MXPA06004746A - Hcv ns3-ns4a protease resistance mutants - Google Patents

Abstract

The present invention is directed to mutants of HCV NS3/4A protease. More particularly, the present invention identifies mutant of HCV NS3/4A protease that are resistant to drug treatment.

Description

HCV PROTEASA NS3-NS4A RESISTANCE MUTANTS Field of the Invention The present invention relates to mutants of NS3 / 4A protease resistance of Hepatitis C virus.

BACKGROUND OF THE INVENTION Infection with the hepatitis C virus ("HCV") is a pressing human and medical problem. HCV is resonoside as the agent that causes the majority of cases of non-A hepatitis, not B, with an estimated human seroprevalence of 3% globally [A. Alberti et al., "Natural History of Hepatitis C," J. Hepatology, 31. (Suppl 1), pp. 17-24 (1999)]. Almost four million. Individuals "may be infected in the United States only [M.J. Alter et al.," The Epidemiology of Viral Hepatitis in the United States, Gastroenterol. Clin. North Am., 23, pp. 437-455 (1994); M. J. Alter "Hepatitis C Virus Infection in the United States," J. Hepatology, 31. (Suppl 1), pp. 88-91 (1999)]. After the first exposure to HCV, only approximately 20% of infected individuals develop acute sphincteric hepatitis, while others appear not to develop significant external symptoms of infection. Ref. 172643 In almost 70% of the cases, however, the virus establishes a chronic infection that persists for decades [S. Iwarson, "The Natural Course of Chronic Hepatitis," FEMS Microbiology Reviews, 14, pp. 201-204 (1994); D. Lavanchy, "Global Surveillanse and Control of Hepatitis C," J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usually results in recurrent liver inflammation and progressively worsens, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [M.C. Kew, "Hepatitis C and Hepatocellular Carcinoma ", FEMS Microbiology Reviews, 14, pp. 211-220 (1994); I. Saito et. al., "Hepatitis C Virus Infection is Associated with the Development of Hepatocellular Carcinoma, " Proc. Nati Acad. Sci. USA, 87, pp. 6547-6549 (1990)]. Unfortunately, there are no very effective treatments to weaken the progress of chronic HCV. The HCV genome codes for a polyprotein of 3010-3033 amino acids [Q.L. Choo, et. al., "Genetic Organization and Diversity of the Hepatitis C Virus." Proc. Nati Acad. Sci. USA, 88, pp. 2451-2455 (1991); ? Kato et al. , "Molesting Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis," Pros. Nati Asad. Sci. USA, 87, pp. 9524-9528 (1990); A. Takamizawa et. al., "Structure and Organization of the Hepatitis C Virus Genome Isolated from Human Carriers," J. Virol., 65, pp. 1105-1113 (1991)]. It is presumed that the non-structural proteins (NS) of HCV provide catalytic machinery essential for viral replication. The NS proteins are derived by the proteolytic cleavage of the polyprotein [R. Bartenschlager et. al., "Nonstructural Protein 3 of the Hepatitis C Virus Encodes to Serine-Type Proteinase Required for Cleavage at the NS3 / 4 and NS4 / 5 Junctions," J. Virol., 67, p. 3835-3844 (1993); A. Grakoui et. al., "Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites," J. Virol., 67, pp. 2832-2843 (1993); A. Grakoui et. al , "Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products," J. Virol., 67, p. 1385-1395 (1993); L. Tomei et. al., "NS3 is a serine protease required for processing of hepatitis C virus polyprotein", J. Virol., 67, p. 4017-4026 (1993)]. The NS 3 protein (NS3) of HCV contains a serine protease activity that processes viral polyprotein to generate the majority of viral enzymes, and is essential for viral replication and infectivity. The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 which processes the four sites downstream of the HCV polyprotein [C. Lin et al., "Hepatitis C Virus NS3 Serine Proteinase: Oraris-Cleavage Requirements and Processing Kinetics", J. Virol., 68, pp. 8147-8157 (1994)]. Substitutions of the catalytic triad of HCV serine protease NS3 resulted in the loss of viral replication and infectivity in chimpanzees [A.A. Kolykhalov et al., "Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3 'nontranslated region are essential for virus replication in vivo", J. Virol., 74: 2046-2051], It is known that sometimes in NS3 protease of yellow fever virus decreases viral infectivity [Chambers, TJ et. al., "Evidence That the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus Serine Protease is a Responsible for Site-Specific cleavages in the polyprotein Viral", Proc. Nati Asad. Ssi. USA, 87, pp. 8898-8902 (1990)]. The NS3 serine protease and its associated cofactor HCV, NS4A, processes the region viral nonstructural protein into individual non-structural proteins, including all viral enzymes [C. Failla, et al., "An amino-terminal domain of hepatitis C virus NS3 protease is essential for interaction with NS4A", J. Virol. 69, pp. 1769-1777; Y. Tanji et al., "Hepatitis C virus-ensoded nonstructural protein NS4A has versatile functions in viral protein processing", J. Virol. 69, pp. 1575-1581; C. Lin et al. , "A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vi tro", J. Virol. 69, pp. 4373-4380], and is essential for viral replication. This processing seems to be analogous to that carried out by the aspartyl protease of the human immunodeficiency virus which is also involved in the processing of viral proteins. HIV protease inhibitors, which inhibit viral protein processing, are potent antiviral agents in man, indicating that the interruption of this stage of the viral life cycle results in therapeutically active agents. Consequently, this is an attractive target for the discovery of drugs or drugs. Several potential HCV protease inhibitors have been described in the prior art [PCT Publications Nos. WO 02/18369, WO 02/08244, WO 00/09558, WO 00/09543, WO 99/64442, WO 99/07733, WO 99/07734, WO 99/50230, WO 98/46630, WO 98/17679 and WO 97/43310, U.S. Patent 5,990,276, M. Llinas-Bruñet et al., Bioorg. Med. Chem. Lett., 8, pp. 1713-18 (1998); W. Han et al., Bioorg. Med. Chem. Lett., 10, 711-13 (2000); R. Dunsdon et al., Bioorg. Med. Chem. Lett., 10, pp. 1571-79 (2000); M. Llinas-Brunet et al., Bioorg. Med. Chem. Lett., 10, pp. 2267-70 (2000); and S. LaPlante et al., Bioorg. Med. Chem. Lett., 10, pp. 2271-74 (2000)]. It is not known if these compounds would have the appropriate profiles to make drugs or drugs acceptable. In addition, it is possible that the HCV protease may become resistant to drug or otherwise acceptable drug. Therefore, the current understanding of HCV has not led to any satisfactory anti-HCV agent or treatment. The only therapy established for the disease caused by HCV is the treatment based on interferon alfa. However, alpha interferons have significant side effects [M. A. Walker et al., "Hepatitis C Virus: An Overview of Current Approaches and Progress," DDT, 4, pp. 518-29 (1999); D. Moradpour et al., "Current and Evolving Therapies for Hepatitis C," Eur. J. Gastroenterol. Hepatol. , 11, pp. 1199-1202 (1999); H. L. A. Janssen et al. "Suicide Associated with Alpha-Inferred Therapy for Chronic Viral Hepatitis," J. Hepatol., 21, p. 241-243 (1994); P.F. Renault et al., "Side Effects of Alpha Inferred," Seminars in Liver Disease, 9, pp. 273-277. (1989)] and induce long-term remission in only a fraction (-25%) of the cases [0. Weiland, "Inferieron Therapy in Chronic Hepatitis C Virus Infection", FEMS Misrobiol. Rev., 14, pp. 279-288 (1994)]. The current standard of care, pegylated interferon alpha in combination with ribavirin, has a sustained viral response (SVR) of approximately 40-50% for patients infected with genotype 1, which constitutes 70% of patients with chronic hepatitis C in the developed countries, and an SVR of 80% in passages infected with HCV genotype 2 or 3 [JG McHutchison, et al., N. Engl. J. Med., 339: 1485-1492 (1998); G.L. Davis et al., N. Engl. J. Med., 339: 1493-1499 (1998)]. In addition, the prospects for effective anti-HCV vaccines remain uncertain. Thus, there is a need for more effective anti-HCV therapies, particularly compounds that inhibit the NS3 protease of HCV. These compounds may be useful as antiviral agents, particularly as anti-HCV agents. An understanding of HCV resistance mutants would also progress to effective treatments against HCV.

SUMMARY OF THE INVENTION The present invention relates to NS3 / 4A protease resistance mutants of Hepatitis C virus. Thus, in certain aspects the invention involves isolated HCV polynucleotides encoding mutant HCV NS3 / 4A proteases. or biologically active analogs or fragments thereof where the codon corresponds to codon 156 of the wild-type polynucleotide and / or the codon corresponding to codon 168 of the wild-type polynucleotide is mutated, so that it does not code for an alanine in 156 and / or aspartic acid at 168. Exemplarily those mutations are described herein through the included polynucleotides in which the codon corresponding to 156 of the wild-type polynucleotide codes for a serine, valine or threonine. Other exemplary embodiments include polynucleotides in which the codon of the polynucleotide corresponding to codon 168 of the native polynucleotide codifies for an aspartic acid, glutamic acid, an alanine, a glycine or a tyrosine. Any combinations of mutations at codons 156 and 168 are specifically contemplated. The natural HCV NS3 / 4A protease is well known to those skilled in the art. This is encoded by a polynucleotide sequence of SEQ ID NO: 1. That polynucleotide sesuensia codes for an amino acid sequence of SEQ ID NO: 2. Polypeptides or biologically active fragments thereof which are encoded by the polynucleotides described herein. In addition, the invention encompasses vectors comprising, host cells that have been transformed or transfected with those polynucleotides and cell lines comprising those polynucleotides. The methods and compositions for producing those vectors, transforming host cells and preparing cell lines are routine and conventional tisnises known to those skilled in the art. Isolated HCV variants comprising the polynucleotides or mutant proteins described herein are also part of the present invention.

Compositions comprising the polynucleotides or proteins alone or in combination with other compositions and components are also contemplated herein. The invention teaches a method for detecting the presence of resistant HCV in a biological sample comprising the depreciation of the presence of a polynucleotide described herein. Typically, those methods comprise obtaining or isolating the polynucleotide from the sample; determine the sequence of the polynucleotide; and evaluating whether a mutation associated with the resistance, with one or more of the mutations described herein (for example, one that codes for serine, valine or threonine in a residue corresponding to a residue 156 and / or codes for a glutamic acid, valine, alanine, glycine or tyrosine in a residue corresponding to residue 168 of the natural HCV NS3 / 4A protease) is present in the polynucleotide. Methods to determine or diagnose whether a HCV infection in a patient is resistant were also contemplated, which involves collecting a biological sample from the patient infected with HCV; and evaluating whether the plasma sample contains the nucleic acid encoding a mutant HCV NS3 / 4A protease, where the presence of the mutant HCV NS3 / 4A protease is indicative that the patient has a resistant HCV infection. Other aspects contemplate methods to evaluate if a patient infected by HCV has a lower sensitivity or susceptibility to VX-950 which comprises evaluating whether the patient has a DNA of NS3 / 4A protease of Hepatitis C virus that has a mutation in the codon coding for residue 156 of the NS3 / 4A protease of the native Hepatitis C virus. Still further aspects are directed to methods for evaluating whether a patient infected with HCV has a lower sensitivity or susceptibility to a protease inhibitor which comprises evaluating whether the patient has a NS3 / 4A protease DNA of Hepatitis C virus that has a mutation in it. the codon coding for residue 156 of the Hepatitis C virus NS3 / 4A protease. The invention further contemplates methods for evaluating a candidate or potential HCV inhibitor comprising introducing the vector comprising a polynucleotide of the invention and a gene indicator that codes for an indicator in a host cell; cultivate the host cell; and measure the indicator in the presence of inhibitor and in the absence of inhibitor. Other methods tested compounds for their activity are HCV providing a mutant protease described herein and a protease substrate; contacting the protease with a candidate or potential inhibitor in the presence of the substrate; and evaluating or measuring the inhibition of the proteolytic activity of the protease. Other aspects provide methods for identifying a compound as an inhibitor of a resistant protease described herein by assaying the activity of that protease in the presence of the compound; assay the activity of the protease in the presence of the compound; compare the results of the test carried out in the presence and absence of the compound; wherein any decrease in protease activity as a result of the presence of the compound indicates that the compound is a protease inhibitor. The methods for identifying the compounds can rescue the astivity of VX-950, where an NS3 / 4A protease that has become resistant to VX-950 in which the resistant protease is contacted with the compound is also taught; and the ability of VX-950 to inhibit protease activity in the presence of that compound was evaluated. The present invention takes advantage of the fact that the three-dimensional structure of the NS3 / 4A protease has been resolved (see for example, WO 98/11134). Using those techniques and the teachings of the present invention, a three-dimensional model of the resistant protease of the invention is obtained; the compounds are designed or selesioned to interact with the three-dimensional structure of the mutant protease and the layering of the compound to bind to or interact with the protease is evaluated (for example through molecular modeling). Exemplary three-dimensional models are based on the crystal structure of x-rays (Figure 1 and Figure 2) of the protease NS3 / 4A. These models can be obtained through computer-implemented methods or through X-ray crystallography. These evaluations can be compared with determinate evaluations of natural protease. The compounds may be one identified from a combined chemical library or prepared through a rational design of drugs or drugs. In exemplary embodiments the compound is a compound prepared through rational drug or drug design and derived from the structure of the VX-950. In exemplary embodiments, the identified compound is formulated into a composition comprising the compound and a pharmaceutically acceptable carrier, adjuvant or carrier. Preferably the composition contains a compound in an amount effective to inhibit the serine protease of NS3 / 4A. Even more preferably, the composition is formulated to be administered to a patient. The composition may also comprise an additional agent selected from an immunomodulatory agent; an antiviral active agent; a second HCV protease inhibitor; an inhibitor of another target in the life cycle of HCV; a cytochrome P-450 inhibitor; or combinations thereof.

Other contemplated methods of the invention that inhibit the activity of the NS3 / 4A protease of Hepatitis C comprising the step of contacting the serine protease with a compound or composition. Additional aspects include methods for treating an HCV infection in a patient comprising the step of administering to the patient a compound of the composition. Additional aspects further contemplate methods for treating or reducing HCV infection in a patient, comprising determining whether the patient has an HCV infection that is resistant to therapy using a method described herein that depends on the detection of described mutations and treating the patient with a composition or therapy directed to the treatment of resistant HCV. Further aspects teach methods for eliminating or reducing the HCS sontamines of a biological sample or of laboratory or medical equipment, comprising the steps of contacting the biological sample or medical or laboratory equipment with a compound identified as described herein. In other embodiments, the biological sample or medical or laboratory equipment is contaminated with a resistant HCV separator according to the determination methods described herein. Other features and advantages of the invention will be apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES The following drawings are part of the present specification and were included to illustrate better aspects of the present invention. The invention can be better understood with reference to the drawings in combination with the detailed description of the specific embodiments presented herein. Figure 1: X-ray structures of the two protease-protease inhibitor complexes (Pl) of HCV NS3 from BILN 2061 and VX-950. The two complex structures were resolved and superimposed (VX-950 in blue and BILN 2061 in red). Three residues shown in the form of circles and bars (R123, R155 and D168) form salt bridges in the structure of the BILN 2061, but not in the structure of the VX-950. The removal of the negative charge in D168 results in the absence of restriction of R155 and the consequent loss of stacking with the large P2 of BILN 2061 and increases the cost of desolvation. The R155 is not restricted by D168 in the structure of the VX-950 and the mutation of D168V does not affect its binding. Figure 2: The A156S mutation produces a loss of union due to spherical shock with the VX-950, but not with the BILN 2061. X-ray structures of VX-950 (upper left, in blue) or BILN 2061 (lower part) left, in red) with the natural A156 highlighted in yellow. Mutation models A156S (in gray) with VX-950 (top right, in blue) or BILN 2061 (bottom right, in red). All allowed torsion angles were considered for the side chain of serine in the residue that mutated. Figure 3: The A156V mutation resulted in a binding loss of Pl due to steric shock with VX-950 or BILN 2061. Mutation models A156V (in green) with VX-950 (left, in blue) or BILN 2061 (right, in Red) . Figure 4: Quiescent strains of VX-950 (A) and BILN 2061 (B) Figures 5A, 5B: Development of HCV replicon cells that are resistant to VX-950. HCV Congen subgenomic replicon cells were serially passaged in the presence of G418 and crescent concentrations of VX-905 Figure 5 (A) as described in the Materials and Methods. The replicon cells were divided and fresh Pl was added to the medium twice a week. The shaded area indicates the period of time in which the replicon cells had little or no total growth accompanied by concurrent massive cell death. The total cellular RNA of the replicon cells at various points in time (indicated by full arrows) during the selection of resistance was extracted and the product of the RT-PCR of the serine protease NS3 of HCV was sequenced directly or after being subcloned in the vector TA. The IC50 values of VX-950 against the A-series of natural replicon T cells at day 56 were determined in the standard 48-hour assay in Figure 5 (B). Figures 6A, 6B: Development of HCV replicon cells that are resistant to BILN 2061. HCV Congen subgenomic replicon cells were serially passed in the presence of G418 and increasing concentrations of BILN 2061 Figure 6 (A) as described in Materials and methods. The replicon cells were divided and fresh Pl was added to the medium twice a week. The shaded area indicates the period of time in which the replicon cells had little or no total growth accompanied by concurrent mass cell death. The total cellular RNA of the replicon cells at various points in time (indicated by filled arrows) during the selection of resistance was extracted and the product of the RT-PCR of the serine protease NS3 of HCV was synthesized directly or after being subslonado in vector TA. The IC50 values of BILN 2061 against the B-series of natural replicon T cells at day 59 were determined in the standard 48-hour assay in Figure 6 (B). Figure 7. The models of protease somplejos: inhibitor. The protein is shown as a caricature based on its secondary structure in a light gray color. The inhibitors are shown in the form of circles and bars (VX-950 in purple and BILN 2061 in yellow) with the nitrogens colored in blue, the oxygens in red and the sulfur in orange. The side chains of the key residues are shown as bars with different colors: Alal56 (green), Aspl68 (orange), and Argl23 (orange). The side chain of Argl55 of the model of BILN 2061: protease is shown in sian and that of the model of VX-950: protease in orange. Those lateral sadenas are highlighted are dotted superfisies. The satalite triad Serl39, His57, and Asp81 are shown in gray. (The figure was prepared by PyMOL Molecular Graphics Systems, DeLano Scientific LLC, San Carlos, California, U.S.A. Copyright ® 1998-2003). Figures 8A-8C. Development of double resistant replicons from VX-950 cells. Figure 8 (A) Replicon cells resistant to 11VX-950 were passed in series in presensia of 0.25 mg / ml of G418, 14 μM of VX-950 and increasing concentrations of BILN 2061. The replicon cells were divided and added VX-950 and BILN 2061 fresh in the middle twice a week, according to the indicated by the restángulos and filled triangles, respec- tively. The total cellular RNA of the replicon cells at day 32 during resistance selection was brought and the product of the RT-PCR covering the HCV NS3 serine protease was sequenced either directly or after being subcloned into the TA vector. . Figure 8 (B) The titration of VX-950 against replicon cells of series A (resistant to VX-950) (full rectangle) or series C (double resistant) (open rectangle) to day 52 by VX was shown -950 The level of HCV RNA was determined after a 48 h incubation with VX-950. Figure 8 (C) BILN 2061 titration against replicon cells of series A (resistant to VX-950) (filled triangle) or series C (double resistant) (open triangle) at day 52 by BILN 2061 was shown. The level of HCV RNA was determined after a 48 h incubation with BILN 2061. Figures 9A-9C. Development of double resistant replicons from BILN 2061 cells. Figure 9 (A) BILN 2061 resistant replicon cells were serially run in the presence of 0.25 mg / ml of G418, 14 μM of VX-950 and concentrated BILN concentrates. 2061. The replicon cells were divided and fresh VX-950 and BILN 2061 were added to the medium twice a week, as indicated by filled rectangles and triangles. The total cellular RNA of the replicon cells at day 32 during the selection of resistance was extracted and the product of the RT-PCR covering the serine protease of HCV NS3 was sequenced either directly or after being subcloned in the vector TA . Figure 9 (B) The VX-950 titration against the B-series replicon cells (BILN 2061 resistant) (full rectangle) or the D-series (double-resistant) (open rectangle) at day 52 by VX- was shown 950 The level of HCV RNA was determined after a 48 h incubation with VX-950. Figure 9 (C) BILN 2061 titration against B-series replicon cells (BILN 2061 resistant) (full triangle) or D-series (double-resistant) (open triangle) at day 52 by BILN 2061 was shown. HCV RNA level was determined after a 48 h incubation with BILN 2061. Figure 10. Development of doubly resistant replicons from replicon cells without prior activation. HCV subgenomic replicon cells were serially run in the presence of 0.25 mg / ml G418, and increasing concentrations of VX-950 and BILN 2061. The replicon cells were divided, and fresh VX-950 and BILN 2061 were added to the medium twice a week, as indicated by the rectangles and full diamonds. The framed areas indicate the period of time in which the replicon cells had little or no total growth accompanied by concurrent massive cell death. The total cellular RNA of the replicon cells at several points in time, indicated by open arrows, during the selection of resistance was extracted and the product of the RT-PCR covering serine protease of HCV NS3 was sequenced directly or after of being subcloned into the TA vector. Figure 11. The scheme of the conformation of the side chain of Thrl56 in relation to the binding of the inhibitor. The thick lines represent the side chain of Thrl56 of the mutant enzyme and the P2 side chain of the inhibitor or substrate. The same three conformations were also considered for the Vall56 side chain. The last conformations (-60 / 180 °) have the lowest energy for any mutation, but they remain repulsive to both inhibitors.

DESCRIPTION OF THE PREFERRED MODALITIES It has been determined by the present inventors that the HCV strains undergo particular mutations in the presence of certain therapeutic compounds that render the HCV strains resistant to the therapeutic potential of those compounds. In particular embodiments, it has been determined that the NS3 / 4A protease of Hepatitis C virus mutates in those HCV resistance mutants so that the mutants become resistant in the protease inhibitor compounds. These discoveries can be exploited in the design of therapies for the treatment of HCV infection. In specific embodiments, it has been determined that residual amino acid 156 of natural HCV NS3 / 4A (the sequence of which is provided as SEQ ID NO: 2) is susceptible to mutation. Mutation of this residue leads to resistance of HCV to therapeutic intervention by protease inhibitors. In one embodiment, it has been shown that natural codon 156, which in natural HCV NS3 / 4A codes for alanine next to a codon which codes for serine in that relative position in HCV NS3 / 4A polypeptide. In another embodiment, the codon is mutated to a codon encoding valine in the relative position in the HCV NS3 / 4A polypeptide. In yet another embodiment, threonine is encoded in that relative position in HCV NS3 / 4A. In view of the above findings, the invention provides an HCV DNA encoding HCV NS3 / 4A protease (or fragment or analog thereof) where codon 156 of the DNA encodes a serine. Another embodiment of this invention provides an HCV DNA encoding a HCV NS3 / 4A protease (or fragment or analog thereof) where codon 156 of the DNA encodes a valine. An even further embodiment provides an HCV DNA encoding an HCV NS3 / 4A protein (or a fragment or analog thereof) where codon 156 of the DNA codes for a threonine. Still in further embodiments, it has been determined that in certain embodiments, the codon at 156 is one that codes for valine, serine or threonine at residue 156, which is normally an alanine residue in native / natural HCV NS3 / 4A. , and there is an additional mutation in which the codon at residue 168 of native / wild type HCV NS3 / 4A, which is normally a residue of aspartic acid mutated to a residue of valine, alanine, glycine or tyrosine. Although in certain embodiments, it was contemplated that mutant HCV NS3 / 4A protease would possess mutations at both positions 156 and 165, it was shown that mutants containing the individual mutations are also part of the present invention. Specific aspects of the invention include HCV DNA encoding an HCV NS3 / 4A protease (or a fragment or analogue thereof) where codon 156 of DNA sodifissa for a valine or a threonine and codon 168 codes for an aspartic acid or glutamic acid. Another embodiment of the present invention provides an HCV DNA encoding a HCV NS3 / 4A protease (or a fragment or analog thereof) wherein codon 168 of the DNA encodes a valine. Another embodiment of this invention provides an HCV DNA encoding an HCV NS3 / 4A protease (or a fragment or analog thereof) wherein codon 168 of the DNA encodes an alanine, a glycine or a tyrosine. The numbering system for the DNA of this invention is according to the sequence SEQ ID NO: 1. The DNA according to this invention can be derived from SEQ ID NO: 1. The DNA can be derived by solid phase synthesis or through recombinant means. In specific embodiments, site-directed mutagenesis of the sequence of SEQ ID NO: 2 is particularly contemplated to generate one or other of the mutants described herein. It should be recognized that mutations of the protein can be complete (ie, all or almost all of the protein is converted to the mutant protein) either partially or absent (ie, without or approximately without mutation). Therefore, a composition or method of this invention may comprise a mixture of natural and mutant protein. According to another embodiment of this invention there is provided an NS3 / 4A protease protein of HCV (or a fragment or analogue thereof) comprising amino acid 156 of the protease, wherein amino acid 156 is serine.

Another embodiment of this invention provides an HCV NS3 / 4A protease protein (or fragment or analog thereof) which comprises amino acid 156 of the protease, when the amino acid is valine. Another embodiment of this invention provides a HCV NS3 / 4A protease protein (or fragment or analog thereof) which comprises amino acid 156 of the protease, when the amino acid is threonine. Another embodiment of this invention provides a HCV NS3 / 4A protease protein (or fragment or analog thereof) which comprises amino acid 156 of the protease, when amino acid 156 is valine or threonine and the amino acid 168 is aspartic acid or glutamic acid. Another embodiment of this invention provides a HCV NS3 / 4A protease protein (or fragment or analog thereof) which comprises amino acid 156 of the protease, when amino acid 168 is valine. Another embodiment of this invention provides a HCV NS3 / 4A protease protein (or fragment or analog thereof) which comprises amino acid 156 of the protease, when the amino acid 168 is alanine, glycine or tyrosine. The DNA and proteins according to this invention can be modified using routine techniques. For example, the DNA may comprise a modification to bind the DNA to a solid support. The proteins may comprise a covalently linked marker compound. The DNA or proteins according to this invention can be in computer readable form, including, but not limited to, computer readable media and / or computer readable databases (see, for example, WO 98/11134). For certain uses, the DNA according to this invention can be inserted into a vector. Any suitable vector would be included within the scope of this invention. The adesuted vestores are known in the art. One modality provides an expression vector. Another embodiment provides a viral vector. A vector can be a sloning tool or additionally regulatory sequences such as a promoter, enhancers and terminators or polyadenylation signals can be additionally displayed. Accordingly, this invention also provides a vector comprising an HCV NS3 / 4A protease DNA (or fragment or analog thereof), wherein: codon 156 of the DNA encodes a serine; the codon 156 of the DNA codifies for a valine; codon 156 of the DNA codifies for a threonine-; the codon 156 of the DNA codes for a valine or a threonine and the sodon 168 sodifies for an aspartic acid or glutamic acid; codon 168 of the DNA codifies for a valine; codon 168 of the DNA codes for an alanine; codon 168 of the DNA codes for a glycine; and / or codon 168 of the DNA encodes a tyrosine. Another embodiment provides an expression vector. Another embodiment provides a viral vector. A vector can be a cloning tool or can additionally comprise regulatory sequences such as promoters, enhancers and terminators or polyadenylation signals. These vectors can be used in any appropriate host cell. Host cells are known in the art. Accordingly, this invention also provides a host cell comprising NS3 / 4A protease DNA where codon 156 of the DNA encodes a serine; the codon 156 of the DNA codes for a valine; codon 156 of the DNA codes for a threonine; the codon 156 of the DNA codes for a valine or a threonine and the codon 168 codes for an aspartic acid or glutamic acid; codon 168 of the DNA codes for a valine; codon 168 of the DNA codes for an alanine; codon 168 of the DNA codes for a glycine; and / or codon 168 of the DNA encodes a tyrosine. DNA expression would provide a host cell comprising a protease having an A156 for the serine mutation; an A156 for the valine mutation; an A156 for the threonine mutation; an A156 for valine or threonine and a D168 for the mutation of glutamic acid; a D168 for the valine mutation; D168 for the alanine mutation; a D168 for the glycine mutation; and / or a D168 for the tyrosine mutation. Cell lines comprising DNA or proteins according to this invention are also provided. The invention also provides a variant of HCV comprising a DNA according to this invention or a protein according to this invention and compositions comprising DNA and proteins. The HCV variants, as well as the DNA and / or proteins according to this invention may be useful in the discovery of drugs or drugs as well in the verification of appropriate HCV therapies. Accordingly, another embodiment of this invention provides a method for detecting the presence of HCV in a biological sample that comprises detecting the presence of a DNA according to this invention. These methods can include the steps of obtaining (or extracting) a DNA; (b) determining the DNA sequence; (c) determining or inferring whether, in the DNA, codon 156 codes for a serine; if codon 156 codes for a valine; if codon 156 of the polynucleotide codes for a threonine, if codon 156 codes for a valine or a threonine and codon 168 codes for an aspartic acid or glutamic acid, if codon 168 codes for a valine, codon 168 codes for a Alanine, a glycine or a tyrosine. In certain embodiments, the biological sample containing the HCV is derived from a mammal that has been infected with HCV. The detection of the presence of that DNA can be used as a diagnosis to guide the practitioner that the individual is one whose HCV infection will likely be resistant or otherwise refractory to the treatment of protease inhibitors. Given that guidance, the skilled person can modify the therapy of the subject having an infection, for example by increasing the dose of the therapy by providing additional therapies using agents to which the HCV strain infects the subject is not resistant. The methods of this invention may require that certain DNA sanctities be obtained. As would be recognized by the experts, the DNA would be obtained and then amplified. Standard techniques (eg, PCR, hybridization) can be used to practice this invention. These techniques are well known to those skilled in the art. Methods for treating or preventing an HCV infection by verifying the mutations provided herein are also provided by this invention. If a resistance mutant is present in the HCV, then the patient can be treated accordingly. That method would comprise: (a) collecting a sample (eg, a plasma sample, PBMC, liver cells or other sample) from the patient infected with HCV; and (b) evaluating whether the plasma sample contains the nucleic acid encoding the HCV NS3 / 4A protein that4 has a mutation at codon 156; where the mutation results in a substitution of alanine with serine. Similar methods could be employed by substituting the mutation 156 from alanine to serine with the other mutations set forth herein. Additionally, similar methods could involve identifying A156 for the mutation of serine (or another mutation identified here) and another protease mutation. These methods would all involve obtaining DNA, amplifying DNA, and determining the DNA sequence. Methods for evaluating the effectiveness of the NS3 / 4A protease inhibitor treatment of a patient infected with HCV are provided by this invention. These methods include: a) collecting a sample (eg, a plasma sample) from the patient infected with HCV; b) assess whether the plasma sample contains nucleic acid encoding the NS3 / 4A protease of HCV if it has a mutation at codon 156; where the mutation results in a substitution of alanine with serine. Similar methods can be carried out with the other mutations of this invention. The methods of this invention are intended to identify resistance mutants in patients who have been given HCV protease inhibitors. This method can be practiced in a patient that is undergoing treatment or has undergone treatment. Those and other diagnostic techniques known in the art (see, for example, US 5,631,128 and US 6489,098). Accordingly, one embodiment provides a method for evaluating whether a patient infected with HCV comprises NS3 / 4A protease DNA of Hepatitis C virus having a mutation at codon 156. It is likely that that patient is resistant to therapy by an agent like the VX-950. Consequently, the patient can be treated with a therapy that uses a substitute for VX-950. Another embodiment provides a method for evaluating whether a patient infected with HCV comprises NS3 / 4A protease DNA of Hepatitis C virus having a mutation at codon 168. Certain of those mutations result in a decrease in sensitivity or susceptibility to the VX-950. Similarly, certain mutations correlate or result in a decrease in sensitivity or susceptibility to BILN 2061 (WO 00/59929; US 6,608,027). Other mutations result in a decrease in sensitivity or susceptibility to both VX-950 and BILN 2061. The decrease in sensitivity or susceptibility to either or both compounds could be evaluated according to this invention. Knowing resistance mutation patterns, more effective treatment regimens can be developed. For example, this invention allows the design and / or discovery of compounds that are active against the resistance mutants set forth herein. Accordingly, this invention provides a method for evaluating a candidate or potential HCV inhibitor comprising: a) introducing a vector comprising the DNA according to this invention and an indicator gene encoding an indicator in a host cell; b) culturing the host cell; and c) measuring the indicator in the presence of the inhibitor and in the absence of the inhibitor. In this method the test compound can be added in any of one or more of steps a) -c). Another embodiment of this invention provides a method for assaying compounds with their anti-HCV activity comprising: a) providing a protease according to this invention and a protease substrate; b) contacting the protease with a candidate or potential inhibitor in the presence of the substrate; and d) evaluating or measuring the inhibition of the proteolytic activity of the protease. Another embodiment of this invention provides a method for identifying an inhibitor of a protease according to this invention, comprising: a) assaying the activity of the protease in the absence of the compound; b) assaying the activity of the protease in the presence of the compound; and c) compare the results of a) and the results of b). That method may further comprise: d) assaying the activity of a natural protease in the absence of the compound; e) assaying the activity of the natural protease in the presence of the compound; and f) compare the results of d) and the results of e). The data of that method could then be analyzed, for example, by comparing the results of a) and / or b) and the results of d) and / or e). Also provided are methods comprising: d) assaying the activity of a second protease of NS3 / 4A comprising amino acid 168 of the protease, wherein amino acid 168 is valine, alanine, glycine, or tyrosine in the absence of the compound; e) assaying the activity of the second protease in the presence of the compound; and f) compare the results of d) and e). The method may further comprise: g) testing the activity of a natural protease in the absence of the compound; h) assaying the activity of the natural protease in the presence of the compound; i) compare the results of g) and the results of h). In a more specific embodiment, the method comprises comparing the results of a) and / or b) and the results of d) and / or e); and / or the results of g) and / or h). After the viruses become resistant to a drug or drug, it is possible that the virus could mutate further and become once again susceptible to the drug or drug. One way this happens is when the virus comes into contact with a second drug or drug. In sonsesuencia, this invention also provides a method to identify a compound capable of rescuing the activity of VX-950, where the NS3 / 4A protease has become resistant to VX-950 comprising: a) contacting a mutant protease described here with the compound of interest; b) assay the ability of VX-950 to inhibit the activity of the protease of a). Similar methods are also provided to rescue the activity of BILN 2061 sontra resistant mutants and / or mutants doubly resistant to VX-950 and BILN 2061. Another aspect of drug-resistant or drug-resistant viruses is that this virus may be treatable with another drug. or drug. Therefore, methods for identifying compounds that are active against the drug-resistant virus are tools for discovering very useful drugs or drugs. The methods described here can be applied in high-throughput screening techniques. Alternatively, the invention also provides methods for carrying out rational drug or drug design techniques. Using the structural information about the HCV NS3 / 4A protease elucidated here (i.e., the mutation of particular residues in 156 and / or 168 of the natural protein) as a basis for the design of effective protease inhibitors. More specifically, the present invention identifies for the first time strains of HCV that are resistant to treatment by protease inhibitors such as VX-950 and BILN 2061. The rational design of drugs or drugs can also be combined with a systematic method of experiments. of large-scale screening where drug targets or potential protease inhibitor drugs are tested with combined library compounds. The rational design of drugs or drugs in a focused method, which uses information about the structure of a receptor of a drug or drug or one of its natural ligands to identify or create drugs or candidate drugs. The three-dimensional structure of a protein can be determined using methods such as X-ray crystallography or nuclear magnetic resonance spectroscopy. In the present invention, the three-dimensional structure of a HCV NS3 / 4A protease of HCV containing one or the other or both of the mutations of residues 156 or 168 can now be easily determined using X-ray crystallographic techniques and / or spectroscopy. NMR Rational drug or drug design can also be combined with a systematic method of large-scale screening experiments, where drug targets or potential protease inhibitor drugs are tested with combined library compounds. Armed with the information provided, one skilled in the art can employ computer programs to search through databases containing the structures of many different chemical compounds. The computer can select those compounds that are most likely to interact with the HCV NS3 / 4A protease of drug or drug resistant mutants and test those compounds identified in routine laboratory tests of protease inhibitors as the tests described here. In certain modalities, it was shown that the structure of the VX-950 or BILN 2061 (see Figure 4) can be used as the initial structure from which additional molecules can be designed. It will be shown here that the mutant proteases are such that the interaction with the VX-950 is reduced. The structures derived from the VX-950 that conform more easily to this, interact with the three-dimensional structure created when the residue 156 is a valine, serine or threonine and / or the residue 168 is a valine, alanine, glycine or glutamic acid will be inhibitors of novel useful proteases that can be employed against resistant strains of HCV in which there is a mutation in the NS3 / 4A proteases of HCV. These compounds can also be effective against natural HCV strains in which the HCV NS3 / 4A protease did not mutate. The teachings of the present invention allow the person skilled in the art to focus and narrow the search as much as possible to limit large-scale selection expenses. In particular embodiments, the structure of the initial compound has the structure of VX-950 (shown below in structure B). Although VX-950 is exemplified, any stereoisomer of 950 can be used with mixtures of D- and L- isomers in the n-propyl side chain being expressly included. The following structure, structure A describes those diastereoisomers. This is a mixture of compounds of Structure B (VX-950) and Structure C, Structure A Structure B Structure C Rational drug or drug design can be used to serially modify the different positions on this molecule to produce derivatives thereof that may be useful as protease inhibitors. The crystalline structures of the NS3 / 4A protease of natural HCV with the VX-950 bound to these, are shown in Figure 1. The data shown in that figure show that removal of the negative charge in D168 of the HCV NS3 / 4A protease results in the absence of restriction of R155 and consequently the large P2 stacking loss of BILN 2061 and increases the cost of desolvation. R155 is not restricted by D168 in the resolved structure of the VX-950 and the D168V mutation does not affect its binding. These binding studies can be easily performed with derivatives elucidated through a rational drug design or drugs to identify agents that have binding capacity and / or therapeutic efficacy in the mutants. The rational design of drugs or drugs has previously been to identify Relenza which was used to treat influenza. The design that led to the discovery of Relenza was developed by choosing molecules that most likely interact with neuraminidase, an enzyme produced by a virus that is required to release newly formed viruses from infected cells. Many drugs or recent drugs for the treatment of HIV infections (eg, Ritonivir ™ 1, Indinavir ™) were also identified through rational drug or drug design schemes in which the drugs or drugs were designed to interact with the protease viral, the enzyme that separates viral proteins and allows them to mount properly. Another well-known drug or drug that was produced by the rational design based on ligands is the Viagra ™. This drug or drug was designed to resemble cGMP, a ligand that binds to phodiesterase. Given that the drug or drug rationales design has proven to be effective once the structure of the drug target or drug is known, it was contemplated that the discoveries of the present invention, which reveal the structures of the NS3 protease / 4A HCV that appears in strains of HCV that are resistant to known HCV protease inhibitors, it was contemplated that those skilled in the art will be able to use rational drug or drug design to identify drugs or drugs useful in the treatment of HCV. Accordingly, this invention also provides a method for identifying an effective compound against a protease of this invention, comprising: a) obtaining a three-dimensional model of the protease; b) design or select a compound; c) evaluate the sapacity of the compound to bind to or interact with the protease. In these methods, the three-dimensional model is based on the crystal structure of x-rays (Figure 1 and Figure 2) of the NS3 / 4A protease. Methods for developing models of the crystal structure are known, for example, by molecular modeling with computer implemented methods (see, for example, US 6,162,613, WO 98/11134, and WO 02/068933). A three-dimensional model can also be obtained by x-ray crystallography and a protein according to this invention. As is known in the art, a protein can be crystallized in the presence or absence of a ligand (such as a compound being evaluated). Assessing the ability of the compound to bind to, or interact with, the protease is known in the art (see, for example, US 6,162,613, WO 98/11134, and / or WO 02/068933). The evaluation can be carried out for example, by molecular modeling. After the compound is selected, it can be tested in standard assays, or assays provided herein, to determine the compound effects of various HCV proteases. Thus, given the teachings of the present inventionIt will be possible to perform screening tests to identify protease inhibitors that are effective against HCV infections resistant to drugs or drugs. The present invention shows that resistance to drugs or drugs is induced in those strains of HCV that have a mutation in any of residues 156 or 168 of the NS3 / 4A protease of HCV. More particularly, it has been shown here that a mutation of A156 to serine, valine, or threonine, and / or a mutation of D168 to a valine, alanine, glycine or glutamic acid, results in HCV being resistant to treatment They are drugs or drugs. It was contemplated that compositions that act as inhibitors of mutant HCV NS3 / 4A proteases comprising one or more of the above-mentioned articular mutations will be useful in therapeutic modalities for the treatment of HCV. The compounds can be those that have been designed to mimic the action of VX-950 or BILN 2061, or are derivatives of VX-950 or BILN 2061. In screening tests to identify those compounds, the candidate substance can be selected first by its basic biochemical activity in vi tro, and then proven by its ability to reduce, alleviate or otherwise therapeutically intervene in HCV infection in an in vivo model of HCV infection. The mutant proteases of the invention possess protease activity. Any of the screening assays can be established to determine the NS3 / 4A protease activity of mutant HCV using any conventional assay used to determine the activity of the natural HCV NS3 / 4A protease. In preferred embodiments, the activity of inhibitors against the mutant HCV NS3 / 4A protease is compared to the activity of inhibitors against the natural HCV NS3 / 4A protease. The ability of the candidate substance to inhibit the protease is determined by obtaining a sample comprising an HCV NS3 / 4A protease; and contacting the sample with the candidate substance. The activity of the HCV protease is determined in the presence and absence of the candidate substance. The protease can be an isolated protein, a membrane fraction that contains the isolated protein, or it can be inside a cell that expresses the protease. Thus, the activity of the protease is typically determined in a sample containing protease in the absence of the candidate substance. The candidate substance would then be added to it or a similar composition of the sample containing protease and the activity of the protease is determined. Any candidate substance that decreases the activity of the protease in the sample is indicative that the candidate substance has the desired inhibitory activity. In the in vivo screening assays, it is monitored that the compound is administered to an animal model, over a period of time and in several doses, and relief of symptoms associated with HCV infection. Any improvement in one or more of those symptoms will be indicative of the candidate substance in a useful agent. As used herein the term "candidate substance" refers to any molecule that can potentially act as an inhibitor of HCV proteases, regardless of whether the proteases are of the natural or mutant variety. That agent can be a protein or fragment thereof, a small molecule inhibitor, or even a nucleic acid molecule. This may prove to be the case in which the most useful pharmacological compounds will be compounds that are structurally related to other known inhibitors of HCV proteases, such as, for example, VX-950 or BILN2061 or other inhibitors discussed herein. The rational design of drugs or drugs includes not only comparisons with those known inhibitors, but predictions related to the structure of target molecules of those inhibitors. On the other hand, they can simply be purchased, from several commercial sources, libraries of small molecules that are believed to meet the basic criteria of drugs or useful drugs in a "brute force" effort to identify useful compounds. The selection of such libraries, including libraries generated in combination (for example, peptide libraries) is a quick and efficient way to select a large number of related (and unrelated) compounds for their activity. The combined methods are also endowed by themselves with a rapid evolution of drugs or potential drugs by the creation of a second, third and fourth generation of modeled compounds of active compounds, but in other undesirable circumstances. The candidate compounds may include fragments or parts of natural compounds or may be found as active combinations of known compounds that are otherwise inactive. It was proposed that the isolates isolated from natural sources such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples can be tested as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be selected could also be derived or synthesized from chemical compositions or man-made compounds. The "effective amounts" of the candidate agent in certain circumstances are those amounts effective to reproducibly produce an alteration to the inhibition of the expression or activity of the HCV NS3 / 4A protease, inhibition of HCV production or virulence, inhibition. of HCV infection, or a decrease or alleviation of one or more of the symptoms of HCV infection compared to the levels of those parameters in the absence of that agent. Compounds that reach significant appropriate sambios in those parameters will be used. Significant changes in activity and / or expression will be those that are represented by alterations in activity and at least approximately 30% -40%, and more preferably, by changes of at least 50%, with higher values of course being possible. The dominant VX-950 resistant mutant, A156S, remains susceptible to BILN 2061. To confirm whether mutations observed in either Alal56 or Aspl68 are sufficient to confer resistance against VX-950 or BILN 2061, respectively, mutagenesis was used. directed to the site to introduce each individual mutation at position 156 or 168 in the protease domain of NS3. Site-specific mutagenesis is another technique useful in the preparation of mutant protease proteins used in the methods of the invention. This technique employs specific mutagenesis of the underlying DNA (which codes for the amino acid sequence that is targeted by the modification). The technique also provides an easy ability to prepare and test sequence variants, incorporating one or more of the above considerations, introducing one or more changes in the nucleotide sequence in the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that sodify for the DNA sequence of the desired mutation, as well as the sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the suppression junction that is being traversed. Typically, a primer of about 17 to 25 nucleotides in length, with about 5 to 10 residues on both sides of the junction of the sequence being altered, is preferred. The technique typically employs a bacteriophage vector that exists in the form of a single strand and double strand. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid. In general, site-directed mutagenesis is effected by first obtaining a single-stranded vector, or by fusing the two strands of a two-strand vector that includes within its sequence a DNA sequence coding for the desired protein. An oligonucleotide primer that contains the desired mutant sesuensia is prepared synthetically. The primer is then annealed with the single stranded DNA preparation, taking into account the degree of incongruence when the hybridization conditions are selected (annealing), and is subjected to DNA polymerizing enzymes such as the Klenow fragment of polymerase I __. coli, to complete the synthesis of the strand that contains the mutation. In this way, a heteroduplex is formed where one strand codes for the original non-mutant sequence and the second strand contains the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells and clones are selected which include the recombinant vectors containing the array of the mutant sequence. Of course, the method described above for site-directed mutagenesis is not the only method to generate potentially useful mutant protease species and therefore does not mean that it is limiting. The present invention also contemplates other methods to achieve mutagenesis such as, for example, treating recombinant vectors containing the gene of interest of mutagenic agents, such as hydroxylamine, to obtain sequence variants. The kinetic parameters for the FRET substrate for the natural NS3 protease domains of genotype la and Ib were identical [Tables 1A and IB] under our assay conditions. Although the cofactor of the peptide NS4A was of the HCV genotype, no discernible differences were observed in the kinetic parameters. This is consistent with molecular modeling, which suggests that the conservative variations in the central region of NS4A between the la and lb genotypes do not affect the interaction between the central peptide of NS4A and the protease domain of NS3. The Ki values of VX-950 and BILN 2061 were determined using the natural protease Ib and Ib genotypes, and there were no statistically significant differences between the two natural proteases (Table 2). The kinetic parameters of the FRET substrate for the A156S mutant protease were virtually the same as those of the natural protease (Tables 1A and IB). However, the Ki value of the VX-950 was 2.9 μM against that of the A156S mutant protease, which is 29 times higher than that of the natural protease (0.1 μM) (Table 2). BILN 2061 has a Ki value of 112 nM against that of the A156S mutant, which is 6 times higher than that of the natural protease, 19 nM (Table 2). The level of HCV RNA in the replicon cells containing the A156S substitution that is similar to that of the natural replicon cells (data not shown), which is consistent with the similar enzymatic catalytic efficiency of the mutant A156S and serine proteases of natural NS3. The IC 50 value of the VX-950 against the A156S replicon cells was 4.65 μM, which is 12 times higher than against the natural repletion cells (0.40 μM) (Table 3). The difference between IC? 0 values of BILN 2061 against A156S (7 nM) and natural replicon cells (4 nM) was not significant (Table 3). The mutants resistant to BILN 2061 major, D168V and D168A, remain completely susceptible to VX-950. The kinetic parameters of the substrate were not affected by the D168V mutation, and showed only minor changes (less than 10 times) for the D168A mutant, as indicated by the comparison of the ksat and kcat / Km values of the two serine proteases of NS3 (Table 1A and IB). Similarly, no significant effect was observed on any substitution in Aspl68 on the Ki value of VX-950 (Table 2). However, the substitution of valine or alanine by aspartic aspart at position 168 resulted in a mutant NS3 protease that was not inhibited until 1.2 μM BILN 2061 (Table 2). These data indicate that any mutant protease is at least 63 times less susceptible to BILN 2061 in somparation with the natural protease. The actual magnitude of resistance can not be determined since the BILN 2061 was not soluble at concentrations greater than 1.2 μM in the assay buffer, as measured by the absorbance at 650 nm. The D168V or D168A mutation was also introduced into the natural HCV replicon by site-directed mutagenesis and a stable replicon cell line containing any substitution was generated. The BILN 2061 had an IC50 of 5.09 μM against the D168V replicon cells, which is greater than 1300 times compared to the natural replicon cells (4 nM) (Table 3). The IC50 of the BILN 2061 was 1.86 μM in the mutant replicon D168A. There was little change in the CI_o values against the VX-950 and the D168V replicon cells (Table 3). Accordingly, compounds identified by the methods of this invention are also provided, where the compound is an inhibitor of an NS3 / 4A protease of HCV. Those compounds can be generated for example through the rational design of drugs or drugs as discussed above. The invention also provides compositions comprising the above compounds and the use thereof. These compositions can be used to pre-treat invasive devices to be inserted into a patient, to treat biological samples, such as blood, before administration to a patient, and for direct administration to a patient. In each case the composition will be used to inhibit the HCV replicon and to decrease the risk of severity of HCV infection. Another embodiment of this invention provides a composition comprising a compound identified according to this invention or a pharmaceutically acceptable salt thereof. According to a preferred modality, the identified compound of agreement is this invention is present in an effective amount to decrease the viral load in a sample or in a patient, where the virus codes for a serine protease necessary for the viral life cycle, and a carrier or excipient pharmaceutically acceptable. If pharmaceutically acceptable salts of the compounds are used this invention in those compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among the acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorrate, camphor sulfonate, cyclopentan-propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate , semisulf to, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl propionate, picrate, pivalate, propionate, succinate , tartrate, thiocyanate, tosylate and undesanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as the salts of dicyclohexylamine, N-methyl-D -glucamine and salts with amino acids such as arginine, lysine and so on. Also, groups containing basic nitrogen can be quaternized as agents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides.; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as stearyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Therefore, soluble or dispersible products are obtained in water or oil. The structures used in the sompositions and methods of this invention can also be modified by adding appropriate functionalities to improve the selective biological properties. Such modifications are known in the art and include those that increase biological penetration in a given biological system (eg, blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter the metabolism and alter the speed of expression. The pharmaceutically acceptable carriers or excipients that may be used in such compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffering substances, phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, trisilicate of magnesium, polyvinyl pyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polymers of polyethylene and polyoxypropylene blocks, polyethylene glycol and wool grease. According to a preferred embodiment, the compositions of this invention are formulated for pharmaceutical administration to a mammal, preferably a human. The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation, topical, rectal, nasal, buccal, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasenobial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally or intravenously. The sterile injectable forms of the compositions of this invention can be aqueous or oleaginous suspensions. These suspensions can be formulated to all those known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be accepted are water, Ringer's solution and isotonic sodium solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspension medium. For this purpose, any soft fixed material including mono or synthetic diglycerides can be employed. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethylcellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers that are commonly used in the manufacture of solid, liquid and other pharmaceutically acceptable dosage forms can also be used for formulation purposes.

Dose levels of between about 0.01 and about 100 mg / kg of body weight per day, preferably between about 0.5 and 75 mg / kg of body weight per day of protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of antiviral disease, particularly mediated by anti-HCV. Typically the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day, or alternatively, as a continuous infusion. That administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the support materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Preferably, those preparations contain from about 20% to about 80% active compound. When the sompositions of this invention involve a combination of a substance identified according to this invention and one or more additional prophylactic therapeutic agents, both the compound and the additional agent will be present at dose levels of between about 10 to 100%, and most preferably between about 10 to 80% of the dose normally administered in a monotherapy regimen. The pharmaceutical compositions of this invention can be administered orally in any dosage form. orally acceptable including, but not limited to, capsules, tablets, suspensions or aqueous solutions. In the case of tablets for oral use, carriers or excipients that are commonly used include lactose or corn starch. Typically, lubricating agents, such as magnesium stearate, can also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring and coloring agents can also be added. Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at the rectal temperature and therefore melts in the rectum to release the drug or drug. These materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions of this invention may also be administered topically, especially when the target of the treatment includes areas or organs easily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are easily prepared for each of those areas or organs. Topical application to the lower intestinal tract can be done in a rectal suppository formulation (see above) or a suitable enema formulation. Topical transdermal patches may also be used. For topical applications, the pharmaceutical compositions can be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers or exsipients. Carriers or exsipients for topical administration of the compounds of this invention include, but are not limited to, oil, liquid petrolatum, white petrolatum, propylene glycol compounds, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in an anioned solution or sage that contains the active components suspended or dissolved in one or more pharmaceutically acceptable carriers or excipients. Suitable carriers or excipients include, but are not limited to mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water. For ophthalmic use / the pharmaceutical compositions can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline solution, or preferably, as solosions in isotonic sterile saline solution, adjusted pH, either with or without a preservative as a slurry of Benzalsonium Alternatively, for ophthalmic uses, the pharmaceutical compositions can be formulated into an ointment such as petrolatum. The pharmaceutical compositions of this invention can also be administered by aerosol or nasal inhalation. These compositions are prepared according to methods well known in the pharmaceutical composition art and can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. More preferred are pharmaceutical compositions formulated for oral administration. In another embodiment, the compositions of this invention additionally comprise another antiviral agent, preferably an anti-HCV agent. These antiviral agents include, but are not limited to, immunomodulatory agents such as α-, β-, and β-interferons, interferon O derivatives, pegylated compounds, and thymosin.; other antiviral agents, such as ribavirin, amantadine, and telbivudine; other protease inhibitors of hepatitis C (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase and polymerase inhibitors; inhibitors of internal ribosomal entry; broad-spectrum viral inhibitors are inhibitors of IMPDH (for example, the compositions of U.S. Patent Nos. 5,807,876, 6,498,178, 6,344,465, 6,054,472, WO 97/40028, WO 98/40381, WO 00/56331, and mycophenolic acid and derivatives thereof , and including, but not limited to, the VX-497, VX-148, and / or VX-944); or combinations of any of the above. See also W. Markland et al., Antimicrobial & Antiviral Chemotherapy, 44, p. 859 (2000) and U.S. Patent 6,541,496. The following definitions are used here (they are the trademarks referring to produstos available from the fesha of presentation of the solisitud). "Peg-Intron" means PEG-Intron®, peginteferon alfa-2b, available from Schering Corporation, Kenilworth, NJ; "Intron" means Intron-A®, interferon alfa-2b available from Schering Corporation, Kenilworth, NJ; "ribavirin" means ribavirin (1-beta-D-ribofuranosyl-lH-1, 2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, CA, described in the Merck Index, entry 8365, Twelfth Edition, also available as Rebetol® from Schering Corporation, Kenilworth, NJ, or as Copegus® from Hoffmann-La Roche, Nutley, NJ; "Pagasys" means Pegasys®, peginterferon alfa-2a available from Hoffmann-La Roche, Nutley , "Roferon" means Roferon®, recombinant interferon alfa-2a available from Hoffmann-La Roche, Nutley, NJ; "Berefor" signifies Berefor®, alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT; ®, a purified mixture of natural alpha interferons such as Sumiferon available from Sumitomo, Japan, Wellferon®, interferon alfa neither available from Glaxo_Wellcome LTd., Great Britain, Alferon®, a mixture of natural alpha interferons produced by Inferieron Sciences, and available from Purdue Frederick Co. , CT The term "interferon" as used herein means a member of the family of specific proteins of highly homologous species that inhibit viral replication and cell proliferation, and modulate the immune response, such as interferon alpha, interferon beta, or interferon gamma. From Merck Index, entry 5015, Eleventh Edition. According to one embodiment of the present invention, interferon is a-interferon. According to another embodiment, a therapeutic combination of the present invention uses natural interferon alpha 2a. Alternatively, the therapeutic combination of the present invention utilizes native interferon alpha 2b. In another embodiment, the therapeutic combination of the present invention utilizes recombinant alpha interferon 2a or 2b. In another embodiment, interferon is pegylated interferon 2a or 2b alpha. Interferons suitable for the present invention include: (a) Intron (interferon-alpha 2B, Schering Plow), (b) Peg-Intron, (c) Pegasys, (d) Roferon, (e) Berofor, (f) Sumiferon, (g) Wellferon, (h) consensus interferon alpha available from Amgen, Ins. , Newbury Park, CA, (i) Alferon; (j) Viraferon®; (k) Infergen®. As is recognized by the experts, a protease inhibitor would be administered, preferably orally. Interferon is typically not administered orally. However, there are no limits here to the methods or combinations of this invention to any specific dosage forms or regimens. Thus, each component of a combination according to this invention may be administered separately, together, or in any combination thereof. In one embodiment, the protease inhibitor and interferon are administered in separate dosage forms. In a modality, any additional agent is administered as part of a single dosage form with the protease inhibitor as a separate dosage form. Since this invention involves a combination of compounds, the specific amounts of each compound may depend on the specific amounts of each of the other compounds in the combination. As recognized by an expert, doses of interferon are typically measured in IU (for example from approximately 4 million IU to approximately 12 million IU). Accordingly, the agents, (whether acting as an immunomodulatory agent or otherwise) that can be used in combination with a compound of this invention include, but are not limited to, interferon-alpha 2B (Intron A, Schering Plow ); Rebatron (Schering Plow, Inteferon-alpha 2B + Ribavirin); pegylated interferon alia (Reddy, KR et al. "Efficacy and Safety of Pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C (Hepatology, 33, pp. 433-438 (2001 ), consensus interferon (Kao, JH, et al., "Efficacy of Consensus Interferon in the Treatment of Chronic Hepatitis" J. Gastroenterol, Hepatol 15, pp. 1418-1423 (2000), interferon-alpha 2A (Roferon A Roche), lymphoblastoid or "natural" interferon, interferon tau (Clayette, P. et al., "IFN-tau, A New Interferon Type I with Antiretroviral activity" Pathol. Biol. (Paris) 47, pp. 553-559 (1999), interleukin 2 (Davis, GL et al., "Future Options for the Management of Hepatitis C." Seminars in Liver Disease, 19, pp. 103-112 (1999), Interleukin 6 (Davis et al. "Future Options for the Management of Hepatitis C. "Seminars in Liver Disease 19, pp. 103-112 (1999), interleukin 12 (Davis, GL et al.," Future Options for the Management of Hepatitis C. "Seminars in Liver Disease, 19, pp. 103-112 (1999); Ribavirin; and compounds that enhance the development of type 1 helper T cell response (Davis et al., "Future Options for the Management of Hepatitis C." Seminars in Liver Disease, 19, pp. 103-112 (1999). Viral infections exerting direct antiviral effects and / or modifying the immune response to the infection The antiviral efestos of interferons are medium freshness through the inhibition of penetration or in relation to the viral coat, synthesis of the viral RNA, tradussion of viral proteins, and / or viral assembly and release. *** Compounds that stimulate the synthesis of interferon in cells (Tazulakhova, EB et al., "Russian Experience in Screening, Analysis, and Clinical Application of Novel Interferon Inducers" J Interferon Cytokine Res., 21 pp. 65-73) include, but are not limited to, double-stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, DN "Immunomodulatory an d Pharmacologic Properties of Imiquimod "J. Am. Acad. Dermatol., 43 pp. S6-11 (2000)). Other non-immunomodulatory or immunomodulatory compounds can be used in combination with a compound of this invention including, but not limited to, those specified in WO 02/18369, which is incorporated herein by reference (see for example, page 273, lines 9-22 and page 274, line 4 to page 276, line 11, which is incorporated herein by reference in its entirety). Compounds that stimulate interferon synthesis in cells (Tazulakhova et al., J. Interferon Cytokine Res. 21, 65-73)) include, but are not limited to, double-stranded RNA, alone or in combination with tobramycin and Imiquimod (3M Pharmaceuticals) (Sauder, J. Am. Arad. Dermatol., 43, S6-11 (2000)). Other known compounds that have, or may have antiviral activity against HCV by virtue of non-immunomodulatory mechanisms including, but not limited to Ribavirin (ICN Pharmaceuticals); inhibitors of inosine dehydrogenase 5'-monophosphate (formula of VX-497 provided herein); amantadine and rimantadine (Younossi et al., In Seminars in Liver Disease 19, 95-102 (1999)), - LY217896 (US Patent No. 4,835,168) (Colacino, et al., Antimicrobial Agents &Chemotherapy 34, 2156- 2163 (1990)); and methyl ester of 9-hydroxyimino-6-methoxy-l, 4a-dimethyl-1,2, 3,4,4a, 9,10, lOa-octahydro-phenanthren-1-sarboxylic acid; 6-methoxy-4a-dimethyl-9- (4-methyl-piperazin-1 -limino) -1,2,3,4,4a, 9,10,10a-octahydro-phenanthrene methyl ester hydrochloride l-carboxylyl; 1- (2-chloro-phenyl) -3- (2, 2-biphenyl-ethyl) -urea (Patent United States 6,127,422). 15. The formulations, doses and routes of administration for the above molecules are taught in the references cited below, or are well known in the art as described, for example, in F.G. Hayden, at Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 50, pp. 1191-1223, and the references cited there. Alternatively, once a compound exhibiting anti-HCV antiviral activity, particularly antiviral activity against a resistant HCV strain, has Once identified, a pharmaceutically effective amount of that compound can be determined using techniques that are well known to those skilled in the art. Note, for example, Benet et al., In Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 1, pp. 3-27, and references cited there. Thus, the formulations, dose ranges and suitable dosage regimens of these compounds can be easily determined by routine methods. The drug or drug combinations of the present invention can be provided to a cell or cells, or to a human patient, either in a separate pharmaceutically acceptable formulation, administered simultaneously or sequentially, formulations containing more than one therapeutic agent, or by a single agent selection and multiple agent formulations. Regardless of the route of administration, combinations of drugs or drugs form an effective anti-HCV amount of the components of the pharmaceutically acceptable formulations. A large number of other immunomodulators and immunostimulants may be used in the currently available methods of the present invention and include: AA-2G; adamantyl amide dipeptide; adenosine deaminase, adjuvant of Enzon, Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax; agelasfin-11; therapy for AIDS, Chiron; algal glucan, SRI; alganunulin, Anutech; Anginlyc; anti-cell factors, Jeddah; Anticort; antigastrin-17 immunogen, Ap; antigen delivery system, Vac; antigen formulation, IDBC; Immunogen antiGnRH, Aphton; Antiherpina; Arbidol; azarol; Bay-q-8939; Bay-r-1005; BCH-1393; Betafectin; Biostim; BL-001; BL-009; Broncostat; Cantastim; CDRI-84-246; cefodizima; Chymozyme inhibitors, ICOS; CMV peptides, City of Hope; CN-5888; cytosine releasing agent, St; DHEAS, Paradigm; DISC TA-HSV; J07B; I01A; I01Z; dithiocarb sodium; ECA-10-142; ELS-1; endotoxin, Novartis; FCE-20696; FCE-24089; FCE-24578; ligand of FLT-3, Immunex; FR-900483; FR-900494; FR-901235; FTS-Zn; G proteins, Cadus; gludapsin; glutaurin; glycophosphopeptical; GM-2; GM-53; GMDP; vasuna of the growth factor, EntreM; H-BIG, NABI; H-CIG, NABI; HAB-439; Helicobacter pylori vaccine; herpes specific immune factor; therapy for HIV, United Biomed; HyperGAM + CF; ImmuMax; Immun BCG; immune therapy;, Connective; immunomodulator, Evans; immunomodulators, Novacell; imreg-1; imreg-2; Indomune; inosine pranobex; interferon, Dong-A (alpha2); interferon, Genentech (gamma); interferon, Novartis (alpha); interleukin-12, Genetics Ins; interleukin-15, Immunex; interleukin-16, Research Cor; ISCAR-1; J005X; L-644257; licomarasminic acid; LipoTher; LK-409, LK-410; LP-2307; LT (R1926); LW-50020; MAF, Shionogi; derivatives of MDP, Merck; met-enkefaliña, TNI; Methylfurylbutyrolactones; MIMP; mirimostim; mixed bacterial vaccine; Tem, MM-1; moniliastat; MPLA, Ribi; MS-705; murabutide; marabutida, Vacsyn; derovates of muramyl peptide; mielo peptide derived from muramilo; -563; NACOS-6; NH-765; NISV, Proteus; NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios; peptidoglycan, Pliva; Perthon, Advanced Plant; derived from PGM, Pliva; Pharmaprojects No. 1099; No. 1426; No. 1549; No. 1585; No. 1607; No. 1710; No. 1779; No. 2002; No. 2060; No. 2795; No. 3088; No. 3111; No. 3345; No. 3467; No. 3668; No. 3998; No. 3999; No. 4089; No. 4188; No. 4451; DO NOT. 4500; No. 4689; No. 4833; Do not . 494; No. 5217; No. 530; pidotimod; pimelautide; pinaíida; PMD-589; podoiillotoxin, Conpharm; POL-509; poly-ICLC; poly-ICLC, Yamasa Shoyu; PoliA-PoliU; Polisasárido A; Protein A, Berlux Bioscience; PS34W0; Acum pseudomonas, Teijin; Psomaglobin; PTL-78419; Pyrexol; piriferone; Retrogen; Retropep; RG-003; Rhinostat; rifamaxil; RM-06; Rollin; romurtida; RU-40555; RU-41821; Rubella antibodies, ResCo; S-27649; SB-73; SDZ-280-636; SDZ-MRL953; SK &F-107647; SL04; SL05; SM-4333; Solutein; SRI-62-834; SRL-172; ST-570; ST-789; lysate of this phage; Stimulon; suppressin; T-150R1; T-LCEF; tabilautida; temurtida; Theradigm-HBV; Theradigm-HBV; Theradigm-HSV; THF, Pharm & Upjohn; THF, Jeddah; timalfasin; fractions of thymic hormones; thymosartin; Thymolimotrophin; thymopentin; thymopentin analogues; thymopentin, Peptech; thymosin fractions 5, Alpha; timostimulin; thymotrinan; TMD-232; TO-115; transfer factor, Viragen; tuftsina, Selavo; ubenimex; Ulsastat; ANGG-; CD-4 +; Collag +; COLSF +; C0M +; DA-A +; GAST-; GF-TH +; GP-120 -; - IF +; IF-A +; IF-A-2 +; IF-B +; IF-G +; IF-G-1B +; IL-2 +; IL-12 +; IL-15 +; IM +; LHRH-; LIPCOR + L LYM-B +; LYM-NK +; LYM-T +; OPI +; PEP +; PHG-MA +; RNA-SYN-; SY-CW-; TH-A-I +; TH-5 +; TNF +; A. Representative nucleoside and nucleotide compounds useful in the present invention include, but are not limited to (+) - cis-5-fluoro-1- [2- (hydroxy-methyl) - [1,3-oxathiolan-5-yl] cytosine; (-) -2'-deoxy-3 '-thiocytidin-5'-triphosphate (3TC); (-) -cis-5-fluoro-1- [2 (hydroxy-methyl) - [I, 3-oxathiolan-5-yl] cytosine (FTC); (-) 2 ', 3', dideoxy-3 '-thiasitidine [(-) -SddC]; 1- (2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl) -5-iodocytosine (FIAC); 1- (2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl) -5-iodocytosine triphosphate (FIACTP); 1- (2'-deoxy-2 '-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FMAU); 1-beta-D-ribofuranosyl-l, 2,4-triazole-3-carboxamide; 2 ', 3' -dideoxy-3 '-fluoro-5-methyl-dexocitidine (FddMeCyt); 2 ', 3' -dideoxy-3 '-chloro-5-methyl-dexocitidine (ClddMeCyt); 2 ', 3' -dideoxy-3 '-amino-5-methyl-dexositidine (AddMeCyt); 2 ', 3' -dideoxy ~ 3 '-fluoro-5-metol-sitidin (FddMeCyt); 2 ', 3' -dideoxy-3 '-chloro-5-methyl-cytidin (ClddMeCyt); 2 ', 3' -dideoxy-3 '-amino-5-methyl-cytidine (AddMeCyt); 2 ', 3' dideoxy-3 '-fluorothymidine (FddThd); 2 ', 3' -dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) 2 ', 3' -dideoxy-beta-L-5-thiacytidine; 2 '", 3' -dideoxy-beta-L-5-cytidine (beta-L-ddC); 9- (1, 3-dihydroxy-2-propoxymethyl) guanine; 2'-deoxy-3'-thia-5 -fluorocytosine; 3'-amino-5-methyl-dexocitidine (AddMeCyt); 2-amino-1, 9 - [(2-hydroxymethyl-1- (hydroxymethyl) ethoxy] methyl] -6H-purin-6-one (ganciclovir ); 2- [2- (2-amino-9H-purin-9-yl) ethyl) -1,3-propandyl diacetate (famciclovir); 2-amino-l, 9-dihydro-9 - [(2-hydroxy-ethoxy) methyl] 6H-purin-6-one (acyclovir); 9- (4-hydroxy-3-hydroxymethyl-but-l-yl) guanine (pencislovir); 9- (4-hydroxy-3-hydroxymethyl-but-1-yl) -6-deoxy-guanine diasetate (íamciclovir); 3'-azido-3'-deoxythymidine (AZT); 3'-chloro-5-methyl-dexocitidine (ClddMeCyt); 9- (2-Iosionyl-methoxyethyl) -2 ', 6'-diaminopurin-2', 3'-dideoxyriboside; 9- (2-Iosynonylmethoxyethyl) adenine (PMEA); acyclovir triosylate (ACVTP); D-carbocyclic-2'-deoxyguanosine (CdG); dideoxy-cytidine; dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine (ddl); triosylate of E-5- (2-bromovinyl) -2'-deoxyuridine; fluoro-arabinofuranosyl-iodouracil; 1- (2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl) -5-iodo-uracil (FIAU); stavudine; 9-beta-D-arabinofuranosyl-9H-purin-6-amine monohydrate (Ara-A); 9-beta-D-arabinofuranosyl-9H-purin-6-amin-5'-monophosphate monihydrate (Ara-AMP); 2-deoxy-3 '-thia-5-fluorocytidine; 2 ', 3' -dideoxy-guanine; and 2 ', 3' -dideoxy-guanosine.

Synthetic methods for the preparation of nucleosides and nucleotides useful in the present invention are well known in the art and are described in Acta Biochim Pol., 43, 25-36 (1996); Swed. Nucleosides Nucleotides 15, 361-378 (1996); Synthesis 12, 1465-1479 (1995); Carbohyd. Chem. 27, 242-276 (1995); Chena Nucleosides Nusleotides 3, 421-535 (1994); Ann. Reports in Med. Chena, Asademic Press; and Exp. Opin. Invest. Drugs 4, 95-115 (1995). The chemical reactions described in the references cited above are generally described in terms of their broader application for the preparation of the compounds of this invention. Occasionally, the reactions may not be applicable as disintegrated to the compound included within the scope of the compounds described herein. The compounds for which this occurs will be readily recognized by those skilled in the art. In all those cases, any reactions can be successfully carried out by sonar modifications, known to those skilled in the art, for example by the appropriate protection of the interfering groups, by changing by alternative conventional reagents, by the interrupted modification of the reaction conditions and the like. , or other reactions described herein or other conventional ones that are applicable to the preparation of the corresponding compounds of this invention. In all the preparative methods, all the starting materials are known or easily prepared from known starting materials. Although nucleoside analogs are generally employed, as antiviral agents as such, nusleotides (nusleoside phosphate) are sometimes converted to nucleosides to facilitate their transport through the sealing membranes. An example of a chemically modified nucleotide capable of entering cells is an S-l-3-hydroxy-2-phosphonylmethoxypropyl cytosine (HPMPC, Gilead Sciences). The nucleoside or nucleotide compounds used in this invention that are acidic can form salts. Examples include salts with alkaline metals or alkaline earth metals, such as sodium, potassium, salty, magnesium or with organismal bases or basic ammonium ammonium salts. The person skilled in the art can choose to administer a chrome P450 monooxygenase inhibitor. These inhibitors may be useful to increase the concentrations and / or increase the blood levels of somatids that are inhibited by chrome P450. If one embodiment of this invention involves a CYP inhibitor, any CYP inhibitor that improves the pharmacokinetics of the relevant NS3 / 4A protease can be used in a method of this invention. These CYP inhibitors include, but are not limited to, ritonavir (WO 94/14436), ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944, and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methyl pyrazole, cislosporin, and clomethiazole. For the preferred dosage forms of ritonavir, see U.S. Patent 6,037,157, and the documents cited therein: U.S. Patent 5,484,801, U.S. Application 08 / 402,690, and International Applications WO 95/07696 and WO 95/09614). Methods for measuring the ability of the compounds to inhibit the activity of known chrome P50 monooxygenases (see US 6,037,157 and Yun, et al., Drug Metabolism &; Disposition, vol. 21, pp. 403-407 (1993). Immunomodulators, immunostimulants and other agents useful in the combination therapy methods of the present invention may be administered in amounts less than those conventional in the art. For example, interferon alpha is typically administered to humans for the treatment of HCV infections in an amount of approximately 1 x 106 units / person three times per week to approximately 10 x 106 units / person three times per week (Simón et al. , Hepatology 25: 445-448 (1997)). In the methods and compositions of the present invention, this dose may be in the range of about 0.1 x 106 units / person three times per week to about 7.5 x 106 units / person three times per week; more preferably from about 0.5 x 10e units / person three times per week to about 5 x 106 units / person three times per week, more preferably from approximately 1 x 106 units / person three times per week to approximately 3 x 10e units / person three times a week. Due to the antiviral effectiveness against the improved hepatitis C virus of the immunomodulators, immunostimulants and other anti-HCV agents in the presence of HCV serine protease inhibitors of the present invention, reduced amounts of those immunomodulators / immunostimulants can be employed in the treatment methods and compositions contemplated herein. Similarly, due to the antiviral effectiveness against the improved hepatitis C virus of the HCV serine protease inhibitors herein in the presence of immunostimulants and immunostimulants, reduced amounts of the HCV serine protease inhibitors can be employed in the methods and compositions contemplated here. These reduced amounts can be determined by routine verification of hepatitis C virus titers in infected patients undergoing therapy. This can be brought to taste, for example, by checking HCV RNA in patient's serum by slot staining, spot staining, or RT-PCR techniques, or by measuring the surface of HCV or other antigens. Patients can also be verified during the combination therapy by using the HCV serine protease inhibitors described herein and other compounds having anti-HCV activity, for example nucleoside and / or nucleotide antiviral agents to determine the lowest effective doses of one sada when they are used in combination. The methods of the combination therapy herein, the nucleoside or nucleotide antiviral compounds, mixtures thereof can be administered to humans in an amount in the range of about 0.1 mg / person / day to about 500 mg / person / day; preferably from about 10 mg / person / day to about 300 mg / person / day; more preferably from about 25 mg / person / day to about 200 mg / person / day; even more preferably from about 50 mg / person / day to about 150 mg / person / day; and more preferably in the range of about 1 mg / person / day to about 50 mg / person / day. Doses of compounds can be administered to a patient in a single dose or in appropriate doses. In the latter case, the unit dosage compositions may contain amounts as sub-multiples thereof to constitute the daily dose. Multiple doses per day can also increase the total daily dose if this is desired by the person prescribing the drug or drug. The regimen for the treatment of a patient suffering from an HCV infection is the somatic and / or compositions of the present invention selected according to a variety of factors, including the patient's age, weight, sex, diet and medical condition. , the severity of the infection, the route of administration, pharmacological considerations, the activity, efficacy, pharmacological properties, and toxicological profiles of the individual employees, and whether a drug or drug release system is employed. The administration and combinations of drugs or drugs described here should generally continue for a period of several weeks to several months or years until the virus titers reach an acceptable level, indicating that the infection has been controlled or eradicated. Patients undergoing treatment with the drug or drug combinations described herein can be routinely verified by measuring viral hepatitis RNA in patients' serum by spot staining, spot spotting, or RT-PCR techniques, or by the measurement of viral antigens of hepatitis C, as surface antigens, in serum to determine the effectiveness of the therapy. The continuous analysis of the data obtained by these methods allows the modification of the treatment regimen during therapy, so that optimum quantities of each component are administered in the combination, and so that the treatment durability can be determined as well. Thus, the treatment regimen / dosing schedule can be modified rationally during the course of therapy, so that the lowest amounts of each of the antiviral compounds used in combination are administered, which together exhibit antiviral effectiveness of satisfactory hepatitis C, and so that the administration of these antiviral drugs in combination continues only as long as it is necessary to successfully treat the infection. The present invention encompasses the use of HCV serine protein inhibitors described herein in various combinations with the above and similar types of compounds having anti-HCV astivity to treat or prevent HCV infections in patients, particularly those patients who have HCV infections. who have developed resistance to treatment with VX-950 or other standard protease inhibitors. For example, one or more inhibitors of HCV serine protease may be used in combination with: one or more interferons or interferon derivatives having anti-HCV activity; one or more compounds other than interferon that have anti-HCV activity; or one or more interferons or interferon derivatives having anti-HCV activity and one or more compounds other than interferon having anti-HCV activity. When used in combination to treat or prevent HCV infection in a human patient, any of the HCV serine protease inhibitors described herein and prior compounds having anti-HCV activity may be present in a pharmaceutically or anti-HCV amount. Effective HCV By virtue of their additive or synergistic effects, suando is used in the combinations described above, each may also be present in a subclinically effective effective anti-HCV or pharmaceutically effective amount, ie, in an amount which, if used alone, provides a reduced pharmaceutical effectiveness that inhibits or completely reduces the accumulation of HCV virions and / or reduces or alleviates the conditions or symptoms associated with infection or pathogenesis by HCV in patients compared to those inhibitors of HCV serine protease and compounds having activity anti-HCV when used in pharmaceutically effective amounts. In addition, the present invention encompasses the use of combinations of HCV serine protease inhibitors and compounds having anti-HCV activity as described above for treating or preventing HCV infections, where one or more of those inhibitors or compounds are present in a pharmaceutically effective amount, and the others are present in a pharmaceutically effective or subclinically effective anti-HCV amount due to their additive or synergistic effects. As used herein, the term "additive effect" discloses the combined effect of two (or more) pharmaceutically active agents that is equal to the sum of the effect of each given alone. A synergistic effect is one in which the combined effect of two (or more) pharmaceutically active agents is greater than the sum of the effect of each given agent alone. After improvement of the conditionality in a patient, a maintenance dose of a compound, composition or combination of this invention can be administered, if necessary. Subsequently, the dose or frequency of administration, or both, can be reduced, depending on the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, if the treatment ceases. Patients may, however, require intermittent treatment on a long-term basis following any recurrence of the symptoms of the disease. It should also be understood that a specific dose and treatment regimen for any partiscular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion. , combination of drugs or drugs, and the judgment of the treating physician and the severity of the particular illness that is being treated. The amount of active ingredients will also depend on the particular compound described and the presence or absence and the nature of the additional antiviral agent in the composition. According to another embodiment, the invention provides a method for treating a patient infected with a virus, characterized by a virally-modified serine protease that is necessary for the life cycle of the virus by administering to the patient a pharmaceutically acceptable formulation of this invention. Preferably, the methods of this invention are not used to treat a patient suffering from an HCV infection. This treatment can completely eradicate the viral infection or reduce the severity of it. More preferably, the patient is a human being. In an alternative embodiment, the method of this invention further comprises the step of administering to the patient an antiviral agent preferably an anti-HCV agent. These antiviral agents include, but are not limited to, immunomodulatory agents, such as a-, β- and y-interferons, interferon-a derivatives, pegylated compounds, a thymosin; other antiviral agents, such as ribavirin and amantadine; other protease inhibitors of hepatitis C (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other agents in the life cycle of HCV, including helicase and polymerase inhibitors; inhibitors of internal ribosomal entry; broad-spectrum viral inhibitors, such as inhibitors of IMPDH (eg, VX-497 and other inhibitors of IMPDH described in U.S. Patent 5,807,876, misophenoise acid and derivatives thereof); or synapses of any of the above. These additional agents can be administered to the patient as part of a single dosage form comprising a compound of this invention and an additional antiviral agent. Alternatively, the adisional agent can be administered separately from the compound of this invention, as part of a multiple dose form, where the additional agent is administered before, together with or after a composition that forms a composition of this invention. In still another embodiment, the present invention provides a method for pretreating a biological substance that is intended for administration to a patient comprising the step of contacting the biological substance with a pharmaceutically-aseptable composition comprising a compound of this invention. These biological substances include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like; organs such as the kidney, liver, heart, lung, etc., sperm and ovules; bone marrow and components thereof; and other fluids to be infused in a patient such as saline solution, dextrose, etc. According to another embodiment, the invention provides methods for treating materials that may potentially come into contact with a virus characterized by a virally encoded serine protease is necessary for its life cycle. This method includes the step of putting the material in check, they are a set of agreement to the invention. These materials include, but are not limited to, surgical instruments and garments; instruments and laboratory garments; blood collection equipment and materials; invasive devices, such as shunts, stent devices, etc. In another embodiment, the compound of this invention can be used as a laboratory tool to assist in the isolation of a virally encoded serine protease. This method comprises the steps of providing a compound of this invention bound to a solid support; contacting the solid support with a sample containing a viral serine protease under condi tions that cause the protease to bind to the solid support; and eluting the serine protease from the solid support. Preferably, the serine protease vial isolated by this method is the NS3-NS4A protease of HCV. More particularly, it is a mutant HCV NS3-NS4A protease that is resistant to treatment by VX-905 and / or BILN 2061 as described herein. Examples of those proteases include those described herein as having mutant (i.e., unnatural) residues at positions 156 and / or 168 of a protein of SEQ ID NO: 2. As used herein, unless otherwise required, the term "comprises" and variations thereof indicate the inclusion of the element established, but not the exclusion of any other element. Routine techniques that are known to the experts can be used to practice this invention. These techniques can be found in published documents. For example, recombinant DNA and standard molecular cloning techniques are well known in the art. See, for example, F.M. Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, Ins. , Media, PA; Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989, and the literature documents cited in US Patents 6,617,156, and 6,617,130, all of which are incorporated herein by reference.

Examples For this invention to be more fully understood, the following preparatory examples and tests are exposed. The following examples were included to demonstrate certain preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques described in the following examples represent techniques that the inventor has discovered to work well in the practice of the invention, and thus can be considered to constitute preferred modes for their practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments described and still obtain an equal or similar result without departing from the spirit or scope of the invention.

Example 1: Construction of Plasmids The DNA encoding the Ala1-Ser181 residues of the HCV NS3 protease (GenBank CAB46913) were obtained by PCR from the plasmid of the HCV replicon Conl, I377neo / NS3-3 '/ p ( renamed pBR322-HCV-Neo in this study) [V. Lohmann et al., Science, 285, pp. 110-113 (1999)] and inserted in pBEVll (S. Chamber, et al., Personal communication) for the expression of HCV proteins a C-terminal mark of hexa-histidine in E. coli. Resistance mutations against Pl of NS3 «4A of HCV were introduced into this construct by site-directed PCR-based mutagenesis. To generate the HCV replicon, which contains the PI-resistant mutation, a 1.2-kb Hind IIl / BstX I fragment of the HCV Con 1 replicon was subcloned into a TA cloning vector, pCR2.1 (Invitrogen). Pl-resistant mutations in the NS3 serine protease domain were introduced into the pCR2.1 vector containing the HCV fragment of Hind IIl / BstX I by PCR, and again the fragment of BsrG 1 / BstX I of 579- was subcloned. bp containing the mutant residue in a secondgeneration Conl replison plasmid containing three adaptable mutants, pBR322-HCV-Neo-mADE (see below). All the constructs were confirmed by sequencing.

Example 2: Generation of HCV Replicon Cell Sub-genomic replicon plasmids pBR322-HCV-Neo [Lohmann et al., Science, 285, pp. 110-113 (1999)] was digested with Ssa I (New England Biolabs). The full-length HCV sub-genomic replicon RNA was generated from the linearized DNA template using a T7 Mega-script kit (Ambion) and treated with DNase to remove the DNA from the template. The obtained RNA transcripts were subjected to electrophoresis in Huh-7 cells and stable HCV replicon cell lines were selected with 0.25 or 1 mg per ml G418 (Geneticin) in Dulbecso minimal modified essential medium (DMEM) containing 10% fetal sheep serum (FBS). The stable cells of the HCV replicon were maintained in DMEM, 10% FBS and 0.25 mg per ml G418. During the course of generation of the stable cell lines of the sub-genomic replicon of HCV, several different patterns of adaptive mutations were identified. One pattern has three substitutions in the HCV structural proteins, which were introduced into the original pBR322-HCV-Neo plasmid by site-directed mutagenesis to generate the second generation sub-genomic replicon plasmid, pBR322-HCV-Neo- mADE. When transcripts of T7 RNA from plasmid pBR322-HCV-Neo-mADE linearized with Ssa I were subjected to electrophoresis in Huh7 cells, stable replicon cell colonies were formed at a much higher efficiency than that of the original common replicon RNA. Mutations of resistensia identified in this study were introduced into the replicon plasmid pBR322-HCV-Neo-mADE by site-directed mutagenesis. Stable replicon cell lines were generated using T7 transcripts derived from natural pBR322-HCV-Neo-mADE or with resistance mutations. Example 3: Test Protocol of HCV Replicon Cells The cells containing hepatitis C virus (HCV) replicon were maintained in DMEM containing 10% fetal bovine serum (FBS), 0.25 mg per ml of G418, with appropriate supplements (media A). On day 1, the monolayer of replicon cells was treated with a mixture of trypsin: EDTA, removed, and then media A was diluted to a final concentration of 100,000 cells per ml with 10,000 cells in 100 ul cultured in each well of a 96-well tissue culture layer, and grown overnight in a tissue culture incubator at 37 ° C. On day 2, the compounds (in 100% DMSO) were serially diluted with DMEM containing 2% FBS, 0.5% DMSO, with appropriate supplements (media B). The final concentration of DMSO was maintained at 0.5% through the dilution series. Media on the monolayer of replicon cells were removed, and then B media containing various concentrations of compounds was added. Media B without any compound was added to other wells as controls without compound. The cells were incubated with somatic or 0.5% DMSO in B media for 48 hours in a tissue culture incubator at 37 ° C. At the end of the 48-hour incubation, the media was removed, and the replicon cell monolayer was washed once with PBS and stored at -80 ° C prior to RNA extraction. The culture plates with treated replicon cell monolayers were frozen, and a fixed amount of another RNA virus, such as bovine viral diarrhea virus (BVDV) was added to the cells in each well. RNA extraction reagents (such as reagents from RNeasy equipment) were added to the cells immediately to avoid RNA degradation. The total RNA was extracted according to the manufacturer's instructions with modifications to improve the efficiency and consistency of the extraction. Finally, the total cellular RNA was eluted including the HCV replicon RNA and stored at -80 ° C until its adisional processing. Real time RT-PCR quantification was established with Taiman with two sets of specific probe primers. One was for the HCV and the other for the BVDV. The total RNA extracts of the treated HCV replicon cells were added to the PCR reactions for the quantification of HCV RNA and BVDV in the same PCR well. The experimental failure was rejected and marked on the basis of the level of BVDV RNA in each well. The level of HCV RNA in each well was calculated according to a standard curve made in the same FCR layer. The percentage of inhibition or decrease of HCV RNA due to treatment with the compound was traced using the DMSO or control without compound as 0% inhibition. The cytotoxity of the compound was measured using a cell viability assay based on mitochondrial enzyme, CellTiter 96 an aqueous solution cell proliferation assay (Promega). The IC50 values (concentration at which a 50% inhibition was observed at the level of HCV RNA) and IC50 (concentration at which a 50% reduction in cell viability was observed) were calculated from the curve of titration of any given compound using a four-parameter curve fitting (SoftMax Pro).

Example 4: Selection of PI-HC Resistant Replicon Cells Stable cells of sub-genomic replicon with 1 HCV were serially passed in the presence of 0.25 mg per ml G418 and slowly increasing concentrations of VX-950 (series A), BILN 2061 (series B), or a combination of both VX-950 and BILN 2061 (series C, D, and E). Concentrations of VX-950 fluenced 3.5 μM (or lOx IC50) in the 48-hour trial (see above), at 28 μM (80x ICS0). For BILN 2061, the initial concentration was 80 nM (80x IC50), and the final concentration was 12.5 μM (12,500x IC50). During the course of the selection, the replicon cells were divided twice a week when a confluence of 70-90% was reached. Fresh HCV Pl was added every 3 to 4 days regardless of whether the cell culture was divided or not.

Example 5: Identification of Pl-HCV resistance mutations During the selection of PI-HCV-resistant replicon cells, cell masses or pellets were collected each time the cell culture was divided. The total cellular RNA was extracted using the RNeasy equipment (Qiagen). A 1.7-kb long DNA fragment spanning the NS3 serine protease region was amplified.

HCV with a pair of HCV-specific oligonucleotides (5'-CCTTCTATCGCCTTCTTG-3 '(SEQ ID NO: 3) and 5' -CTTGATGGTCTCGATGG-3 '(SEQ ID N0: 4)) using the Titan One-Step RT-PCR kit (Roche Applied Science). The amplified products were purified using the QIA-quick PCR purification kit (Qiagen). To verify the emergence of PI-HCV-related mutations in the serine protease domain of HCV NS3 during selection, the 1.7-kb purified RT-PCR products of replicons treated with Pl from several points in time of different cultures were subjected to sequence determination. To determine the frequency of PI-resistant mutations, the 1.7-kb RT-PCR products of the HCV RNA of the VX-950 or BILN 2061 resistant replicon cells were ligated into the cloning vector Ta pCR2.1 ( Invitrogen). For each point in time, multiple individual bacterial colons were isolated and the region coding for the HCV NS3 protease of the purified plasmid DNA was sequenced.

Example 6: Expression and purification of the serine protease domain of HCV NS3 Each of the expression constructs for the serine protease domain of HCV NS3 which are the natural sesquence of the resistance mutations (A156S, D168V, or D168A) were transformed into E. coli cell BL21 / DE3 pLysS (Stratagene). Freshly transformed cells were grown at 37 ° C in BHI medium (Difco Laboratories) supplemented with 100 μg per ml of penicillin and 35 μg per ml chloramphenicol up to an optical density of 0.75 to 600 nM. Induction with 1 mM IPTG was effected for 24 hours at 24 ° C. The filler doughs were harvested by centrifugation and frozen instantaneously at -80 ° C before purification of the protein. All the purification steps were carried out at 4 ° C. For each of the NS3 proteases of HCV 100 g of cell paste in 1.5 L of buffer A were used. { 50 mM HEPES (pH 8.0), 300 mM NaCl, 0.1% n-octyl-β-D-glucopyranoside, 5 mM β-mechaptoethanol, 10% (v / v) glycerol} and stirred for 30 min. The lysate was homogenized using a microfluidizer (Microfluidics, Newton, MA), followed by ultracentrifugation at 54,000 x g for 45 min. Imidazole was added to the supernatants at a final concentration of 5 mM together with 2 ml of Ni-NTA resin pre-equilibrated with buffer A containing 5 mM imidazole. The mixtures were stirred for three hours and washed with 20 column volumes of buffer A plus 5 mM imidazole. The mixtures were stirred for three hours and washed with 20 volumes of buffer solnum A plus 5 mM imidazole. The NS3 proteins of HCV were eluted in buffer A which had 300 mM imidazole. The eluates were concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, pre-equilibrated with buffer A. The appropriate fractions of the purified purified HCV proteins were pooled and stored at -80 ° C.

Example 7: Enzyme assays for the serine protease domain of HCV NS3 A Enzymatic assay protocol by CLAP Method for CLAP with rr crdboron to separate the substrate 5AB and products Substrate: NH2-Glu-Asp-Val-Val- (alpha) Abu-Cys-Ser-Met-Ser-Tyr-COOH A 20 mM 5AB standard solution (or solution concentration) was produced in DMSO w / with 0.2 M DTT. This was stored in aliquots at -20 C.

Shock absorber: 50 mM HEPES, pH 7.8; 20% glycerol; 100 mM The total assay volume was 100 μL The shock absorber KK4A, DTT, and tNS3 were combined; 78 μL were distributed in each well in 96-well plate wells. This was incubated at 30 C for -5-10 min. 2.5 μL of the appropriate concentration of test compound in DMSO (DMSO only for control) was dissolved and added to each well. This was cooled to room temperature for 15 min. The reaction was initiated by the addition of 20 μL of 5AB substrate of 250 μM substrate (the concentration of 25 μM is equivalent or slightly less than the Km for 5AB). The reaction was incubated for 20 min at 30 ° C, and then terminated by the addition of 25 μL of 10% TFA. Aliquots of 120 μL of final reaction product were transferred to CLAP flasks. The SMSY product was separated from the substrate and KK4A by the following method.

Microbore separation method; Instrumentation: Agilent 1100 Degasser G1322A Binary pump G1312A Sampler G1313A Camera with column thermostat G1316A Detector of diode array G1315A Column: Phenomenex Jupiter; C18 of 5 micrometers; 300 angstroms; 150x2 mm; P / O 00F-4053-B0. Column thermostat: 40 ° C Injection volume: 100 μL Solvent A = water grade CLAP + 0.1% TFA Solvent B = CLAP grade asetonitrile + 0.1% TFA B FRET Enzyme Assay Protocol Enzyme activity was determined using a modification of the assay described by Taliani et al., [Taliani et al., Anal. Biochem., 240, pp. 60-67 (1996)]. An internally extinguished fluorogenic peptide (FRET substrate), Ac-DED (EDANS) EEaAbuD [C00] ASK (DABCYL) -NH2, was purchased from AnaSpec Incorporated (San José, CA). The assay was performed in a continuous mode in a 96-well microtiter plate format. The buffer was composed of 50 mM HEPES (pH 7.8), 100 mM NaCl, 20% glycerol, 5 mM DTT, and 25 μM KK4A peptide (KKGSWIVGRIVLSGK; SEQ ID NO: 5). The KK4A peptide represents the central region of the NS4A cofactor of the genotype with added lysine residues for better solubility [Landro et al. Biochemistry, 36, pp. 9340-9348 (1997)]. The reaction was initiated by the addition of the FRET substrate after a 10-min preincubation of the buffering components with 2 nM NS3 protease at room temperature. The region was verified at 30 ° C for 20 min using a fmax fluorometric plate reader from Molecular Devices. The filters for the excitation and emission wavelengths were 355 nm and 495 nm, respectively. For the determination of the kinetic parameters of the substrate, the FRET peptide concentrations were varied from 0.5 to 7.0 μM. No intermolecular extinction was observed in this interval. The kinetic parameters of the substrate, Km and V ax, were determined by fitting the data to the Michaelis-Menten equation. The inhibition constants (Ki) were determined by the titration of the activity of the enzyme using the assay described above, except that the compound dissolved in DMSO was added (not more than 2% v / v of DMSO, only solvent was used as control) to the buffering and enzyme components after the initial 10 minute preincubation as described above. This mixture was incubated for an additional 15 min at room temperature before incubation with the FRET substrate for another 20 min at 30 ° C. SE were tested from seven to eight concentrations of compound, and the resulting data were adjusted to the integrated form of the Morrison equation for the inhibition of a strong binding [J.F. Morrison, Biochim. Biophys. Acta, 185 pp. 269-286 (1969)]. All substrate and inhibitor data were adjusted using Marquardt-Levenberg nonlinear regression with the GraphPad Prism programming programs and systems.

Example 8: Development of resistance to VX-950 in HCV replicon cells The VX-950 (Figure 4A, chemical structure) is a clinical candidate for the treatment of Hepatitis C. VX-950 is a covalent, reversible inhibitor, of the serine protease of NS3 * 4A HCV. Although it competes with the peptide substrate at the active site, it exhibits apparent noncompetitive inhibition as a result of its strong binding properties and time-dependent inhibition mechanism (C.Gates and Y-P. Luong). Incubation of HCV Congen subgenomic replicon cells with VX-950 resulted in a concentration-dependent decline in HCV RNA level, as measured by the real-time RT-PCR (Taqman) method ( Figure 5B). The IC50 value of VX-950 is 354 riM in the 48 test. To identify the VX-950 resistance mutations, the Congen subgenomic replicon cells were serially passed (ie, growth in subculture) in presence of 0.25 mg per ml of G418 and gradually increasing the concentrations of VX-950 (series A) (Figure 5A, selection curve). The initial concentration of VX-950 was 3.5 μM or 10 times the IC 50 and the highest concentration was 28 μM or 80 times the IC 50. The replicon cells were divided or the medium was replenished every 3 or 4 days, and fresh VX-950 was added. Since VX-950 inhibits HCV NS3 serine protease activity and consequently blocks the replication of HCV RNA, the steady state level of HCV proteins and neomycin transferase protein gradually declined and eventually became undetectable in the presence of a high concentration of VX-950. Cells with little or no protein neomycin transferase proliferate at a rate that gradually decreases and eventually die in G418 presensia. HCV RNA alone are mutations that are resistant to VX-950, can be repaired in the presence of a high concentration of VX-950 and support the growth of the replicon cells that harbor it. The replicon cells in the A series grew normally during the first 10 days in the presence of 3.5 μM of VX-950. After 10 days, the cells of Serial A were significantly slower and mass selcular death was observed between days 10 and 17 (Figure 5A, surva of selection). Normal growth was not resumed until day 21. It was determined that the IC50 of VX-950 against the replicon cells of series A at day 56 was 8.1 to 12.0 μM, which is 23- to 34 times higher than IC50 (354 nM) against natural replicon cells (Figure 5B, IC50 curve).

The total cellular RNAs of the cells of series A on days 7, 21, and 56 were extracted and subjected to RT-PCR to amplify the coding region of the serine protease domain NS3. The product of the RT-PCR was sequenced in bulk to identify the positions of potential mutations that could be responsible for the observed reduction in sensitivity to VX-950. The nucleotide and amino acid sequences of the natural HCV protease of the original refill cells are shown in SEQ ID NO: l and SEQ ID NO: 2. No mutation related to VX-950 was observed in the serine protease domain of NS3 of the replicon cells of the A series on day 7 when compared with the natural Conl replicon cells cultured in the absence of the VX-950. At days 21 and 56 in series A, substitutions were observed in Alal56 in the protease domain, suggesting that the mutations in residue 156 may be critical for the reduction of sensitivity to VX-950. No mutation was found in any of the four proteolytic sites in the non-structural HCV protein region that are cleaved by the serine protease of NS3 »4A. To delineate the identity and frequency of substitutions, a 1.7-kb RT-PCR product from series A replicon cells was subcloned on days 7 or 98 in the TA vector and multiple clones were sequenced for both samples. All the clones derived from the samples from day 7 contained the natural Alal56. On day 98 the sample of replicon cells of the A series, which had been grown in the presence of 28 μM of VX-950 for 63 days, had a substitution of 79% or 60% of 76 clones of alanine to serine (A156S). In addition, cells resistant to VX-950 have been selected under a constant concentration of VX-950 and G418. In this case, multiple colonies of resistant cells were observed after a prolonged culture period under VX-950 and G418. The HCV NS3 serine protease sequences were determined from those resistant colonies and similar mutations were found at amino acid 156 of the HCV serine protease.

Example 9: Development of BILN 2061 Resistance in HCV Replicon Cells Another inhibitor of HCV NS3 * 4A protease, BILN 2061 (Figure 4B, chemical structure) (WO 00/59929; US 6,608,027) has been shown to be effective in patients with Hepatitis C (lamarre et al., Nature Medicine, 2003). The HCV replicon cells resistant to BILN 2061 (B series) were selected in a similar manner as for the VX-950. Again, subgenomic HCV replicon cells were run in series in the presence of 0.25 mg per ml of G418 and slowly increasing concentrations of BILN 2061 (Figure 6A, selection curve). The B-series replicon cells grew normally during the first 7 days in the presence of BILN 2061 80 nM or 80 times above the IC 50. However, the proliferation of the B-series cells decreased significantly after 7 days and massive cell death was observed between days 7 and 17. As previously, normal growth was not resumed until day 21. The BILN 2061 had a IC50 value of 1.0 to 1.8 μM against the cells of the B series at day 59, which is 1,000 to 1,800 times higher than that of the IC50 (1 nM) against the natural replicon cells (Figure 6B, IC50 curve) ). No relasional mutations were observed in BILN 2061 in the serine protease domain of NS3 at day 7. On day 24, a variety of substitutions were observed at amino acid 168 of the NS3 protein, suggesting that substitutions at residue 168 may contribute to resistance against BILN 2061. No mutations were observed at the four sites in the non-structural HCV protein region that are cleaved by the serine protease of NS3 »4A. To determine the frequency of several substitutions at residue 168 of NS3, the NS3 serine protease of the B-series replicon at day 98, which was cultured in the presence of 3.2 μM BILN 2061, was sequenced. 60 of 94 clones or 64% had a substitution of Aspl68 to Val (D168V), and 23 clones or 24% had a mutation from Aspl68 to Ala (D168A).

In addition, cells resistant to BILN 2061 were selected under a constant concentration of BILN 2061 and G418. In this case, multiple colonies of resistant cells were observed after a prolonged culture period under BILN 2061 and G418. The NS3 serine protease sequences of HCV were determined from those resistant colonies and similar mutations were found at amino acid 168 of the HCV serine protease.

Example 10: Selection of the Replicon of Resistant Cells to Both of the VX-950 and BILN 2061 A. Development of Cross-Resistance HCV Replicons from VX-950 Resistant Cells To identify resistance mutations that cross-resist both the VX -950 and the BILN 2061, several selection schemes were used. The first was serially passed a replicon cell line resistant to VX-950 [series A in C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)] in the presence of 0.25 mg / ml of G418, 14 μM VX-950, and slowly increasing the BILN concentrations 2061 (series C) (Figure 8A). For BILN 2061, the initial concentration was 40 nM and the final concentration was 6.4 μM. The replicon cells were divided or the medium was replenished every 3 or 4 days, and fresh VX-950 and BILN 2061 were added. Since HCV Pl inhibits the serine protease activity of NS3 »4A and consequently blocks the replication of HCV RNA, the steady-state level of HCV protein and neomycin transferase protein gradually declined and eventually became undetectable in the presence of a high concentration of HCV Pl (data not shown). Cells with little or no neomycin transferase protein proliferate at a rate that gradually decreases and eventually die in the presence of G418. It is expected that the replicon cells with the major VX-950 resistant mutation, A156S, die in the presence of increasing concentrations of BILN 2061 since they have been shown to be susceptible to inhibition of BILN 2061 [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)]. Only the HCV RNA with mutations that show squirrel resistensia to both the VX-950 and BIL? 2061 can replicate in the presence of high concentrations of HCV Pl and support the growth of the replicon cells that harbor them. However, the replicon cells in the C series grew normally during the entire selection process, which lasted 56 days. It was determined that the IC50 values of the BIL? 2061 against the C-series replicon cells at day 52 were -3 μM, which are 300 times greater than those of the IC50 against the A-series replicon cells (VX-950 resistant) (~ 10 nM ) (Figure 8B). Since the 30 μM VX-950 did not result in a greater than 50% reduction of HCV RNA in the C-series replicon cells at day 52, the actual IC 50 values of the VX-950 could not be determined. which indicates that the C-day replicon cells at day 52 are still resistant to VX-950 (Figure 8C). The total cellular RNA of the cells of the C series at day 32, to which they had been cultured in the presence of VX-950 14 μM and 0.32 μM of BILN 2061, was extracted and subjected to RT-PCR to amplify the coding region of the serine protease domain of NS3. The product of the RT-PCR was massively sequenced to identify the positions of potential mutations that could be responsible for the observed reduction in sensitivity to both of HCV Pl. Substitutions were observed in Alal56 in the protease domain, suggesting that mutations in residue 156 may be critical for the reduction of sensitivity to both Pl. This observation was somewhat unexpected since it was found that the mutation resistant to the larger VX-950 is A156S [C. Lin et al. J. Biol. Chem. 279, 17508-17514 (2004)]. No mutation was found in any of the four proteolytic sites in the non-strutural HCV protein region that are cleaved by the serine protease of NS3 »4A. To delineate the identity and frequency of the sustitus, a 1.7-kb RT-PCR product of C-series replicon cells was subcloned at day 32 into the TA vector and 10 individual colonies were subjected to sequencing. 6 clones had a substitution of Alal56 to Thr (A156T), and 3 clones had a substitution of Alal56 with Val (A156V). The lOma clone retains the A156S mutation.

B. Development of Cross-Resistance HCV Replicons from 2061 BILN Resistant Cells The second sequestration scheme was to grow BILN 2061-resistant replicon cells in the presence of BILN 2061 and VX-950. In this case, a replicon line resistant to BILN 2061 [B series in C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)] in the presence of 0.25 mg / ml of G418, and slowly increasing the consentrations of VX-950 and BILN 2061 (series D) (Figure 9A). For the BILN 2061, the initial concentration was 160 nM and the final concentration was 6.4 μM. Only two concentrations of VX-950 were used: 7 μM and 14 μM. It was expected that the replicon cells with the older 2061 BILN-resistant mutations, D168V or D168A, would die in the presence of high concentrations of VX-950 since they had been shown to be susceptible to VX-950 inhibition.

[C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)].

Again, only HCV RNA with mutations that had cross-resistance to both VX-950 and BILN 2061 can replicate in the presence of high concentrations of both PI Plots of HCV and support the growth of the replicon cells harboring them. However, the replicon cells in the D series normally were found for most of the selection processes, which also lasted 56 days. Since the 30 μM VX-950 did not result in a greater than 50% reduction of HCV RNA in the D-series replicon cells at day 52, the actual IC50 values of VX-950 could not be determined, but it will be at least greater than 100 veses than the IC50 (-0.3 μM) against the B-series replicon cells (resistant to BILN 2061) (Figure 9B). It was determined that the IC50 values of BILN 2061 against the D-series replicon cells at day 52 were -4 μM, which indicates that the D-series replicon cells at day 52 are still resistant to BILN 2061 ( Figure 9C). The total cellular RNA of the D series cells at day 32, which had also been cultured in the presence of 14 μM VX-950 and 0.32 μM BILN 2061, was extracted and subjected to RT-PCR to amplify the serine coding region NS3 protease. The RT-PCR product was massively sequenced to identify the positions of potential mutations that could be responsible for the observed mutation to the sensitivity of both HCV Pl. Again, substitutions were observed in Alal56 in the protease domain, confirming that mutations in residue 156 may be critical for reduced sensitivity to both Pl. No mutation was found in any of the four proteolytic sites in the non-structural HCV protein region that are cleaved by the serine protease of NS3 * 4A. To delineate the identity and frequency of the substitutions, a 1.7-kb RT-PCR product from the A-series replicon cells was subcloned on day 32 into the TA vector and 14 individual colonies were subjected to sequencing. 12 clones had the A156V substitution, while 1 clone had the A156T mutation. The 14th clone had two mutations, A156S and D168V.

C. Development of Cross-Resistance HCV Replicons from Replicon Cells without Prior Activation In our previous studies of resistance mutations against a single PI of HCV, cell growth was prevented for several days by VX-950 or BILN 2061, during which massive cell death was observed [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)], which indicated the emergence of resistant mutant replicon cells and concurrent death of non-resistant replicons. However, no such cell death or decrease in cell growth was observed at the selection of the cross-resistance replicon of the C or D series as described above. It is possible that cross-resistance mutations, A156T and A156V, may already have existed in replicon cells resistant to VX-950 (series A) or BILN 2061 (series B) as a minor population. If so, those two selection schemes could select a deviation towards the mutation A156T or A156V over other potential cross resistance mutations. In this way, a third selection scheme was carried out using candid HCV replicon cells that are sensitive to any inhibitor. Congel subgenomic replicon cells derived from pBR322-HCV-Neo-mADE [C. Lin et al. J. Biol. Chem. 279, p. 17508-17514 (2004)] were serially run in the presence of 0.25 mg / ml of G418 and slowly increasing the concentrations of VX-950 and BILN 2061 (E series) (Figure 10). The initial concentration of VX-950 was 3.5 μM and the highest concentration was 14 μM. For the BILN 2061, the initial concentration was 80 nM and the final consentration was 3.2 μM. The replicon cells were divided or the medium was replenished every 3 or 4 days, and fresh VX-950 and BILN 2061 were added. Replicon cells in the E series grew normally during the first 10 days in the presence of 3.5 μM VX-950 and 160 nM BILN 2061. After 10 days, the cells of the E series grew significantly more slowly and massive cell death was observed between days 10 and 21 (Figure 10). Normal growth was not resumed until day 21. The total cellular RNA of the C-series cells on days 10, 21, and 48 was extracted and subjected to RT-PCR to amplify the coding region of the serine protease domain. of NS3. No HCV Pl related mutation was observed in the serine protease domain of NS3 of the E-series replicon cells at day 10 when compared to natural Conl replicon cells cultured in the absence of both HCV Pl. To delineate the identity and frequency of the substitutions, a 1.7-kb RT-PCR product from E-series replison cells was subcloned on day 21 or 48 in the TA vector and multiple clones were sequenced for each of the samples On day 21 the sample of replicon cells of the E series, which had been cultured in the presence of 3.5 μM VX-950 and 0.32 μM of BILN 2061 for 14 days, 65% or 30 of 46 clones had a substitution of Alal56 at Thr (A156T), while the other substitution of Alal56 with Val (A156V) was found in 35% or 16 of 46 clones. For the day 48 the E-series sample, which had been sultivated in the presence of 14 μM VX-950 and 1.6 μM of 2061 BILN for 14 days, 80% or 35 of 44 clones had the A156T substitution, while the A156V substitution was found in 20% or 9 of 44 clones. In any case, no other mutations were found in the serine protease domain of NS3 in more than 10% of the plasmid clones of TA, indicating that the A156T and A156V are the only two mutations that contain cross-resistance to both of VX-950 and BILN 2061.

Example 11: Demonstration and Confirmation of Mutations Resistant in Amino Acid 156 or 168 in Enzymatic and Replicon Cell Assays. To confirm whether the mutations observed in Alal56 or Aspl68 are sufficient to confer resistance against VX-950 or BILN 2061, respectively, site-directed mutagenesis was used to introduce each individual mutation at position 156 or 168 in the natural NS3 protease domain . Site-directed mutagenesis is another specific technique useful in the preparation of mutant protease proteins used in the methods of the invention. This technique employs the mutagenesis specific to the subjacent DNA (which codes for the amino acid sequence that is targeted by the modification). The technique also provides an easy ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more changes in the nucleotide sequence in the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as well as sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient sequence size and complexity to form a stable duplex on both sides of the suppression junction that is being traversed. Typically, a primer of about 17 to 25 nucleotides in length, with about 5 to 10 residues on either side of the junction of the sequence being altered, is preferred. The technique typically employs a bacteriophage vector that exists in the form of a single strand and double strand. Typical vectors useful in site-directed mutagenesis include vectors such as f or M13. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid. In general, site-directed mutagenesis is effected by first obtaining a single-stranded vector, or by fusing the two strands of a double-stranded vector that includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer containing the desired mutant sequence is prepared synthetically. This primer is then annealed with the preparation of single-stranded DNA taking into account the degree of mismatch when the hybridization conditions are selected (annealing), and is subjected to enzymes that polymerize DNA as the Klenow fragment of E polymerase I coli, to complete the synthesis of the strand that contains the mutation. In this way, a heteroduplex is formed where one strand codes for the original non-mutant sequence and the second strand contains the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include the vectors containing the array of the mutant sequence. Of course, the method described above for site-directed mutagenesis is not the only method to generate potentially useful mutant protease species and therefore does not mean that it is limiting. The present invention also contemplates other methods to achieve mutagenesis, for example, treating recombinant vectors containing the gene of interest of mutagenic agents, such as hydroxylamine to obtain sequence variants.

A. The dominant VX-950 resistant mutant, A156S, remains susceptible to 2061 BILN. To confirm whether the observed substitution of Alal56 with Ser is sufficient to confer resistance against VX-950 but not against BILN 2061, site-directed mutagenesis was used. to replace Alal56 with Ser in the natural NS3 protease domain. The kinetic parameters for the FRET substrate for the natural NS3 protease domains of genotype la and Ib were identical (Table 1) under our assay conditions. Although the cofactor of the NS4A peptide was of the HCV genotype, no discernible differences in the kinetic parameters were observed. This is consistent with molecular modeling, which suggests that the conservative variations in the central region of NS4A between the genotypes la and Ib do not affect the interaction between the central peptide of NS4A and the protease domain of NS3. The Ki values of VX-950 and BILN 2061 were determined using the genotypes la and lb of natural protease, and there were no statistically significant differences between the two natural proteases (Table 2). The synthetic parameters of the FRET substrate for the A156S mutant protease were virtually the same as those of the natural protease (Table 1). However, the Ki value of the VX-950 was 2.9 μM against that of the A156S mutant protease, which is 29 times higher than that of the natural protease (0.1 μM) (Table 2). BILN 2061 has a Ki value of 112 nM against that of the A156S mutant, which is 6 times higher than that of the natural protease, 19 nM (Table 2).

The level of HCV RNA in the replicon cells containing the A156S substitution that is similar to that of the natural replicon cells (data not shown), or which is consistent with the similar enzymatic satatytic efficiency of the mutant A156S and the serine proteases of natural NS3. The IC50 value of VX-950 against the A156S replicon cells was 4.65 μM, which is 12 times higher than against natural replicon cells (0.40 μM) (Table 3). The difference between IC50 values of BILN 2061 against A156S (7 nM) and natural replicon cells (4 nM) was not significant (Table 3).

B. The older 2061 BILN resistant mutants, D168V and D168A, remain fully susceptible to VX-950. To confirm if the substitutions observed in either Aspl68 with Val or Ala are sufficient to confer resistance against BILN 2061 but not with VX-950, site-directed mutagenesis was used to replace Aspl68 with either Val or Ala in the Protease domain of natural NS3. The kinetic parameters of the substrate were not affected by the D168V mutation, and showed only minor changes (less than 10 times) for the mutant D168A as indicated by the comparison of the kcat and kcat / Km values of the two serine proteases of natural NS3 and mutant (Table 1). Similarly, no significant effect was observed on any substitution in Aspl68 over the Ki value of VX-950 (Table 2). However, the substitution of valine or alanine for aspartic aspart at position 168 resulted in a mutant NS3 protease that was not inhibited until 1.2 μM of BILN 2061 (Table 2). These data indicate that any mutant protease is at least 63 times less susceptible to BILN 2061 compared to the natural protease. The actual magnitude of resistance can not be determined since the BILN 2061 was not soluble at concentrations higher than 1.2 μM in the assay buffer, as measured by the absorbance at 650 nm. The D168V or D168A mutation was also introduced into the natural HCV replicon by site-directed mutagenesis and a stable replicon cell line containing any substitution was generated. The BILN 2061 had an IC50 of 5.09 μM against the D168V replicon cells, which is greater than 1300 times than against the natural replicon cells (4 nM) (Table 3). The IC50 of the BILN 2061 was 1.86 μM against the mutant replicon D168A. There was little change in the IC50 values of VX-950 against D168V and the natural replicon cells (Table 3). Table 1 shows the characterization of the enzymatic properties of natural or mutant HCV NS3 serine protease domains. Five proteins of the serine protease domain of HCV NS3, including the natural proteases of genotype la and Ib (wt), and three mutants (A156S, D168V, and D168A) of the genotype Ib, were expressed and purified as described in the Materials and Methods. The kcat and Km values of these NS3 proteases were determined using the core peptide of KK-NS4A and the substrate FRET.

Table 1. Enzymatic characterization of natural or mutant HCV NS3 serine proteases Table 2 shows a confirmation of resistance in the enzymatic assay. The Ki values of VX-950 and BILN 2061 were determined against the five serine protease domains of purified HCV NS3, including the natural proteases (p) of the genotype la or lb, as well as three mutants, A156S, D168V, and D168A in the lb genotype, using the KK-NS4A peptide and the FRET substrate. The solubility of BILN 2061 in the buffer was limited to sonsentrasions greater than 1.2 μM. No inhibition was observed by either the mutant NS3 protease D168V or D168A in the presence of 1.2 μM BILN 2061.

Table 2. Ki values of VX-950 and BILN 2061 against natural and mutant NS3 proteases Table 3 shows the confirmation of resistance to HCV replicons. Four stable subgenomic HCV replicon cell lines, including natural gas, and three mutants A156S, D168V and D168A were generated, using T7 RNA assay transcripts of the replicon plasmids are one of corresponding high efisiensia. The IC50 values of VX-950 and BILN 2061 were determined against the four HCV replicon cell lines in the standard 48-hour assay.

Table 3. IC50 values of VX-950 and BILN 2061 against HCV replicon cells with natural and mutant NS3 proteases C. The A156T and A156V mutations present cross-resistance to both VX-950 and 2061 BILN. To confirm whether the mutations observed in Alal56 are sufficient to confer cross-resistance against both the VX-950 and BILN 2061, site-directed mutagenesis was used to replace Alal56 with Val or Thr in the protease domain of NS3. The catalytic efficiency (kcat / Km) of the mutant protease A156T or A156V against the FRET substrate was approximately 5 to 7 times lower than that of the natural protease (Table 4). The Ki value of VX-950 was less than 9.9 μM or 33 μM against the mutant protease at A156T or A156V, respectively, which is 99- or 330-fold higher than against the natural protease (0.1 μM), respectively (Table 5). The mutant protease was not inhibited until BILN 2061 1.2 μM (Table 5). These data indicate that the mutant protease is at least 63 times less susceptible to BILN 2061 compared to the natural protease. The actual magnitude of resistance could not be determined since the BILN 2061 was not soluble at concentrations higher than 1.2 μM in the assay buffer, as measured by the absorbance at 650 nm (the data is not shown).

Table 4 Enzymatic properties of natural and mutant HCV NS3 serine protease domains Summary of Table 4: 3 HCV NS3 serine protease domain proteins from the Conl strain, including the natural proteases and two mutants, A156T and A156V, were expressed and purified. The kcat and Km values of these NS3 proteases were determined using the central peptide KK-NS4A and the substrate FRET, and the average of two independent assays is shown.

Table 5 Confirmation of the resistance in the enzyme assay Summary of Table 5: Ki values of VX-950 and BILN 2061 were determined against five purified HCV NS3 serine protease domains, including the natural protease, as well as two mutants, A156T and A156V, using the KK- peptide NS4A and the substrate FRET. The solubility of BILN 2061 in the reaction buffer was limited to concentrations higher than 1.2 μM. No inhibition was observed for any mutant NS3 protease A156T or A156V in the presence of 1.2 μM BILN 2061. The level of The RNA in the replicon cells containing the substitutions A156T or A156V was lower than the natural replicon cells (data not shown), which is consistent with a lower catalytic enzyme efficiency of the two mutants in comparison with that of the natural NS3 serine proteases. No significant reduction of HCV replicon RNA to 30 μM VX-950 was observed in any mutant replicon cell line, indicating a decrease of at least 75 times in the sensitivity conferred by the mutation (Table 6). The IC50 value of BILN 2061 against replicon A156T cells was 1.09 μM, which is approximately 272 times higher than against natural replicon cells (4 nM). For the A156V mutant replicons, the BILN 2061 had an IC50 value of 5.76 μM, indicating a decrease of more than 1,400 fold in the sensitivity conferred by the A156V mutation (Table 6).

Table 6: Confirmation of resistance in HCV replicons Summary of Table 6: Three stable HCV subgenomic replicon cell lines were generated, including the wild type, and two mutants, A156T and A156V, using the T7 RNA transcripts of the replicon plasmids with one of corresponding high efficiency. The IC50 values of VX-950 and BILN 2061 were determined against the three HCV replicon cell lines in the standard 48-hour assay, and the average of two dependent assays was shown.

Example 12: Modeling - I The VX-950 and BILN 2061 were modeled at the active site of the serine protease domain of NS3 using the structure of the full-length HCV NS3 protein published by Yao et al., [Yao., Et. al., Structure Fold Des., 7, pp. 1353-1363 (1999)] (PDB code: 1CU1). The coordinates of the protease domain of segment A in this structure demonstrated that the C-terminal strand of the NS3 protein binds at the substrate binding site of the protease. The carboxyl terminal group of this strand is located near the residues of the active site His57, Asp81, and Serl39 so that it forms hydrogen bonds with the side chains of His57 and Serl39 as well as the structural amides of residues 137 and 139, which they form the oxyanionic orifice. Addition- ally, the last six residues (626 to 631) of the NS3 protein form an antiparallel ß strand, extended along the edge of the E2 strand of the β-protease barrel and produce 12 skeletal or skeletal hydrogen bonds or structure to structure. An inhibitor based on the product such as BILN 2061 is expected to bind to the NS3 protease in a similar manner. Therefore, using the coordinates of this crystal structure to build our models of inhibitor-protease complexes. The 2061 BILN molecule was constructed in the QUANTA molecular model programming and programming systems (Acselrys Inc., San Diego, California, USA), and manually marked at the active site so that its carboxyl group overlaps with the carboxylate C -terminal of NS3 of the full length NS3 protein. The inhibitor molecule was then rotated so as to produce all the following hydrogen bonds in the structure or backbone: the NH of Pl with the carbonyl of Argl55, the carbonyl of P3 with the NH Alal57, and the NH of P3 with the carbonyl from Alal57. This binding mode placed the large P2 group of BILN 2061 in direct shock with the side chain of Argl55 to avoid shock, the side chain was modeled in an extended conformation as suggested by the description of the crystalline structure of a NS3 protease complex with an inhibitor that is analogous to BILN 2061 [YS Tsantrizos, Angew. Chem. Int. Ed. Engl. 42, pp. 1356-1360 (2003)]. The inhibitor was minimized in energy in two stages. In the first stage, only the inhibitor and the side chain atoms of Argl55, Aspl68 and Argl23 of the protease were given the freedom to move during energy minimization during 1000 steps. In the second step, all the atoms of the side chain of the active site were allowed to move along with the inhibitor for an additional 1000 steps. This modeled structure closely mimics the published structure of the BILN analogue 2061 [Y.S. Tsantrizos, Angew. Chem. Int. Ed. Engl. 42, p. 1356-1360 (2003)]. A similar procedure was adopted to model VX-950 at the active site of the protease. The inhibitor was modeled as a covalent adduct with a si-side junction of the Serl39 side chain to the keto carbonyl of the inhibitor. This mode of binding has been observed for analogous ketoamide inhibitors [Perni et al., Bioorg. Med. Chem. Lett. 14, in press (2004)] and ketoacid inhibitors [Di Marco et al., J. Biol. Chem., 275, p. 7152-7157 (2000)]. The main chain of the inhibitor was superimposed by residues 626 to 631 of the C-terminal strand of NS3 so that all of the following hydrogen bonds were produced with the backbone or structure: NH of Pl with the carbonyl of Argl55, the sarbonyl of P3 with the NH of Alal57, NH of P3 with the carbonyl of Alal57, and the carbonyl of the crown of P4 with the NH of Cysl59. In this binding mode, group P2 of the VX-950 was placed in the cavity of S2 without any need to move the side chain of Argl55. The t-butyl and the cyclohexyl groups were placed in the cavities of S3 and S4, respectively. To be consistent, we used the same two-stage energy minimization protocol used for the BILN 2061 model. These two complex models were used to predict the effect of Alal56 and Aspl68 mutations or the binding of protease inhibitors. The side chain of Aspl68 is exposed to solvent. The valine side chain of mutant D168V can adopt three conical conformations with? X = 60 °, -60 ° or 180 °. The variations after the Vall68 side chain were modeled. The interaction energy of the mutant enzyme D168V and the inhibitor was minimized by allowing the inhibitor and the Vall68 atoms to move while fixing the positions of all the other atoms of the protein molecule. In all cases, the Vall68 side chain does not produce any spherical shock with the inhibitor atoms. The mutation of serine in Alal56 was modeled by the following procedure. The Alal56 side chain is in contact of van der Waals with the P2 group of both inhibitors (Figure 9). The serine side chain of mutant A156S was modeled into three conical conformations of? I = 60 °, -60 ° and 180 °, and the energy was minimized by assembling the conformation of the rest of the fixed protein. These models were used to examine the effects of this mutation on the binding of the inhibitor. It was found that the -60 ° conformation has the lowest energy since it forms a hydrogen bond with the neighbor Argl55 carbonyl, but produces the maximum number of unfavorable contacts with both inhibitors. The 60 ° and 180 ° conformations are energetically equivalent, but the 60 ° conformation has less favorable counts and was used in our analysis. Alal56 is located on strand E2 in a structure of the NS3 protease «4a of HCV [R.A. Love at al, Cell 87, pp. 331-342 (1996)]. Several atoms of the structure of this strand (mainly the carbonyl of Argl55 and both of the nitrogen and carbonyl of the Alal57 backbone) produce hydrogen bonds with the atoms of the structure or skeleton of the substrates inhibitors based on the substrate. In our structural model of the VX-950 complex: NN3 protease (Figure 9), three hydrogen bonds were formed between the NH of Pl in the carbonyl of Argl55, the carbonyl of P3 and the NH of Alal57, and the NH of the P3 and the carbonyl of Alal57. The same hydrogen bonds were formed in the model by BILN 2061 complex. The Alal56 side chain is a van der Waals contact with a P2 group of those inhibitors. Our mutant model A156S the terminal oxygen of Serl56 is too close to the cyclohexyl group P2 of VX-950, and is also close to the terminal cyclopentyl crown of BILN 2061. Since the cyclopentyl crown of BILN 2061 is at the flexible end of the inhibitor, can be removed from this unfavorable contact without losing much of the union. A similar movement of the cyclohexyl group of P4 of the VX-950 causes destabilization of the interactions between the inhibitor and the S4 and S5 subsidies of the protease. Therefore, greater binding loss is expected for the VX-950 than for the BILN 2061 with the A156S mutant protease. Aspl68 is located in strand F2 and the structure of the protease of NS3 and is involved in the interactions of the salt bridge with the side chains of Argl23 and Argl55 (Figure 9) [R.A. Love at al, Cell 87, pp. 331-342 (1996)]. It is also part of the S4 connection cavity. The aliphatic part of this side chain is in contact of van der Waals with the terminal cyclopentyl group of BILN 2061, which is expected not to be affected by the mutation of D168V, since a valine side chain in this position does not produce any sterile shocks that are the inhibitor. However, this D168V substitution results in the loss of the interaction of the salt bridge with the side chain of Argl55 on E-2 (Figure 1), which in turn makes multiple contants with the long P2 group and the BILN 2061 in the model described here. The conformation of Argl55 (Figure 9, encoded in cyan) in the model of the BILN 2061: natural NS3 protease complex is no longer favored energetically in the mutant D168V for two reasons. First, it can not remain close to the structure of strand E-2 in the absence of the salt bridge interaction between Argl55 and Aspl68. Second, a positive charge not compensated and exposed to the solvent, the Argl55 side chain will look for a larger solvation nucleus, as observed in the crystalline structures of the protease and the two protease: inhibitor complexes that are available in the Protein Data Ban (codes: 1DY8 and 1DY9) [S. Di Marco et al., J. Biol. Chem., 275 pp. 7152-7157 (2000)]. These conformations of the Argl55 are in shoque diresto with the quinoline group of P2 of the BILN 2061 and destabilize their union. Therefore, the substitution of Aspl68 with any amino acid, with different glutamate, disrupts the interactions of the salt bridge are Argl55 and will result in the redussion of BILN 2061 binding. On the other hand, the conformation of Argl55 of the two crystalline structures published of NS3 protease complex: inhibitor and similar to that of the VX-950 complex model: protease (encoded in orange in the in orange in Figure 9). In addition, this Argl55 conformation confers the stabilization of the VX-950 junction since it allows the maximum number of van der Waals contacts between the Argl55 side chain and the inhibitor. Therefore, the VX-950 is not expected to be afflicted by the substitution in Aspl68 compared to the BILN 2061.

Example 13: Modeling - II Modeling of VX-950 and BILN 2061 at the active site of the serine protease domain of NS3 using the crystal structure of the NS3 NS3 protein of full length [N. Yao et al., Strusture Fold Des. 7, pp. 1353-1363 (1999)] (Protein Data Base sode: 1CU1) was previously described [C. Lin et al. J. Biol. Chem. 279, 17508-17514 (2004)]. The side chain of Alal56 on the strand of ß-of E2 on the protease NS3 * 4A of HCV separates the savities S4 and S2 from the active site of the enzyme and is in contact of van der Waals with the P2 group of the two inhibitors ( Figure 7). The replacement of Val or Thr of Alal56 extends the side chain with two additional groups (methyl or hydroxyl) into the compact space between the natural enzyme and the inhibitors. The lateral chain of Vall56 or Thrl56 was modeled in the three possible conical conformations = 60 °, -60 ° and 180 ° following the procedure previously described for modeling the mutation A156S [C. Lin et al., J. Biol. Chem., 279 pp. 17508-17514 (2004)]. The conformations of the side chain were minimized in energy maintaining the conformation of the rest of the fixed protein. The inhibitors, of VX-950 and BILN 2061, were labeled in those active sites of the mutant enzyme to elucidate the effect of the mutation on the binding of the inhibitor. Of the three possible conformations of the side chain of Ser in position 156 (Figure 11), the conformation with? _ = 60 ° has the least number of unfavorable contacts with VX-950 and BILN 2061. The other two conformers (with = 180 ° and -60 °) has multiple unfavorable contacts with both inhibitors of the side chain of P2 or the carbonyl group of P3. In the mutation A156T or A156V, the additional group in the Cß atom of the side chain is forced to occupy one of these two positions with x = 180 ° or -60 °, which produces unfavorable interactions with the inhibitors. The three possible conformations of Thr are shown schematically in Figure 11. In all cases, the additional group has recursive interassion with the inhibitor and / or atoms of the structure of the enzyme. Minimizing the energy, -60 / 180 ° has at least one repulsive interaction and the main cause of repulsion is the close shock between the methyl or terminal hydroxyl group. Therefore, mutations A156T and A156V are resistant to both inhibitors. All compositions and / or methods described and claimed herein may be produced and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and in the steps or sequence of the method described herein. without departing from the concept, spirit and scope of the present invention. Specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein although the same or similar results would be achieved. All substitutes and similar modifications evident to those skilled in the art are considered within the spirit, scope and concept of the invention as defined in the appended claims. The references cited herein in their entirety, to the extent that they may provide exemplary expedits or other supplementary details herein, are hereby specifically incorporated by reference. The following list of references cited herein in their entirety is specifically incorporated herein by reference. Alter, H. J., and Seeff, L. B. (2000) Semin. Liver Dis. 20, 17-35. Alter, M.A. (1997) Hepatology 26:62. (disease) Babine, R. E., et al. (2002) in WO 0218369, Eli Lilly and Company Barbato et al., (1999) J. Mol. Biol. 289: 371-384 (NMR structure of the protease NS3-4A) Bartenschlager, R., et al. 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Engl. 42, 1356-1360 Wasley, A. & M.J. Alter (2000) Semin. Liver Dis. 20, 1-16 (epidemiology of HepC) Yan et al., (1998) Protein Sci. 7: 837-847 (structure of the protease of NS3-4A) Yao, N., et al. (1999) Structure Fold Des. 7, 1353-1363. It should be noted that with relasion to this fesha, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (79)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. An isolated HCV polynucleotide encoding an NS3 / 4A protease of HCV or a biologically active analogue thereof, characterized in that the codon corresponding to codon 156 of the native polynucleotide and / or the codon corresponding to codon 168 of the The natural polynucleotide is mutated so that it does not sodifish for an alanine 156 and / or aspartic acid at 168.
2. 2. The isolated HCV polynucleotide according to claim 1, characterized in that the codon of the polynucleotide corresponding to codon 156 of the wild-type polynucleotide codifies for serine.
3. 3. The HCV polynucleotide isolated from the sonformity is claim 1, characterized in that the codon of the polynucleotide corresponding to codon 156 of the wild-type polynucleotide codes for valine.
4. 4. The isolated HCV polynucleotide according to claim 1, characterized in that the codon of the polynucleotide corresponding to codon 156 of the native polynucleotide codes for a threonine.
5. 5. The HCV polynucleotide isolated from sonification is claim 1, characterized in that the codon of the polynucleotide corresponding to codon 156 of the wild-type polynucleotide codes for a valine or a threonine and the codon of the polynucleotide corresponding to codon 168 of the wild-type polynucleotide codes for an aspartic acid or glutamic acid.
6. 6. The isolated HCV polynucleotide according to claim 1, characterized in that the codon of the polynucleotide corresponding to codon 168 of the wild-type polynucleotide codes for valine.
7. 7. The isolated HCV polynucleotide according to claim 1, characterized in that the codon 168 of the polynucleotide encodes an alanine, a glycine or a tyrosine.
8. 8. The HCV polynucleotide isolated in accordance with claim 1, characterized in that the natural HCV polynucleotide has a sequence of SEQ ID NO: 1.
9. 9. The polynucleotide that sodifies for a biologically active fragment of an NS3 / 4A protease. of HCV, the polynucleotide fragment is characterized in that it encodes an NS3 / 4A protease domain of HCV containing a sodium which corresponds to codon 156 of the natural polynucleotide and / or the codon corresponding to codon 168 of the natural polynucleotide, where the codon corresponding to codon 156 and / or the codon corresponding to codon 168 is mutated so that it does not code for an alanine 156 and / or aspartic acid 168.
10. 10. The polynucleotide according to claim 9, characterized in that it comprises the codon 156 of the NS3 / 4A protease domain of HCV, where codon 156 codes for a residue of valine, threonine or serine in place of an alanine residue.
11. The polynucleotide according to claim 9, characterized in that it comprises codons corresponding to Sodons 156 and 168 of the HCV NS3 / 4A protease domain where Sodium 156 codes for a valine or threonine instead of an alanine and the codon 168 codes for an aspartic acid or glutamic acid.
12. 12. The polynucleotide according to claim 9, characterized in that it comprises a codon corresponding to codon 168 of the NS3 / 4A protease domain of HCV where codon 168 codes for a valine.
13. 13. The polynucleotide according to claim 9, characterized in that it comprises a codon corresponding to codon 168 of the NS3 / 4A protease domain of HCV where codon 168 codes for an alanine, glycine or tyrosine.
14. 14. The isolated HCV polynucleotide according to claim 9, sarasterized in that the natural HCV polynucleotide has a sequence of SEQ ID NO: 1.
15. 15. An isolated HCV NS3 / 4A protease protein or biologically active fragment or the like biologically active thereof, characterized in that it comprises a sequence in which the residual amino acid corresponding to amino acid 156 of the natural HCV NS3 / 4A protease is not an alanine residue and / or the residual amino acid corresponding to amino acid 168 of HCV NS3 / 4A protease is not a residue of aspartic acid.
16. 16. The isolated HCV NS3 / 4A protease protein or fragment or analog according to claim 15, characterized in that the amino acid corresponding to amino acid 156 of the natural protease is valine, threonine or serine.
17. 17. The isolated HCV NS3 / 4A protease protein or fragment or analog according to claim 15, characterized in that the amino acid corresponding to amino acid 156 of the protease is serine, valine or threonine and amino acid 168 is aspartic acid or acid glutamic
18. 18. The isolated HCV NS3 / 4A protease protein or conformational fragment or analogue is claim 15, characterized in that the amino acid corresponding to amino acid 168 of the natural protease is valine.
19. 19. The isolated HC3 NS3 / 4A protease protein or fragment or analog according to claim 15, characterized in that the amino acid corresponding to amino acid 168 of the natural protease is alanine, glycine or tyrosine.
20. The isolated HCV NS3 / 4A protease protein according to claim 15, characterized in that the HCV NS3 / 4A protease has a sequence of SEQ ID NO: 2.
21. 21. A vector, characterized in that it comprises the polynucleotide in accordance with any of claims 1-14.
22. 22. A host cell or cell line, characterized in that it comprises the polynucleotide according to any of claims 1-14.
23. 23. A host cell characterized in that it is transformed or transfected with a vector according to claim 21.
24. 24. A cell or host cell line, characterized in that it comprises the protein according to any of claims 15-21.
25. 25. An isolated HCV variant, characterized in that it comprises a polynucleotide according to any of claims 1-14.
26. 26. An isolated HCV variant, characterized in that it comprises a protein according to any of claims 15-23.
27. 27. A composition, characterized in that it comprises the polynucleotide according to any of claims 1-14.
28. 28. A composition, characterized in that it comprises the polynucleotide according to any of claims 1-4 or 7-10 and the polynucleotide according to any of claims 5, 6, 11 or 12.
29. 29. A composition, characterized in that it comprises a protein according to any of claims 15-21.
30. 30. A composition, characterized in that it comprises a protein according to any of claims 15-18 and a compliance protein is claim 19 or claim 23.
31. 31. A method for detecting the presence of resistant HCV in a biological sample, facesterized because it comprises detecting the presence of a polynucleotide according to any of claims 1-4 in the biological sample.
32. 32. The method according to claim 31, characterized in that it comprises: a) obtaining the polynucleotide of the sample; b) determining the sequence of the polynucleotide; c) determining whether the polynucleotide, codon 156 and / or codon 168 are codons that are not natural codons and because the codons do not code for aspartic acid.
33. The method according to claim 31, characterized in that the non-natural codons code for serine, valine or threonine in the residue corresponding to residue 156 of the natural HCV NS3 / 4A protease or code for a glutamic acid, valine, alanine, glycine or tyrosine in a residue corresponding to residue 168 of the natural HCV NS3 / 4A protease.
34. 34. The method of compliance with the claim 33, characterized in that it comprises determining or inferring whether, in the polynucleotide, a) sodon 156 sodifies for a serine, codon 156 codes for a valine, codon 156 codes for a threonine, codon 156 codes for a valine or threonine and codon 156 codes for an aspartic acid or glutamic acid; and b) codon 168 codes for a valine or codon 168 codes for an alanine, a glycine or a tyrosine.
35. 35. A method to determine if infection by HCV in a patient is resistant, characterized in that it comprises: a) collecting a biological sample from the patient infected with HCV; and b) evaluating whether the plasma sample contains the nucleic acid encoding a mutant HCV NS3 / 4A protease, where the presence of the mutant HCV NS3 / 4A protease is indicative that the patient has resistant HCV infection.
36. 36. The method of compliance with the claim 35, characterized in that the nucleic acid encoding a mutant HCV NS3 / 4A protease having a mutation in an amino acid corresponding to residue 156 and / or residue 168 of HCV NS3 / 4A, where the presence of a amino acid that is not alanine in the residue corresponding to residue 156 of natural HCV NS3 / 4A, and / or an amino acid that is not aspartic acid in the residue corresponding to residue 168 of natural HCV NS3 / 4A, is indicative that the patient has a resistant HCV infection.
37. 37. The method according to the claim 35, characterized in that the mutation comprises the presence of a serine, valine or threonine in the residual amino acid corresponding to residue 156 of the natural HCV NS3 / 4A protease.
38. 38. The method according to any of claims 35, 36 or 37, characterized in that the NS3 / 4A protease of HCV comprises the presence of an aspartic acid such as valine, alanine, glycine or tyrosine in the residual amino acid corresponding to the residue 168 of the natural HCV NS3 / 4A protease.
39. 39. The method according to any of claims 35, 36 or 37, characterized in that the nucleic acid encoding the HCV NS3 / 4A protease present in the biological sample comprises a mutation at codon 156; wherein the mutation results in a substitution of alanine with serine, valine or threonine and / or the mutation comprises a substitution of aspartic acid at residue 168 with glutamic acid.
40. 40. The method according to any of claims 35-39 characterized in that the nucleic acid encoding the HCV NS3 / 4A protease present in the biological sample comprises a mutation at codon 168; wherein the mutation results in a substitution of aspartic acid with alanine, glycine, valine or tyrosine.
41. 41. A method for evaluating whether the HCV-infected patient has a decreased sensitivity or susceptibility to VX-950, characterized in that it comprises evaluating whether the patient has a NS3 / 4A protease DNA of the Hepatitis C virus that has a mutation in the patient. the codon coding for residue 156 of the HCV NS3 / 4A protease of the native Hepatitis C virus.
42. 42. A method for evaluating whether the HCV-infected patient has a reduced sensitivity or susceptibility to a protease inhibitor, characterized in that it comprises evaluating whether the patient has a NS3 / 4A protease DNA of the Hepatitis C virus that has a mutation. in the codon coding for residue 156 of the HCV NS3 / 4A protease of the native Hepatitis C virus.
43. 43. The method according to any of claims 41-42, characterized in that the mutation correlates with the results of decreased sensitivity or susceptibility to BILN 2061. 4.
44. The method according to any of claims 41-42, characterized in that the mutation correlates with the results of decreased sensitivity or susceptibility to VX-950 and BILN 2061.
45. 45. A method for evaluating a candidate or potential HCV inhibitor. , characterized in that it comprises: a) introducing a vector comprising a polynucleotide according to any of claims 1-14 and a reporter gene encoding an indicator in a host cell; b) culturing the host cell; and c) measuring the indicator in the presence of an inhibitor and in the absence of an inhibitor.
46. 46. A method for assaying compounds for activity against HCV, characterized in that it comprises: a) providing a protease according to any of claims 15-23 and a protease substrate; b) contacting the protease with a candidate or potential inhibitor in the presence of the substrate; c) evaluate or measure the inhibition of the proteolytic activity of the protease.
47. 47. A method for identifying a compound as a protease inhibitor according to any of claims 15-23, characterized in that it comprises: a) assaying the activity of the protease in the absence of the compound; b) assaying the activity of the protease in the presence of the compound; s) compare the results of a) and the results of b).
48. 48. The method of conformity is claim 47, characterized in that it comprises: d) assaying the activity of a natural protease in the absence of the compound; e) assaying the activity of the natural protease in the presence of the compound; f) compare the results of d) and the results of e).
49. 49. The method of conformity is claim 48, characterized in that it comprises the results of a) and / or b) and the results of d) and / or e).
50. 50. The method according to claim 47, characterized in that it comprises d) assaying, in the absence of the compound, the activity of a second NS3 / 4A protease, wherein the second protease comprises an amino acid corresponding to residue 168 of the natural protease , where the amino acid is mutated to valine, alanine, glycine or tyrosine; e) assaying the activity of the second protease in the presence of the compound; and f) compare the results of d) and e).
51. 51. The method of compliance with the claim Characterized in that it comprises: g) assaying the activity of a natural protease in the absence of the compound; h) assaying the activity of the natural protease in the presence of the compound; i) compare the results of g) and the results of h).
52. 52. The method according to claim 51, characterized in that it comprises comparing the results of a) and / or b) and the results of d) and / or e); and / or the results of g) and / or h).
53. 53. A method for identifying a compound capable of rescuing the activity of VX-950, where the NS3 / 4A protease has become resistant to VX-950, characterized in that it comprises: a) contacting a protease in accordance with any of the claims 15-23 with the compound; b) assay the ability of VX-950 to inhibit the activity of the protease of a).
54. 54. A method for identifying an effective compound against a protease according to any of claims 15-23, characterized in that it comprises: a) obtaining a three-dimensional model of the protease; b) design or select a compound; c) evaluate the ability of the compound to bind to interact with the protease.
55. 55. The method of compliance with the claim 54, characterized in that the three-dimensional model is based on the X-ray crystal structure (Figure 1 and Figure 2) of the NS3 / 4A protease.
56. 56. The method of compliance with the claim 55, characterized in that the model is obtained by methods implemented in computer.
57. 57. The method of compliance with the claim 54, characterized in that the three-dimensional model is obtained by X-ray crystallography of the protein according to any of claims 15-21.
58. 58. The method according to any of claims 54-57 characterized in that the evaluation is by molecular modeling.
59. 59. The method according to any of claims 54-58, characterized in that the compound is contacted with a natural protease.
60. 60. The method according to any of claims 54-59 characterized in that the compound is contacted with a protein according to any of claims 15-19.
61. 61. The method according to any of claims 54-60 characterized in that the compound is contacted with a protein according to any of claims 20-21.
62. 62. The method according to any of the claims 54-61 characterized in that the compound is an identified compound of a combined chemical library.
63. 63. The method according to any of claims 54-62 characterized in that the compound is a compound prepared through the rational design of drugs or drugs.
64. 64. The method according to any of claims 54-62 characterized in that the compound is a compound prepared through the rational design of drugs or drugs and derived from the structure of VX-950.
65. 65. A compound characterized in that it is identified according to any of claims 56-61.
66. 66. A composition characterized in that it comprises the compound according to claim 65, and an adjuvant support or pharmaceutically acceptable carrier.
67. 67. The compliance composition is claim 66, characterized in that the compound is in an amount effective to inhibit the serine protease of NS3 / 4A.
68. 68. The composition according to claim 67, sarasterized because the composition is formulated to be administered to a patient.
69. 69. The composition according to claim 68, characterized in that the composition comprises an additional agent selected from an immunomodulatory agent.; an antiviral agent; a second HCV protease inhibitor; an inhibitor of another target in the life cycle of HCV; a cytosome inhibitor P-450; or combinations thereof.
70. 70. The compliance composition is claim 69, characterized in that the immunomodulatory agent is a, ß- or? interferon or thymosin, the antiviral agent is ribavirin, amantadite, or thymosin; or the inhibitor of another target in the life cycle of HCV is a helicase, polymerase, or metalloprotease inhibitor of HCV.
71. 71. The composition according to claim 70, characterized in that the inhibitor of citrochrome P-450 is ritonavir.
72. 72. A method for inhibiting the activity of an NS3 / 4A protease of Hepatitis C, characterized in that it comprises the step of contacting the serine protease with a compound according to claim 65.
73. 73. A method for treating an infection for HCV in a patient, characterized in that it comprises the step of administering to the patient a somatic package with claim 65.
74. 74. A method for treating or reducing an HCV infection in a patient, characterized in that it comprises: a) determining whether the patient has an HCV infection that is resistant to therapies using a method according to any of claims 35-44; b) treat the patient with a therapy composition directed to the treatment of resistant HCV.
75. 75. A method according to claim 73 or 74, characterized in that it comprises the additional step of administering to the patient an additional agent selected from an immunomodulatory agent; an antiviral agent; a second HCV protease inhibitor; an inhibitor of another target in the HCV life cycle, or combinations thereof, where the additional agent is administered to the patient as part of an individual or separate dosage form.
76. 76. The method of conformity is claim 75, characterized in that the immunomodulatory agent is, ß, o? interferon; or thymosin; the antiviral agent is ribavirin or amantadite, or the inhibitor of another target in the life cycle of HCV is an inhibitor of helicase, polymerase, or metalloprotease of HCV.
77. 77. A method for eliminating or reducing HCV contamination of a biological sample or medical or laboratory equipment, characterized in that it comprises the step of contacting the biological sample or medical laboratory equipment with a compound according to claim 65.
78. 78. The method of conformity is claim 77, characterized in that the biological sample or the medical or laboratory equipment is contaminated with a strain of HCV resistant to the method according to claims 31-34.
79. 79. The method according to claim 77, characterized in that the sample or equipment is selected from blood, other body fluids, biological tissue, a surgical instrument, a surgical garment, a laboratory instrument, a laboratory garment, an apparatus for collection of blood or other body fluid; a material to store blood or other body fluid.