AU2545099A - Peptide inhibitors of the serine protease activity associated to the ns3 proteinof hcv, relevant uses and process of production - Google Patents

Description

WO 99/38888 PCT/IT99/00022 -1 PEPTIDE INHIBITORS OF THE SERINE PROTEASE ACTIVITY ASSOCIATED TO THE NS3 PROTEIN OF HCV, RELEVANT USES AND PROCESS OF PRODUCTION. DESCRIPTION 5 Field of the invention The present invention relates to the molecular biology and to the virology of the human hepatitis C virus (HCV) . In particular, it relates to the research of molecules that could potentially be adopted in the 10 therapy of the variety of hepatitis consequent to the infection of this virus. State of the Art Presently, the method most frequently adopted in art in order to generate molecules with therapeutical 15 potentialities towards viral pathologies, is that of subjecting collections of compounds, containing a large number of single chemical entities of high molecular diversity, to an automatized program to detect the existence of single active agents. Those agents are then 20 subjected to further chemical modifications aimed at improving their therapeutical potential. In the specific case of HCV, methods allowing in vitro culture and the passage of infective particles in cellular cultures have not been described in art. 25 Moreover, the only animal infection models utilise primates. The high cost of these animal models drastically limits the number of preparations that can be assayed for their antiviral capability. In practice, identification of molecules having a therapeutic 30 potential is at present limited to the identification of molecules capable of interfering with the biological activity of viral proteins somehow expressed outside of the complete viral context. Study of the HCV biology, although heavily hindered by the limitations discussed 35 above, has allowed identification of viral proteins whose biological activity is deemed essential for the viral replication, and whose inhibition is therefore deemed to WO 99/38888 PCT/IT99/00022 -2 have a probable therapeutic usefulness. The HCV virus is the principal etiologic agent of non-A non-B hepatitis (NANB), whose chronic infection in serum is often a cause of liver cirrhosis and may 5 progress in 20 - 30 years time to hepatocellular carcinoma. Regarding the molecular biology of HCV, as is known, it is a *virus with a membrane, containing an encapsidized RNA+ genoma of approximately 9.4 Kb. The genomic organisation of the HCV virus comprises 10 a structural region, coding for proteins concurring to form the virus structure, and a non-structural region NS, coding for functional proteins (helicase/protease;

RNA

dependant RNA polymerase). Both regions are placed in a single open reading 15 frame (ORF) variable between 9030 and 9099 nucleotides that is translated in a single viral polyprotein, whose length may vary between 3010 and 3033 amino acids, only afterwards, during the viral infection cycle, proteolytically processed in individual genic products. 20 Different molecular biology studies have indicated that the polyprotein ripening is due to different enzymes. In particular, the processing of the nonstructural portion of the HCV polyprotein, comprising the NS2-NS3-NS4A NS4B-NS5A-NS5B proteins (placed in this order), is due 25 to the activity of two different proteases, on of which is a serine-protease contained inside of the N-terminal region (amino acids 1-181) of the NS3 protein (therefore named NS3 protease), responsible of the cleaving at NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B sites 30 (Bartenschlager, R. Antiviral Chemistry & Chemotherapy 1997). NS3 is a 68 KDa protein, in fact showing 2 functional domains, one serine protease domain in the first 200 amino-terminal amino acids and a RNA-dependant 35 ATPase domain at the carboxy- terminus. Initially the substrate specificity of NS3 protease has been qualitatively investigated using transient WO 99/38888 PCT/IT99/00022 -3 transfection (Kolykhalov, A. et al. J. Virol. 1994; Bartenschlager, R., et al. J. Virol. 69, 198-205, 1995), in vitro translation (Leinbach, S., et al Virology 1994), or intracellular processing of fusion proteins in E.coli 5 (Komoda, Y., et al. J. Virol. 1994). More recently, efficient heterologous expression and purification of the enzymatically active protease domain have been described (Shimizu, Y., et al. J. Virol. 1996; Steinkthler, C., et al. J. Biol. Chem. 1996; Kakiuchi, N., et al. Biochem. 10 Biophys. Res. Commun. 1995; Overton, H., et al. J. Gen. Virol. 1995; D'Souza, E. D. A., et al. J. Gen. Virol. 1995; Suzuki, T., et al. J. Gen. Virol. 1995; Shoji, I., et al. Hepathology 1996; Mori, A., et al. FEBS Lett. 1996; Hong, Z., et al. Anal. Biochem. 1996; Steinkujhler, 15 C., et al. J. Virol. 1996), and optimal conditions for the determination of protease activity have been established (Steinkuhler, C., et al. J. Virol. 1996; Urbani, A., et al. J. Biol. Chem. 1997; Bianchi, E., et al. Anal. Biochem. 1996; Taliani, M., et al. Anal. 20 Biochem. 1996). According to what has been described on the virus biology and on the infection and viral replication cycles, it is evident that a substance capable of interfering with the NS3 protein associated proteolytic 25 activity might constitute a new therapeutical agent. In fact, inhibition of this protease activity would entail the stopping of the proteolytic processing of the non structural region of the HCV polyprotein and would, therefore, hinder viral replication in infected cells. 30 The development of methods enabling the production of enzymatically active NS3 and of enzymatic activity assay methods allowed the setting-up of research programs of new chemical entities, capable of interfering with the NS3 protease activity. These programs essentially consist 35 of the introduction in the enzymatic activity assays of a large number of single chemical entities in order to determine their specific activity on protease. Compounds WO 99/38888 PCT/IT99/00022 -4 thus defined as active, are then subjected to further chemical modifications, aimed at improving their therapeutic potential. A second commonly adopted approach comprises the rational modification of substrates ligands 5 of the protease, in order to develop compounds, capable of altering or abolishing biological activity, with a high binding affinity. Summary description of the invention The subject of the present invention are peptides 10 capable of inhibiting protease activity associated to the HCV NS3 enzyme. They have been identified during studies on NS3 enzyme substrate specificity, due to the identification among products of NS3 proteolytic action on the viral polyprotein, of some peptides capable of 15 acting as inhibitors of the protease itself. In particular, it was found that proteolysis derived peptides bearing in the C-terminal portion of their sequence the amino acids naturally occurring in P4, P3, P2 and Pl positions (according to the definition 20 of Schechter, I. and Berger, A., 1967) of the junction sites NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B, exhibit an inhibitory capacity towards the NS3 protease itself. The sequences of the abovementioned four cleaving sites of the NS3 enzyme are listed in table I. 25 TABLE I: Sequence of the NS3 cleaving sites Cleaving site Sequence NS3/NS4A D L E V V T S T W V NS4A/NS4B D E M E E C A S H L NS4B/NS5A D C S T P C S G S W NS5A/NS5b E D V V C C S M S Y

P

6

-P'

4 residues of HCV Bk strain polyprotein cleaving sites. Amino acids in the sequences are indicated with the one-letter code.

P

1 and P' 1 are bolded. Among peptides of viral origin, a particular 30 inhibitory effectiveness was evidenced in the two peptides indicated in the sequence listing as SEQ ID NO:1 and SEQ ID NO: 8, the sequence thereof corresponds WO 99/38888 PCT/IT99/00022 -5 to the P6-Pl residues respectively of sites NS4A/NS4B and NS5A/NS5B. The fact that products of the enzymatic action are capable of acting as competitive inhibitors of the 5 enzyme responsible of their production is very unusual for a serine protease as NS3, and therefore unexpected, opening new perspectives for the development of more effective drugs against nonA-nonB hepatitis. Following the characterization of such peptides, it 10 was further assessed that such inhibitory capability can be specifically ascribed to the presence of at least a free acid function in the C-terminal position of such peptides. The amino acids in the other positions of the peptides, although significantly affecting the level of 15 inhibitory capability of the peptides, can not by themselves confer inhibitory properties to the same peptides. Accordingly, in correspondence to each position have been identified the amino acid or the amino acids 20 increasing the relevant inhibitory capability. Hence further peptides, presenting at their C-terminal position an acid function, have been chemically synthesised, whose amino acidic sequence is partly obtained by viral peptides sequences, characterised in 25 that they show a remarkable increase of inhibitory capacity. In any case, as the sequence of the peptides of viral origin corresponds to the P6-Pl residues of the viral sites, which they are derived from, the positions 30 occupied by each amino acid residue in all the peptides obtained have been conventionally denominated from P6 to P1, P6 being the the position of the N-terminal end and P1 being the position of the C-terminal end. In relation to that, and to what will be disclosed 35 hereinafter, subject of the present invention is first of all peptides consisting in six amino acid residues arranged in positions from P6 to P1, P6 being the WO 99/38888 PCT/IT99/00022 -6 position of the N-terminal end and P1 being the position of the C-terminal end, characterized in that the amino acid in the P1 position has at least a free acid function and in that they are capable of inhibiting the 5 protease activity of the HCV virus associated to the NS3 protein. In particular subject of the invention are: - the peptides wherein the amino acid in P1 position is a cysteine, an analog or a derivative 10 thereof, and in particular an amino acid selected from the group comprising L-cysteine, D-cysteine, homocysteine, S-methylcysteine, alanine, s ethylcysteine, threonine, methionine, serine and penicillamine; 15 - the above mentioned peptides having in P6 position an acid function, in particular selected from the group comprising aspartic acid, succinic acid and acylsulfonamide; - the. above mentioned peptides having in PS 20 position an acid function, in particular selected from the group comprising aspartic acid, succinic acid, acylsulfonamide; - the above mentioned peptides having in the P4 position an hydrophobic amino acid, in particular 25 selected from the group comprising 3,3,diphenilalanine, leucine, isoleucine and phenylglicine; - the above mentioned peptides having in the position P3 an amino acid selected from the group comprising glutamic acid, valine and isoleucine, and in 30 a realization form having in the position P5 an amino acid selected from the group comprising aspartic acid, p-nitrophenylalanine, tyrosine, g-carboxyglutamic acid, D-phenylalanine, D-tyrosine, D-valine, D-iscleucine, D 3,3-diphenylalanine, D-aspartic acid, D-glutamic acid 35 and D-g-carboxyglutamic acid, in another realization form, together with such amino acid in P5 position or not, in the position P1 an amino acid selected from the WO 99/38888 PCT/IT99/00022 -7 group comprising aminobutyric acid, norvaline and valine. Cases of particular relevance are the one wherein the peptides are capable of inhibiting 50% of the NS3 5 enzymatic activity at a concentration lower than or equal to 2 pM (IC 5 0 ) , and the one wherein the peptides have in the positions P4, P3, P2 and P1, the amino acids naturally occurring respectively in P4, P3, P2 and P1 positions of one of the junction sites of the HCV virus, 10 said junction sites being selected from the group comprising NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B. Further subject of the present invention are the peptides obtainable by the proteolysis reaction of 15 polipeptides containing at least one of the junction sites of the polyprotein of said HCV virus, said junction sites being selected from the group consisting of NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junction sites. 20 Thus, are of particular relevance the case wherein the junction sites consist of decapeptides, containing the amino acids naturally occurring in the positions P4, P3, P2 and P1 of NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junction sites; the case wherein the HCV 25 viruses is selected from the group comprising HCV virus of la, 1b, 1c, 2a, 2b, 2c, 2d, 2e, 2f, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 5a, 5a, 6b, 7a, 7b, 7c, 7d, 8a, 8b, 9a, 9b, 9c, 10a and la genotype, described as non limiting examples in Tokita, M. et al J. of Gen. Virol. 30 1996; and in Myakawa, Y., et al, Molecular Med. Today, 1995, and the case wherein the virus is of H-FDA, H-AP, HCV-1, HCV-J, HCV-BK, HC-J6, HCV-T, HC-J8, HCV-JT and/or HCV-JT' strain described as non-limiting examples in Grakou et al, J. of Virol., 1993. 35 In a preferred embodiment, the peptides according to the present invention are those having an amino acid sequence selected from the group comprising the WO 99/38888 PCT/IT99/00022 -8 sequences reported in the annexed sequence listing as from SEQ ID.NO:1 to SEQ ID NO:69. A further subject of the present invention is the use of the abovementioned peptides for derivation of 5 binding or inhibition assays of the enzymatic activity of HCV NS3 protease, but above all the utilisation of those peptides for the preparation of drugs for the treatment of non-A non-B hepatitis. Moreover, of particular relevance is the use that 10 may be done of this peptide inhibitors in the "co crystallisation" with the enzyme, to obtain structural information on the enzyme active site , thereby facilitating the discovery of new enzymatic activity modulators, of peptidic nature or not. 15 All peptides as described above can be used to prepare pharmaceutical compositions, characterised in that they . comprise beside at least one of the aforedescribed peptides, a pharmaceutically effective carrier, vehicle or auxiliary agent, as well as 20 compositions that likewise comprise at least one of said peptides. A further subject of the present invention is a process for the production of at least one of the afore mentioned peptide characterized by the step of carrying 25 out the the proteolysis of polypeptides containing at least one among the sequences of the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and/or NS5A/NS5B junction sites of the polyprotein of HCV virus. In particular, cases wherein the proteolysis 30 reaction is operated by NS3 protease of the HCV virus are considered wherein HCV displays a genotype la, lb, 1c, 2a, 2b,. 2c, 2d, 2e, 2f, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, Sa, 5a, 6b, 7a, 7b, 7c, 7d, Ba, 8b, 9a, 9b, 9c, 10a and/or lla, described as non-limiting examples 35 in Tokita, M. et al J. of Gen. Virol. 1996; and in Myakawa, Y., et al, Molecular Med. Today, 1995, and the case wherein the virus is of H-FDA, H-AP, HCV-1, HCV-J, WO 99/38888 PCT/IT99/00022 -9 HCV-BK, HC-J6, HCV-T, HC-J8, HCV-JT and/or HCV-JT' strain described as non-limiting examples in Grakou et al, J. of Virol., 1993. Another case of particular relevance is the one 5 wherein the junction sites, contained in the NS3 polypeptide substrate, consist of decapeptides, containing the amino acids naturally occurring in P4, P3, P2 and P1 positions of the same junction sites themselves. 10 The invention will be better understood with the aid of the annexed figures. Brief description of the drawings Figure 1 shows the reaction kinetics of the NS4A/NS4B substrate cleaving catalysed by NS3 protease. 15 Figure 2 shows the determination of the IC50 of peptide SEQ ID NO:l by displacement of the fluorescent marker derived from peptide SEQ ID NO: 69. In fig. 2a the intensity decrease of the fluorescence spectrum of the NS3 protease-peptide complex SEQ ID NO: 69 is 20 plotted against the increasing concentration of the peptide SEQ ID NO: 1. In fig. 2b the variation of intensity of the fluorescence spectrum at 520nm is plotted against the peptide SEQ ID NO: 1 concentration for the IC50 assessment. 25 Detailed description of the invention The subject of the present invention are peptides having a relevant inhibitory capacity towards of the NS3-associated protease activity, some of which correspond to those of viral origin, others thereby 30 obtained by modifications of one or more amino acid residues. In table II are particularly reported, as a non limiting example, codes and features of 69 peptide inhibitors obtained from the study on NS3 enzyme 35 substrate specificity, and the concentration in pM of compound is indicated, whereto 50% inhibition of NS3 enzymatic activity (IC50) is obtained, as a reference WO 99/38888 PCT/IT99/00022 -10 parameter for the assessment of the higher or lower efficiency of inhibitory capacity of the single peptides. TABLE II: Summary list of sequences of peptide inhibitors 5 according to the invention SEQ ID Amino acid sequence IC50 NO:1 Asp Glu Met Glu Glu Cys 1.0 NO:2 Asp Glu Met Glu Glu (D)Cys 4.0 NO:3 Asp Glu Met Glu Glu Abu 5.8 NO:4 Asp Glu Met Glu Glu Ser 41 NO:5 Asp Glu Met Glu Glu Gly 62 NO:6 Met Glu Glu Cys 150 NO:7 Glu Met Glu Glu Cys 21 NO:8 Glu Asp Val Val Cys Cys 5.3 NO:9 Glu Asp Val Val Abu Cys 2.8 NO:10 As'p Glu Val Val Cys Cys 2.1 NO:11 Glu Asp Val Val Gly Cys 20 NO:12 Asp Glu Met Glu Glu Alg 12 NO:13 Glu Asp Val Val MeGly Cys 21 NO:14 Asp Glu Met Glu Glu CysN 30%@64pM NO:15 Glu Asp Val MeVal Abu Cys 230 NO:16 Glu Asp MeVal Val Abu Cys 1,3 NO:17 Asp Glu Met Glu Glu Cys(ol) 130 NO:18 GluS Met Glu Glu Cys 1.3 NO:19 MetS Glu Glu Cys 77 NO:20 AsGlu Met Glu Glu Cys 0.6 NO:21 Asp Glu Met Glu Glu VGly 38 NO:22 Asp Glu Met Glu Leu Cys 1.1 NO:23 Asp Glu Met Glu Cha Cys 0.3 NO:24 Asp Glu Met Glu Nap Cys 0.8 NO:25 AspS Val Val Abu Cys 4.6 NO:26 Glu Asp Val Val Abu (D)Cys 194 NO:27 Asp Glu Met Glu Glu Cys(Me) 16.7 NO:28 Asp Glu Val Glu Cha Cys 0.33 NO:29 Asp Glu Ile Glu Cha Cys 0.12 WO 99/38888 PCT/IT99/00022 -11 NO:30 Asp Glu Tyr Glu Cha Cys 0.2 NO:31 Asp Glu Phe Giu Cha Cys 0.42 NO:32 Asp Glu Leu Glu Cha Cys 0.12 NO:33 Asp Glu Cha Glu Cha Cys 0.14 NO:34 Asp Glu Nie Glu Cha Cys 0.22 NO:35 Asp Glu Dif Glu Cha Cys 0.05 NO:36 Asp Glu Tha Glu Cha Cys 0.8 NO:37 Asp Glu FCI Glu Cha Cys 0.3 NO:38 Asp Glu Phg Glu Cha Cys 01 NO:39 Asp Glu Dif Glu Cha (D)Cys 3.4 NO:40 Asp Glu Met Glu Glu bAla 20%@20011M NO:41 Asp Giu Met Glu Giu CysAs 4.0 NO:42 Giu Dif Glu Cha Cys 1.4 NO:43 Dif Giu Cha Cys 30 NO:44 Asp Glu Leu Val Cha Cys 0.08 NO:45 Asp Giu Leu Ile Cha Cys 0.06 NO:46 Asp MeGlu Leu Glu Cha Cys 1.0 NO:47 Asp Giu Dif Giu Cha AAia 7.1 NO:48 Asp Glu Met Glu Giu Cpc 9.0 NO:49 Asp Glu Duff Ile Cha 46 NO:50 Glu Dif Ile Cha Cys 2.5 NO:51 Dif Ile Cha Cys 100 NO:52 Asp Glu Met Glu Glu CnAia 19 NO:53 Asp Glu Dif le Cha Cys 0.06 NO:54 Asp Glu Leu Glu Cha Abu 1.6 NO:55 Asp Glu Leu Glu Cha Val 4.0 NO:56 Asp Glu Leu Glu Cha Nva 1.3 NO:57 Asp Asp Leu Glu Cha Cys 0.290 NO:58 Asp Fno Leu Gu Cha Cys 0.240 NO:59 Asp Tyr Leu Glu Cha Cys 0.135 NO:60 Asp Gia Leu Glu Cha Cys 0.05 NO:61 Asp (D)Phe Leu Gilu Cha Cys 0.820 NO:62 Asp (D)Tyr Leu Glu Cha Cys 0.680 NO:63 Asp (D)Vai Leu Glu Cha Cys 0.470 WO 99/38888 PCT/IT99/00022 -12 NO:64 Asp (D)Ile Leu Giu Cha Cys 0.330 NO:65 Asp (D)Dif Leu Glu Cha Cys 0.276 NO:66 Asp (D)Asp Leu Giu Cha Cys 0.122 NO:67 Asp (D)Glu Leu Giu Cha Cys 0.045 NO:68 Asp (D)Gla Leu Ile Cha Cys 0.0015 NO:69 Asp Glu Dpr(N-b-Dns)Glu Cha Cys 0.4 Abu = 2-aminobutyric acid Alg = allylglycine AsGlu = Glu presenting an acylsulfonamide in N-terminus 5 position AspS = Asp whereto a succinil group is bound bAla = beta-alanine Cha = beta-cyclohexylalanine CnAla = cyanoalanine 10 Cpc = 1-amino-1-cyclopentan-carboxylic acid CysAs = Cys presenting in C-terminal position an acylsulfonamide Cys(Me)= S-methylcysteine Cys(ol) = cysteinol 15 CysN = cysteamine Dpr = b-diaminopropionic acid AAla= dehydroalanine Dif = 3,3-diphenylalanine Dns = Dansyl (5-Dimethylamino-1-naftalensulfonyl) 20 FCI = 4-clorophenylalanine Fno = 4-nitrophenylalanine Gla = g-carboxyglutamic acid GluS = Glu whereto a succinyl group is bound MetS = Met whereto a succinyl group is bound 25 MeGlu = N-methyl-glutamic acia MeGly = methyl-glycine MeVal = methyl-Val Nap = naphtylalanine Nle = norleucine 30 Nva = norvaline WO 99/38888 PCT/IT99/00022 -13 Phg = phenylglycine Tha = 2-tienylalanine VGly = Vinylglycine Of the peptides listed in Table II, as already 5 said, two (SEQ ID NOS:1 and 8) are produced directly from the cleaving of the NS3 itself, respectively on the 4A/4B site (SEQ ID NO:l) and on the 5A/5B site (SEQ ID NO:8) of the viral polyprotein. This inhibition can be evidenced studying the time-dependence of the 10 proteolytic cleaving reaction, mediated by the NS3 enzymatic activity, of a substrate corresponding to site 4A/4B (see Table.I). Figure 1 shows that the enzymatic conversion of this peptide in its cleaving products decreases over time. Using methods known in the art it 15 is possible to estimate that this NS3 protease activity decrease is consistent with the forming, during the proteolytic cleaving reaction, of a product that inhibits the enzyme with a Ki constant, defined as the dissociation constant of the enzyme-inhibitor complex, 20 of 600 nM. Comparing values indicated in the above table, a remarkable increase is clearly evident of the inhibitory capacity of the most part of the synthetic peptides, as compared to the capacity related to peptides of viral origin. 25 Results are reported in detail hereinafter, with reference to substitutions of amino acids in P1-P6 positions of viral peptides sequence (SEQ ID NO:1, SEQ ID NO:8). P1 Residue 30 The substitution of a cysteine in the P1 position with a cysteamine, as in SEQ ID NO:14, or its reduction to an alcohol as in SEQ ID NO:17 (both belonging to the series derived from SEQ ID NO:1) entails a decrease of the inhibitory capacity of a >100-fold factor. The 35 carboxylic group of the cysteine was substituted by an acylsulfonamide group in the peptide represented by SEQ ID NO:41.

WO 99/38888 PCT/IT99/00022 -14 P5 and P6 residues With reference to both the series derived from SEQ ID NO:1 (IC50 = 1.0 IM) and from SEQ ID NO:8 (IC50 = 5.3 pM), the presence of an acid seems to be important, 5 in P5 as well as in P6. Actually, if P6 deletion from SEQ ID NO:1 causes a significant decrease of the inhibitory activity(SEQ ID NO:7, TC5C = 21 pM), the deletion of both residues causes a 10>-fold decrease (SEQ ID NO:6, IC50 = 150 pM). 10 This result is confirmed also when operating the same modifications in more potent analogs like SEQ ID NO:35 (IC50 = 0.055 p.M) and SEQ ID NO:53 (IC50 = 0.063 t M) . In the first case SEQ ID NO:42 (IC50 = 1.4 iM) and SEQ ID NO:43 (IC50 = 30 ptM) are obtained; in the second 15 case, SEQ ID NO:50 (IC50 = 2.5 pM) and SEQ ID NO:51 which yields 50% inhibition at a 100 pM concentration. However, aspartic acid in P6 of SEQ ID NO:1 can be replaced with a simple carboxylic acid, like succinic acid (with loss 'f the acetylaminic moiety), or with an 20 acylsulfonamide without observing a significant decrease of the inhibitory capacity (compare SEQ ID NO:18, IC50 = 1.3 gM e SEQ ID NO:20, IC50 = 0.6 4M). This has also been verified for the series derived from SEQ ID NO:8, with SEQ ID NO:25 (IC50 = 2.8 pM) active as SEQ ID NO:9 25 (IC50 = 2.8 pM). Lastly, the two acids in P5 and P6 SEQ ID NO:1 are interchangeable (compare SEQ ID NO:8, IC50 = 5.3 FM, with SEQ ID NO:10, IC50 = 2.1 pM). P2 substitutions 30 The effect of the P2 substitutions was studied in both the series derived from the original viral peptides. As for the series derived from SEQ ID NO:8 it was observed that while the substitution of the P2 cysteine with ami'nobutyric acid as in SEQ ID NO:9 (IC50 35 = 2.8 pM) is tolerated, Gly in the same position results in a peptide 10-fold less active (SEQ ID NO:11, IC50 = 20 pM).

WO 99/38888 PCT/IT99/00022 -15 A more dramatic effect is observed in the SEQ ID NO:1 derived series, where substitution of the glutamic acid in P2 with an hydrophobic residue maintains or even improves the inhibitory activity (SEQ ID NO:22, IC50 = 5 1.1 ptM; SEQ ID NO:24 IC50 = 0.8 pLM; SEQ ID NO:23, IC50 = 0.3 pM). P4 substitutions SEQ ID NO:23 was taken as a starting point to optimise the P4 position. This was realised synthesising 10 a series of analogs with the general structure of the starting sequence, presenting modifications only on the P4 position Results showed that the P4 position has a strong preference for hydrophobic amino acids, both with 15 aliphatic and aromatic side chains, the best residue being 3,3-diphenylalanine (SEQ ID NO:35, IC50 = 0.055 pt M), followed by leucine (SEQ ID NO:32, IC50 = 0.118 pM), isoleucine (SEQ ID NO:29, IC50 = 0.122 iM) and phenylglycine (SEQ ID NO:38, IC50 = 0.120 piM). 20 P3 substitutions SEQ ID NO:32 was taken in turn as a starting point to optimise the P3 position. As for the P4 position, the result was obtained systematically by synthesising a series of analogs that, though presenting the same 25 structure of the SEQ ID NO:32, were modified in P3 position only. Only two residues yielded a potency comparable with the glutamic acid in P3 of the SEQ ID NO:32, i.e. valine and isoleucine in P3. 30 P5 substitutions SEQ ID NO:32 was again taken as a starting point to optimise P3 position. As for P3 and P4 positions, the result was obtained systematically by the synthesis of a series of analogs that, though presenting the same 35 structure of the SEQ ID NO:32, were modified in P5 position only. The most notable L-amino acids in this position are P5 = aspartic acid (SEQ ID NO:57, IC50 = WO 99/38888 PCT/IT99/00022 -16 0.290 ptM), P5 = p-nitrophenylalanine (SEQ ID NO:58, IC50 = 0.240 pM), P5 = tyrosine, (SEQ ID NO:59, IC50 = 0.135 pM) e P5 = g-carboxyglutamic acid (SEQ ID NO:60, IC50 = 0.055 pM). Also amino acids with a D chirality are well 5 tolerated in this position, and in fact the two more potent compounds show this chirality: P 5 = D phenylalanine (SEQ ID NO:61, IC50 = 0.820 ptM), P5 =D tyrosine (SEQ ID NO:62, IC50 = 0.680 pM), P5 = D-valine (SEQ ID NO:.63, IC50 = 0. 470 pM) , P5 = D-isoleucine (SEQ 10 ID NO:64, IC50 = 0.330 pM), P5 = D-3,3-diphenylalanine (SEQ ID NO:65, IC50 = 0.276 pM), P5 = D-aspartic acid (SEQ ID NO:66, IC50 = 0.122 JM), P5 = D-glutamic acid (SEQ ID NO:67, IC50 = 0.045 pM) and P5 = D-g carboxyglutamic acid (SEQ ID NO:68, IC50 = 0.0015 JIM). 15 P1 substitutions The effects of the P1 residue in the SEQ ID NO:1 derived inhibitor series also parallels the trend observed for the substrate. In order of decreasing IC50 the residues are: cysteine(SEQ ID NO:1, IC50 = 1 JM), 20 aminobutyric acid (SEQ ID NO:3, IC50 = 5.8 pM), 1-amino 1-cyclopentancarboxylic acid(SEQ ID NO:48, IC50 = 9 JM), allylglycine (SEQ ID NO:12, IC50 = 12 pM), S-methyl cysteine(SEQ ID NO:27, IC50 = 17 JM), cyanoalanine (SEQ ID NO:52, IC50 = 19 9M), vinylglycine (SEQ ID NO:21, 25 IC50 = 38 pM), serine (SEQ ID NO:4, IC50 = 41 pM), glycine (SEQ ID NO:5, IC50 = 62 pM), -alanine (SEQ ID NO:40, 20% inhibition at a 200 JIM concentration). The chirality of the Pi cysteine must be L- in the SEQ ID NO:8, since inversion of chirality yields a 70 30 fold decrease in activity (SEQ ID NO:26, IC50 = 194 pM). Likewise, the D-cysteine for L-cysteine exchange is highly detrimental of the inhibitory capacity in the more potent analogs modified in P2 and P4 positions (compare SEQ ID NO:35, IC50 = 0.05 JIM and SEQ ID NO:39, 35 IC50 = 3.4 pM). L-cysteine cannot be exchanged with D-cysteine in SEQ ID NO: 8 (compare SEQ ID NO:9, IC50 = 2.8 JIM and SEQ WO 99/38888 PCT/IT99/00022 -17 ID NO:26, IC50 = 194 pM). Further analysis were carried out using as a basis the more potent analog SEQ ID NO:32 (IC50 = 118 nM) . These analysis confirmed that cysteine substitution 5 causes anyhow a 10-fold decrease in inhibitory activity; the best substitute is aminobutyric acid (SEQ ID NO:54, IC50 = 1.6 pM) together with norvaline (SEQ ID NO:56, IC50 = 1.3 p4M), followed by valine (SEQ ID NO:55, IC50 = 4.0 4M). 10 Deletion of the Pi residue in SEQ ID NO:49 yields a >700-fold decrease in activity. N-methylated Peptidomimetics derived from SEQ ID NO:1 and SEQ ID NO:8 As already said, beside having examined the effects 15 of the substitution of the amino acid residues in positions P1 and P6 of the original viral peptides, we have systematically examined also the effects of N methylation of the bond peptide in a series of analogs always derived from sequences SEQ ID NO:l and SEQ ID 20 NO:8. So far, only a general description has been given of the present invention. With the aid of the following examples, a more detailed description will now be given of specific embodiments thereof, with the purpose of 25 giving a clearer understanding of objects, features, advantages and methods of application of the invention. For the sake of simplicity, in the examples the amino acid residues are also indicated with the one-letter code. 30 Example 1 Enzyme preparation Escherichia coli BL21(DE3) cells were transformed with a plasmid containing the cDNA coding for the serine protease domain of the HCV BK strain NS3 protein (amino 35 acids 1-180) under the control of bacteriophage T7 gene 10 promoter. The protease domain was purified as previously described (SteinkUhler, C. et al., J. Biol.

WO 99/38888 PCT/IT99/00022 -18 Chem. 1996) . The enzyme was homogenous as assessed with electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulphate (SDS-PAGE) using as detector the silver stain, and over 95% pure as assessed from 5 reversed phase HPLC carried out using a 4.6 x 250 mm Vydac C4 column. Enzyme preparations were routinely checked by mass spectrometry done on HPLC purified samples, using a Perkin Elmer API 100 instrument, and N terminal sequence analysis carried out using Edman 10 degradation on an Applied Biosystems model 470A gas phase sequencer. Both techniques indicated that in more than 90% of the enzyme molecules the N-terminus methionine and alanine have been removed, yielding an enzyme starting with proline in position 2. Enzyme 15 stocks were quantitated by quantitative analysis of the amino acidic content, shock-frozen in liquid nitrogen and kept in aliquots at -80*C until use. Control experiments have proved that this freezing procedure does not interfere with the specific activity of the 20 enzyme. Peptide synthesis Peptide synthesis was performed by Fmoc chemistry(Phluorenhylmethyl-oxycarboiiyl) /t-Bu (tert buthyl) chemistry , essentially as described in Atherton 25 and Sheppard. (19-89) . Peptides were assembled on a Novasyn® TGA (Novabiochem) resin and cleaved off the polymer at the end of the synthesis with TFA 88%, phenol 5%, triisopropylsilane 2%, water 5% (Sole, N. A. and Barany, G. J. Org. Chem. 1992). 30 Crude peptides were purified by reversed-phase HPLC on a Nucleosyl C18, 250 x 21 mm, 100 A, 7 pm using water, 0.1% TFA and acetonitrile 0.1% TFA as eluents. Analytical HPLC was performed on Ultrasphere C18, 250 x 4.6 mm, 80 A, 5 pm (Beckman) . Purified peptides were 35 characterised by mass spectrometry, [ H]-NMR and amino acid analysis. HPLC protease activity assay WO 99/38888 PCT/IT99/00022 -19 Concentration on stock solutions of peptides, prepared in DMSO or in buffered aqueous solution and kept at -80oC until use, was determined by quantitative amino acid analysis performed on azeotropic HCl 5 hydrolysed samples. If not differently specified, cleaving assay wa8 performed in 57 p1 50 mM Tris pH 7.5, 2% CHAPS, 50% glycerol, 10 mM in DTT (buffer A), to which 3 pl of the substrate peptide Ac DEMEECASHLPYK(Ac)-NH2 were additioned. As protease co 10 factor a peptide spanning the central hydrophobic core (residues 21-34) was used of the NS4A protein, with a three-lysine tag at the N-terminus to increase solubility (Bianchi, E. et al., Biochemistry 1997), Pep4AK (KKKGSVVIVGRIILSGR-NH2) . PeP4AK was pre-incubated 15 for 10 minutes with 10-50 nM protease prior to the addition of the substrate. Incubation time was chosen in order to obtain a substrate conversion of less than 7%. The reaction was stopped by addition of 40 pl 1% TFA, and the extent of substrate cleaving was determined by 20 HPLC using a Merck-Hitachi chromatograph equipped with an autosampler. '80 p1 of sample was injected on a Lichrospher C-18 reversed phase cartridge column (4 x 75 mm, 5 pm, Merck) and fragments were separated using a 10-40% acetonitrile gradient at 5%/min using a flow rate 25 of 2.5 ml/min. Peak detection was accomplished by monitoring both absorbance at 220 nm and fluorescence of the tyrosine residue (%ex = 260 nm, Xem = 305 nm). Cleaving products were quantitated by integration of chromatograms with respect to appropriate standards. 30 Initial rates of cleaving were determined on samples characterized by a substrate conversion rate of less than 7%. Kinetic parameters were calculated from the initial rates as a function of substrate concentration with the help of KaleidographO software, assuming 35 Michaelis-Menten kinetics. Microplate p-otease activity assay The HCV-protease (J strain) was stored until use at WO 99/38888 PCT/IT99/00022 -20 -80 0 C in 250 mM NaCl, phosphate buffer pH 6.5, 50% glycerol, 0.1% CHAPS; PeP4AK was stored at -80 0 C in DMSO; the tritiated substrate Ac-DEMEECASHLPYK ( 3 H-Ac)

NH

2 and the corresponding cold substrate Ac 5 DEMEECASHLPYK(Ac)-NH2 were stored at -80 0 C in DMSO/DTT. The assay was run in Costar polypropilene 96-well plates. The composition of the reaction mixture was as follows (100 pl): Glycerol 15% 10 DTT 30 mM Hepes pH 7,5 50 nm Triton X-100 0.05% Protease 10 rM hot + cold substrate S pM (300.000 cpm) 15 PeP4AK 15 pM The reaction mixture was diluted in DMSO (final concentration 10% DMSO) PeP4AK was pre-incubated with protease for 5 min prior to addition of substrate mix. In these conditions, 20 the substrate Km was 7±2 pM. Plates were shaken for 30 minutes at room temperature, then a ionic exchange resin (100 41 of 20% Fractogel TSK-DEAE@ 650S, Merck) was added to capture unprocessed substrate and plates shaken for another 10 minutes. After allowing the resin to 25 settle by gravity, 30 p.l of the reaction mix were transferred in a 96-well plate (Picoplate, Packard), admixed with 250 p.l of scintillation cocktail Microscint 40, and the radioactivity measured in a scintillation Packard Top Count $-counter. 30 Example 2 Competition assay based on product inhibitors The property of peptides, derived from the cleaving of the NS3 protease substrates, of binding to the active site of the enzyme, is exploitable for the development 35 of competition assays wherein an inhibitor peptide specifically marked is replaced by another molecule binding to the same site. This technology is exploitable WO 99/38888 PCT/IT99/00022 -21 for the identification of NS3 protease competitive inhibitors. The marking of the inhibitor peptide can be obtained with the introduction of functionalities chemical, radioactive, fluorescent, luminescent or 5 coloured using techniques known in art. For instance, introduction techniques of 12sI atoms iT: peptides containing tyrosine residues are known. It is also possible to synthesise peptides binding the active site of NS3 protease using amino acid residues marked with 10 radioactive isotopes like 3H, 14C or 3S. In art, even chemical modification techniques are known of peptides that can be adopted to introduce a radioactive marker in a peptide using reagents containing radioisotopes. For example, it is possible to mark with 3H a peptide 15 sequence containing primary aminic groups by reaction of said groups with acetic anhydride containing 3H. Peptides binding NS3 active site containing radioisotopes can be adopted to find other compounds binding the same site using techniques known in art. For 20 instance, a peptide having a sequence that binds to NS3 protease active site marked using the abovedescribed techniques can be added to i buffered solution containing NS3 protease or NS3 protease and. its cofactor NS4A, or peptides deriving from the sequence of this 25 cofactor. The protease bound to the peptide can be isolated using filtration techniques, chromatographic resins bonding, or precipitation using saline solutions or organic reagents. The amount of marked peptide can easily be determined using detecting techniques of the 30 radioactive decay process as scintillation. In this process, the addition of a substance capable of binding to the NS3 active site prior to protease isolation using said techniques, entails the displacing of the marked peptide and therefore a reduction in the emission of the 35 radioactive decay products. It is also possible to introduce in a peptide having a sequence binding to the NS3 protease active WO 99/38888 PCT/IT99/00022 -22 site a chemical functionality with fluorescent properties. It is known in art that the spectroscopic properties of some chemical functionalities undergo alterations depending on the physic-chemical conditions 5 wherein the spectroscopic propterties are determined. These conditions comprise pH, ionic strength, dielectric constant and the specific solvent wherein spectroscopic measurements are carried out. In particular, it is known that some molecules, once bound to proteins undergo 10 spectroscopically detectable changes. Some of these molecules are described in "Handbook of Fluorescent Probes and Research Chemicals" and are commercially available. Others are obtainable with chemical modifications of molecules of known spectroscopic 15 properties, capable of placing them in the context of a peptide binding to the NS3 protease active site. Examples of chemical functionalities that can be used with this aim are: fluorescein, mansyl, coumarin, rhodamine and dansyl. Particularly, dansyl was proved 20 capable of an interaction with tryptophan residues of resonance energy transfer. In this process, tryptophan is excited at a wavelength of between 280 and 295 nm and transfers its excitation energy to the dansyl molecule, that in turn emits energy at a wavelength of between 510 25 and 540 nm. The phenomenon of resonance energy transfer decays with the sixth power of the distance and is operative at distances of between 10 and 100 A, making it extremely sensitive to determine the bond between two molecules. 30 The molecule in SEQ ID NO:69 is a hesapeptide derived by the optimization of the sequence of a NS3 protease cleaving product, SEQ ID NO:23, wherein the methionine residue was replaced with an 2,3 diaminopropionic acid residue, derivatized on P3 amino 35 group with the dansyl group. It has been proved that the molecule in SEQ ID NO:69 binds to the NS3 protease active site with a WO 99/38888 PCT/IT99/00022 -23 Ki=200 nM. Its bond with protease can be determined with fluorescence spectroscopy. In particular, it is possible to excite the functionality of the dansyl present in the molecule both directly, using light with a 335 nm 5 wavelength or, exploiting the presence of two tryptophans in the NS3 protease, indirectly using the aforementioned phenomenon of resonance energy transfer between NS3 tryptophans and the molecule SEQ ID NO:69 bound to the enzyme. In both cases the bond is directly 10 observable by virtue of the different spectroscopic properties of free and bound molecules. However, utilisation of the resonance energy transfer phenomen is to be preferred as more sensitive. The SEQ ID NO:69 molecule can be utilised to 15 determine the binding of other molecules to NS3 protease active site, capable therefore of displacing it from the interaction with - the enzyme. A typical experiment is shown in Fig. 2. To a buffered solution containing NS3 protease 200 nM complexed with Pep4AK were added SEQ ID 20 NO:69 200 nM. The bond of the two molecules was measured exciting NS3 tryptophans at a 280 nm wavelength and recording emission spectrum around 520 nm. Addition of the NS3 protease competitive inhibitor SEQ ID NO:l causes a deplacement of SEQ ID NO:69 from NS3 active 25 site and a concomitant reduction of the phenomenon of fluorescence energy transfer. From this experiment it is possible to determine an IC5 0 value for SEQ ID NO:1 of 1 pM, that is the same value found assaying the effect of this molecule on the NS3 protease activity.

WO 99/38888 PCT/IT99/00022 -24 BIBLIOGRAPHICAL REFERENCES Atherton, E. and Sheppard, R. C. (1989) Solid phase peptide synthesis, a practical approach, IRL Press, Oxford. 5 Bartenschlager, R. (1997) Antiviral Chemistry & Chemotherapy 8(4), 281-301. Bartenschlager, R., Ahlborn-Laake, L., Yasargil, K., Mous, J. and Jacobsen, H. (1995) J. Virol. 69, 198-203. Bianchi, E., Steinkuhler, C., Taliani, M., Urbani, A., 10 De Francesco, R. and Pessi, A. (1996) Anal. Biochem. 237, 239 244. Bianchi, E., Urbani, A., Biasol, G., Brunetti, M., Pessi, A., De Francesco, R. and Steinkuhler, C. (1997) Biochemistry 36, 7890-7897. 15 D'Souza, E. D. A., Grace, K., Sangar, D. V., Rowlands, D. J. and Clarke, B. E. (1995) J. Gen. Virol. 76, 1729-1739. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. and Rice, C. M. (1993) Proc. Natl. Acad. Sci. USA 90, 10583-10587. 20 Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K. and Shimotono, K. (1993) J. Virol. 67, 4665-4675. Hong, Z., Ferrari, E., Wright-Minogue, J., Chase, R., Risano, C., Seelig, G., Lee, C. and Kwong, A. D. (1996) Anal. 25 Biochem. 240, 60-67. Kakiuchi, N., Hijikata, M., Komoda, Y., Tanji, Y., Hirowatari, Y. and Shimotohno, K. (1995) Biochem. Biophys. Res. Commun. 210, 1059-1065. Kolykhalov, A. A., Agapov, E. and Rice, C. (1994) J. 30 Virol. 68, 7525-7533. Komoda,. Y., Hijikata, M., Sato, S., Asabe, S. I., Kimura, K. and Shimotohno, K. (1994) J. Virol. 68, 7351-7357. Leinbach, S., Bhat, R., Xia, S. M., Hum, W. T., Stauffer, B., Davis, A., Hung, P. P. and Mizutani, S. (1994) 35 Virology 204, 163-169. Mori, A., Yamada, K., Kimura, J., Koide, T., Yuasa, S., Yamada, E. and Miyamura, T. (1996) FEBS Lett. 378, 37-42.

WO 99/38888 PCT/IT99/00022 -25 Myakava,Y., Okamoto, H. and Mayumi, M. (1995) Molecular Medicine Today 1, 20-25 Overton, H., McMillan, D., Gillespie, F. and Mills, J. (1995) J. Gen. Virol. 76, 3009-3019. 5 Schechter, I. and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162 Shimizu, Y., Yamaji, K., Masuh', Y., Yokota, T., Inoue, H., Sudo, S. and Shimotohno, K. (1996) J. Virol. 70, 127-132. Shoji, I., Suzuki, T. Chieda, S., Sato, M., Harada, T., 10 Yamakawa, Y. watabe, S., Matsuura, Y. and Miyamura T. (1996) Hepathology 22, 1648-1655. Sole, N. A. and Barany, G. (1992) J. Org. Chem. 57, 5399-5403. Steinktihler, C., Tomei, L. and De Francesco, R. (1996) 15 J. Biol. Chem. 271,' 6367-6373. Steinkthler, C., Urbani, A., Tomei, L., Biasol, G., Sardana, M., Bianchi, E., Pessi, A. and de Francesco, R. (1996) J. Virol. 70, 6694-6700. Suzuki, T., Sato, M. Cjieda, S., Shoji, I., Harada, T., 20 Yamakawa, Y., Watabe, S., Matsuura, Y. and Miyamura, T. (1995) J. Gen. Virol. 76, 3021-3029. Taliani, M., Bianchi, E., Narjes, F., Fossatelli, M., Urbani, A., Steinkuhler, C., De Francesco, R. ana Pessi, A. (1996) Anal. Biochem. 240, 60-67. 25 Tokita, H., Okamoto, H., Iizuka, H., Kishimoto, J., Tsuda, F., Lesmana, L.A., Myakava,Y. and Mayumi, M. (1996) J. of Gen. Virol. 77, 293-301. Urbani, A., Bianchi, E., Narjes, F., tramontano, A., De Francesco, R., Steinkuhler, C. and Pessi, A. (1997) J. Biol. 30 Chem. 272, 9204-920-9. Zang, R., Durkin, J., Windsor, W.T., McNemar, C., Ramanathan, L. and Le, H.V. (1997) J. of Virol. 71/8 6208 6213.

WO 99/38888 PCT/IT99/00022 -26 ABBREVIATIONS AND SYMBOLS USED IN THE TEXT CHAPS = 3-[ (3-colamidopropyl) -dimethyl-ammonium] -1 propan-sulfonate; HPLC = high-performance liquid chromatography; 5 TFA = Trifluoroacetic acid; ORF = Open Reading Frame; NMR = Nuclear Magnetic Resonance DMSO = Dimethylsulfoxide DTT = Ditiotreithol

Claims (28)

1. Peptides consisting in six amino acid residues arranged in positions from P6 to P1, P6 being the position of the N-terminal end and P1 being the position 5 of the C-terminal end, characterized in that the amino acid in the P1 position has at least a free acid function and in that they are capable of inhibiting the protease activity of the HCV virus associated to the NS3 protein.

2. The peptides according to claim 1, wherein the 10 amino acid in P1 position is a cysteine, an analog or a derivative thereof.

3. The peptides according to claim 2, wherein the amino acid in P1 position is selected from the group comprising L-cysteine, D-cysteine, homocysteine, S 15 methylcysteine, alanine, S-ethylcysteine, threonine, methionine, serine and penicillamine.

4. The peptides according to any of claims 1 to 3, having in P6 position an acid function.

5. The peptides according to claim 4, said acid 20 function in P6 position being selected from the group comprising aspartic acid, succinic acid and acylsulfonamide.

6. The peptides according to any of claims 1 to 5, having in P5 position an acid function. 25

7. The peptides according to claim 6, said acid function in P5 position being selected from the group comprising aspartic acid, succinic acid, acylsulfonamide.

8. The peptides according to any of claims 1 to 7, 30 having in the P4 position an hydrophobic amino acid.

9. The peptides according to claim 8, said amino acid in P4 position being selected from the group comprising 3,3,diphenilalanine, leucine, isoleucine and phenylglicine. 35

10. The peptides according to any of claims 1 to 9, having in the position P3 an amino acid selected from the group comprising glutamic acid, valine and WO 99/38888 PCT/IT99/00022 -28 isoleucine.

11. The peptides according to claim 10, having in the position P5 an amino acid selected from the group comprising aspartic acid, p-nitrophenylalanine, 5 tyrosine, g-carboxyglutamic acid, D-phenylalanine, D tyrosine, D-valine, D-isoleucine, D-3,3-diphenylalanine, D-aspartic acid, D-glutamic acid and D-g-carboxyglutamic acid.

12. The peptides according to claim 10 or 11, 10 having in the position P1 an amino acid selected from the group comprising aminobutyric acid, norvaline and valine.

13. The peptides according to any of claims 1 to 12, wherein said peptides are capable of inhibiting 50% 15 of the NS3 enzymatic activity at a concentration lower than or equal to 2 pM (ICso)

14. The peptides according to any of claims 1 to 13, having in the positions P4, P3, P2 and P1, the amino acids naturally occurring respectively in P4, P3, P2 and 20 P1 positions of one of the junction sites of the HCV virus, said junction sites being selected from the group comprising NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B.

15. The peptides according to claim 14, said 25 peptides being obtainable by the proteolysis reaction of polipeptides containing at least one of the junction sites of the polyprotein of said HCV virus, said junction sites being selected from the group consisting of NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junction 30 sites.

16. The peptides according to claim 15, wherein said junction sites consist of decapeptides, containing the amino acids naturally occurring in the positions P4, P3, P2 and P1 of said junction sites. 35

17. The peptides according to any of claims 14 to 16, wherein said HCV virus is selected from the group comprising the HICV viruses of la, lb, 1c, 2a, 2b, 2c, WO 99/38888 PCT/IT99/00022 -29 2d, 2e, 2f, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 5a, 5a, 6b, 7a, 7b, 7c, 7d, 8a, 8b, 9a, 9b, 9c, 10a and 11a genotype.

18. The peptides according to claim 17, wherein 5 said HCV virus is selected from the group comprising the HCV viruses of H-FDA, H-AP, HCV-1, HCV-J, HCV-BK, HC-J6, HCV-T, HC-J8, HCV-JT and HCV-JT' strain.

19. Peptides having an amino acid sequen-e selected from the group comprising the sequences reported in the 10 annexed sequence listing as from SEQ ID NO:1 to SEQ ID NO:69.

20. Use of the peptides according to any of the claims from 1 to 19, for the derivation of binding or inhibition assays of the enzymatic activity of the NS3 15 protease of the HCV virus

21. Use of the peptides according to any of the claims from 1 to 19, for the preparation of drugs for the treatment of the non-A non-B hepatitis.

22. Pharmaceutical compositions for the treatment of 20 the non-A non-B hepatitis, characterized in that they comprise at least one peptide according to any of claims 1 to 19 and a pharmaceutically effective carrier, vheicle or auxiliary agent.

23. Compositions for inhibiting the protease 25 activity of the HCV virus associated to the NS3 protein, characterized in 'that they comprise at least one peptide according to any of claims 1 to 19.

24. A process for the production of at least a peptide according to any one of claims 1 to 19 30 characterized by the step of carrying out the the proteolysis of polypeptides containing at least one among the sequences of the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and/or NS5A/NS5B junction sites of the polyprotein of HCV virus. 35

25. The process according to claim 24, wherein the proteolysis reaction is operated by NS3 protease of the HCV virus. WO 99/38888 PCT/IT99/00022 -30

26. The process according to claim 24 or 25, wherein the HCV. virus is selected from the group comprising the HCV viruses of la, lb, 1c, 2a, 2b, 2c, 2d, 2e, 2f, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 5a, 5 Sa, 6b, 7a, 7b, 7c, 7d, 8a, 8b, 9a, 9b, 9c, 10a and 11a genotype.

27. The process according to claim 26, wherein the HCV virus is selected from the group comprising the HCV viruses of H-FDA, H-AP, HCV-1, HCV-J, HCV-BK, HC-J6, 10 HCV-T, HC-J8, HCV-JT and HCV-JT' strain.

28. The process according to any one of the claims from 24 to 27, wherein the junction sites consist of decapeptides, containing the amino acids naturally occurring in the positions P4, P3, P2 and P1 of said 15 junction sites.