peptide with ether.

 The synthesis of N307-pNA was performed as follows.

1. Dissolve Fragment 1 (3g) and Cys (trt) -Al a—Me t—Al a—Leu—pNA (2.1 g) and HOBT (0.4 g) in 40 ml of DMF. 2. Add lg DCC dissolved in DMF at 0 ° C.

 3. After stirring at room temperature for 6 hours, remove the urea with a filter.

 4. Add 10 ml piperidine to the filtrate.

 5. After treating for 20 minutes, evaporate the solvent and precipitate the peptide with ether.

6. Dissolve the peptide in 40 ml of cleavage solution (reagent K) and treat at room temperature for 2 hours.

7. Precipitate the peptide with ether.

 (II) Synthesis of N 307—MC A (shown in Figure 4)

 Using Leu-MCAHC1 in the same manner as above.

 (I I I) Synthesis and purification of peptides

 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%).

 (IV) Confirmation of completed synthetic substrate

 1) Mass spectrometry

 The molecular weight of the substrate was confirmed by “Electron spray (ESI) mass spectrometry”. Both Ν 307—ρΝΑ (FIG. 5) and N 307—MCA (FIG. 6) agreed with the target molecular weight.

2) Amino acid composition analysis

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

N 307-NA N 307 -MCA

Ala 1.90 (2) 1.91 (2)

A s x 0.94 (1) 0.94 (1)

Cy s nd nd

 G 1 x 1.00 (1) 0.99 (1)

Leu 0.97 (1) 0.96 (1)

L y s 0.93 (1) 0.94 (1)

Met 0.95 (1) 0.95 (1)

Pro 1, 33 (1) 1.30 (1)

Va 1 1.79 (2) 1.80 (2)

Both N307-pNA and N307-MCA had the amino acid composition predicted from the amino acid sequence.

 (V) Activity measurement of N S 3 protease using N307-pNA substrate

The reaction was performed using a 96-well plate (Maximum immunoplate / Nunc). L to 12 µg of MBP-NS3 and 4A18-40 (final concentration: 44 µM) were added to PBS buffer (containing 2 mM DTT) to give 99 µl. 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).

 (VI) Activity measurement of NS3 protease using N307-MCA substrate (Method 1

)

 The reaction was performed using a 96-well plate (Black Cliniplate Solid, Labsystems, Finland). 4 µg of MBP-NS 3 and 4A 18-40 (final concentration 22 µM) were added to a PBS buffer (containing 2 mM DTT) to give 99 µl. 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). After the reaction was completed, 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.

(VI I) N 307—Measurement of NS 3 protease activity using MCA substrate (Method 2)

The reaction was performed using a 96-well plate as in (VI). To a PBS buffer (including 2mM DTT) containing N307-MCA at a final concentration of 20〃M, add 0.3〃g of NS3ANS4A and 0.05M of APM at the same time to make a total volume of 100〃1. . NS3ΔΝS4A 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).

Industrial applicability

By measuring the activity of NS3 protease using the synthetic substrate of the present invention, 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.

Sequence listing

SEQ ID NO: 1

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Lys Glu Asp Val Val Pro Cys Ala Met Ala Leu

 1 5 10

Array features

 Location: 11

 Other features: Leu may be associated with a fluorophore or luminophore. SEQ ID NO: 2

Array length: 20

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Gly Glu Ala Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr Thr Trp

1 5 10 15

Thr Gly Ala Leu

 20 SEQ ID NO: 3

Array length: 18 Sequence type: amino acid

 Topology: linear

Sequence type: peptide sequence

 Glu Ala Gly Asp Asp He Val Pro Cys Ser Met Ser Tyr Thr Trp Thr

1 5 10 15

Gly Ala SEQ ID NO: 4

Array length: 16

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Ala Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr Thr Trp Thr Gly 1 5 10 15 SEQ ID NO: 5

Array length: 14

Sequence type: amino acid

 Topology: linear

Sequence type: ぺ butide

Array

 Gly Asp Asp He Val Pro Cys Ser Met Ser Tyr Thr Trp Thr

1 5 10 SEQ ID NO: 6 Array length: 13

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Array

 Gly Asp Asp He Val Pro Cys Ser Met Ser Tyr Thr Trp 1 5 10 SEQ ID NO: 7:

Array length: 14

Sequence type: amino acid

 Topology: linear

Sequence type: Beptide

Array

 Lys Lys Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr Thr 1 5 10 SEQ ID NO: 8

Array length: 13

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

Lys Lys Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr 1 5 10 SEQ ID NO: 9

Array length 10

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Gly Asp Asp He Val Pro Cys Ser Met Ser

 1 5 10 SEQ ID NO: 1 0

Array length: 13

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Asp Asp lie Val Pro Cys Ser Met Ser Tyr Lys Asp Lys 1 5 10 SEQ ID NO: 1 1

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Beptide

Array

l ie Val Pro Cys Ser Met Ser Tyr Lys Asp Lys

1 5 10 SEQ ID NO: 1 2

Array length: 9

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Val Pro Cys Ser Met Ser Tyr Lys Asp 15 SEQ ID NO: 1 3

Array length: 9

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Array

 He Val Pro Cys Ser Met Ser Tyr Lys 1 5 SEQ ID NO: 1 4

Array length: 8

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array He Val Pro Cys Ser Met Ser Tyr

 1 5 SEQ ID NO: 1 5

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Lys Asp Lys lie Val Pro Cys Ser Met Ser Tyr 1 5 10 SEQ ID NO: 1 6

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Beptide

Array

 Lys Asp Lys lie Val Pro Cys Ser Met Ser Trp 1 5 10 SEQ ID NO: 1 7

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide Array

 Lys Asp Lys He Val Pro Cys Ala Met Ala Tyr 1 5 10

SEQ ID NO: 1 8

Array length: 11

Sequence type: amino acid

 Topology: linear

Sequence type: Beptide

Array

 Lys Glu Asp lie Val Pro Cys Ser Met Ser Tyr 1 5 10

SEQ ID NO: 1 9

Array length: 11

Sequence type: amino acid

 Topology: linear

 Sequence type: Peptide

 Array

 Lys Asp Lys He Val Pro Cys Ala Met Ala Leu 1 5 10

SEQ ID NO: 20

 Array length: 5

 Sequence type: amino acid

Topology: linear Sequence type: peptide

Array

 Cys Ala Met Ala Leu

 1 5

Array features

 Location: 1

 Other features: Cys is linked to a trityl group <Sequence features

 Location: 5

 Other features: Leu binds to pNA.

Claims

The scope of the claims 1. A synthetic substrate represented by the following formula (I).  (N-terminal) Z—Cys-A1a—Met—A1a—X—A—Y (C-terminal)  (I)  (Wherein, Z represents an amino acid or peptide, X represents Leu, Tp or Ty, A represents a bond or peptide, Y represents a fluorophore or a chromophore, and must be present in the Z-Cys region. At least one of the peptide bonds is not easily digested by aminopeptidase, and any peptide bonds present in the XA region are destroyed by aminopeptidase.)  2. The chromophore or fluorophore is 7-amino-4-methylcoumarin (7-amino-4-methyl-coumarin), 7-amino-14-trifluoromethylcoumarin (7-amino-4-trif 1 uoromethyl-). 2. The synthetic substrate according to claim 1, which is coumarin), noranitroaniline (para-mtroaniline) or 3-naphthylamine (? -naphthylamine).  3. The amino acid or amino acid residue which is hardly digested by aminopeptidase existing in the region of Z-Cys is Asp, Ser, Pro, I1e or Va1. 3. The synthetic group according to 1 or 2.  4. Claim 1 which is “: Lys—Glu—Asp—Val—Val—Pro—Cys—Ala—Met—Ala—Leu—Y” (SEQ ID NO: 1) Synthesis of 5. The synthetic substrate according to claim 4, wherein the chromophore bound to the C-terminal is paranitroaniline.  6. The synthetic substrate according to claim 4, wherein the chromophore bound to the C-terminal is 7-amino-4-methylcoumarin. 7. The energy of performing double digestion of hepatitis C virus NS3 protease and aminopeptidase on the synthetic substrate described in any of the range of claims 1 to 6, A method for measuring the activity of hepatitis C virus NS3 protease.  8. A step of double digestion with HC3-derived NS3 protease and aminobeptidase in the presence of the hepatitis C virus NS4A-derived peptide. The method of range 7.  9. The method according to claim 8, wherein the NS4A-derived peptide is a peptide containing an amino acid sequence at positions 18 to 40 from the N-terminus of NS4A.