RU2365624C2 - Resistant mutants of protease ns3-ns4a hcv - Google Patents

Abstract

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology and concerns protein of protease NS3/4A HCV or its biologically active fragment, which contains succession, in which residue of amino acid, corresponding to amino acid 156 of protease NS3/4A HCV of wild type, is not the residue of alanine and, not obligatorily, residue of amino acid, which corresponds to aminocacid 168 of protease NS3/4A HCV of wild type, is not asparaginic acid. Invention also relates to polynucleotide, coding said protein, as well as application of said protein in detection of HCV presence in biological sample, as well as in method of determination if the infection, mediated by HCV, in patient is resistant to medication against HCV, in estimation method, if the infected by HCV patient has lower sensitivity or susceptibility to VX-950, as well as in method of candidate examination for inhibitor or potential inhibitor of HCV.

EFFECT: expansion of arsenal of medications for HCV treatment.

68 cl, 17 dwg, 6 tbl, 13 ex

Description

BACKGROUND

FIELD OF THE INVENTION

This invention relates to resistant hepatitis C virus NS3 / 4A protease mutants.

Related Art

Hepatitis C virus infection (“HCV”) is an insurmountable human medicine problem. HCV is considered an etiological factor in most cases of hepatitis, neither A nor B, with a serologically established prevalence among people of 3% worldwide [A. Alberti et al., “Natural History of Hepatitis C,” J. Hepatology, 31., (Suppl.), Pp. 17-24 (1999)]. Nearly four million individuals can only be infected in the United States [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.), Pp. 88-91 (1999)].

On first contact with HCV, only about 20% of infected individuals develop acute clinical hepatitis, while it turned out that other individuals do not appear to have significant visible symptoms of an infectious disease. However, in almost 70% of cases, the virus causes 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 Surveillance and Control of Hepatitis C,“ J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usually leads to recurrent and progressively worsening inflammation of the liver, which often leads to more serious pathological conditions, such as cirrhosis and hepatic cell 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. Natl. Acad. Sci. USA, 87, pp. 6547-6549 (1990)]. Unfortunately, there are no sufficiently effective treatments for reducing the progression of chronic HCV.

The HCV genome encodes a polyprotein of 3010-3033 amino acids [Q.L. Choo, et. al., "Genetic Organization and Diversity of the Hepatitis C Virus." Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); N. Kato et al., "Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis," Proc. Natl. Acad. 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)]. Non-structural (NS) HCV proteins are believed to provide the necessary catalytic mechanism for virus replication. NS proteins are formed as a result of proteolytic cleavage of a polyprotein [R. Bartenschlager et. al., "Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3 / 4 and NS4 / 5 Junctions," J. Virol, 67, pp. 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, pp. 1385-1395 (1993); L. Tomei et. al., "NS3 is a serine protease required for processing of hepatitis C virus polyprotein", J. Virol., 67, pp. 4017-4026 (1993)].

NSV protein 3 (NS3) HCV exhibits serine protease activity, which processes the viral polyprotein to form the majority of viral enzymes, and is necessary for the replication and infectivity of the virus. The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) were shown to comprise the NS3 serine protease domain, which processes all four subsequent sections of the HCV polyprotein [C. Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”, J. Virol., 68, pp. 8147-8157 (1994)]. Substitutions of the catalytic triad of serine protease NS3 HCV lead to impaired replication and infectivity of the virus 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]. Mutations in yellow fever virus NS3 protease are known to reduce the infectivity of the virus [Chambers, T.J. et. al., "Evidence that the N-terminal Domain of Nonstractural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein", Proc. Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)].

The serine protease NS3 HCV and its associated cofactor, NS4A, process the region of the viral non-structural protein into individual non-structural proteins, including all viral enzymes [C. Failla, et al., "An ammo-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A", J. Virol. 69, pp. 1769-1777; Y. Tanji et al., "Hepatitits C virus-encoded 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 vitro", J. Virol. 69, pp. 4373-4380], and are necessary for viral replication. It turned out that the mentioned processing is similar to the processing carried out by the aspartyl protease of the human immunodeficiency virus, which is also involved in the processing of viral proteins. HIV protease inhibitors (HIV), which inhibit the processing of viral proteins, are potent antiviral agents for humans, indicating that interruption of this stage of the viral life cycle is the basis for the development of therapeutically effective agents. Therefore, it is an attractive target for drug development.

Some potential HCV protease inhibitors have been described in the prior art in this area [PCT Publication No. 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, United States Patent 5,990,276, M. Llinas-Brunet 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)]. However, it is not known whether the named compounds have the appropriate profiles to be acceptable drugs. In addition, it is possible that HCV protease may become resistant to another suitable drug.

Therefore, current understanding of HCV does not lead to any satisfactory anti-HCV agents or methods of treating HCV infection. The only recognized treatment for HCV disease is alpha interferon-based therapy. However, alpha interferons exhibit 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 Alfa-Interferon Therapy for Chronic Viral Hepatitis," J. Hepatol., 21, pp. 241-243 (1994); P.F. Renault et al., "Side Effects of Alpha Interferon," Seminars in Liver Disease, 9, pp. 273-277. (1989)] and induce long-term remission only in some cases (~ 25%) [O. Weiland, “Interferon Therapy in Chronic Hepatitis C Virus Infection”, FEMS Microbiol. Rev. 14, pp. 279-288 (1994)]. The existing standard of treatment, pegylated with alpha-interferon in combination with ribavirin, leads to a long-term viral response (SVR) in approximately 40-50% of cases in patients infected with genotype 1, which is important for 70% of patients with chronic hepatitis C in developing countries, and to SVR in 80% in patients infected with 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 unclear.

Thus, there is a need for more effective anti-HCV treatments, in particular a need for compounds that inhibit HCV NS3 protease. Such compounds can be used as antiviral agents, in particular as anti-HCV agents. Understanding of HCV-resistant mutants should be further improved towards the development of effective treatments for HCV.

SUMMARY OF THE INVENTION

This invention relates to resistant mutant hepatitis C virus NS3 / 4A protease.

Thus, in some aspects, the invention includes isolated HCV polynucleotides that encode mutant HCV NS3 / 4A proteases or biologically active analogs and fragments thereof, in which a codon that corresponds to codon 156 of a wild-type polynucleotide and / or a codon that corresponds to a codon 168 wild-type polynucleotide mutated so that it does not encode alanine at position 156 and / or aspartic acid at position 168. Examples of such mutations described herein include polynucleotides in which a codon that matches codon 156 of the wild-type polynucleotide encodes serine, valine or threonine. Other exemplary embodiments encompass polynucleotides in which a codon of a polynucleotide that corresponds to codon 168 of a wild-type polynucleotide encodes aspartic acid, glutamic acid, alanine, glycine or tyrosine. Any combination of mutations in codon 156 and 168 is considered separately. The wild-type HCV NS3 / 4A protease is well known to those skilled in the art. It is encoded by the polynucleotide sequence of SEQ ID NO: 1. Such a polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 2.

Polypeptides or biologically active fragments thereof that are encoded by the polynucleotides contemplated herein are also contemplated herein. In addition, the invention encompasses vectors that contain host cells transformed or transfected with such polynucleotides, and cell lines that contain said polynucleotides. Methods and compositions for creating such vectors, transforming host cells and producing cell lines are standard and generally accepted methods known to those skilled in the art. Isolated HCV variants that contain the described mutant polynucleotides or proteins are also part of this invention.

Compositions containing polynucleotides or proteins, either alone or in combination with other compositions and components, are also contemplated.

The invention describes a method for detecting the presence of drug-resistant HCV in a biological sample, including determining the presence of the described polynucleotide. Typically, such methods include obtaining or isolating a polynucleotide from a sample, determining the polynucleotide sequence, and evaluating whether a mutation associated with resistance is present in the polynucleotide, such as one or more of the mutations discussed in the description (for example, a mutation that encodes serine, valine, or threonine in the residue that corresponds to residue 156, and / or encodes glutamic acid, valine, alanine, glycine or tyrosine in the residue that corresponds to residue 168 of the wild type HCV NS3 / 4A protease).

Methods for detecting or detecting whether a patient's HCV infection is drug resistant are also contemplated, including taking a biological sample from an infected HCV patient and determining whether the plasma sample contains nucleic acid encoding a mutant HCV NS3 / 4A protease, wherein HCV NS3 / 4A mutant protease indicates that the patient has drug-resistant HCV infection.

In other aspects, methods for evaluating whether an HCV-infected patient has a reduced sensitivity or susceptibility to VX-950, including evaluating whether the patient has hepatitis C virus NS3 / 4A DNA having a mutation in the codon that encodes a 153 NS3 protease residue, are contemplated. / 4A wild type hepatitis C virus.

The following aspects are directed to methods for evaluating whether an HCV infected patient has a reduced sensitivity or susceptibility to a protease inhibitor, including evaluating whether the patient has hepatitis C virus protease NS3 / 4A DNA having a mutation in the codon that encodes a 156 NS3 / protease residue Wild-type hepatitis C virus 4A.

The invention also contemplates methods for evaluating an inhibitor candidate or potential HCV inhibitor, comprising administering a vector containing a polynucleotide of the invention and an indicator gene encoding an indicator into the host cell, culturing the host cell and determining the indicator in the presence and absence of the inhibitor.

Other methods for studying the activity of compounds against HCV include providing a mutant protease, as discussed herein, and a protease substrate; contacting a candidate or potential inhibitor in the presence of a substrate; and evaluating or quantifying the inhibition of the proteolytic activity of the protease.

In other aspects, methods of identifying a compound as a drug-resistant protease inhibitor of the disclosure are provided by testing the activity of such a protease in the absence of a compound; testing protease activity in the presence of a compound; comparing the results of studies conducted in the presence and absence of the compound, and any decrease in protease activity as a result of the presence of the compound indicates that the compound is a protease inhibitor.

Methods for identifying compounds that are capable of restoring the activity of VX-950 are also described, wherein the NS3 / 4A protease becomes resistant to VX-950, methods in which a resistant protease is in contact with a compound, and the ability of VX-950 to inhibit protease activity are evaluated in the presence of such a compound .

The fact that the three-dimensional structure of the NS3 / 4A protease is established is used in this invention (see, for example, WO 98/11134). Using such methods and recommendations of the present invention, a three-dimensional model of the resistant protease of the invention is obtained; the compounds are designed or selected to interact with the three-dimensional structure of the mutant protease, and the ability of the compound to bind to or interact with the protease (for example, molecular modeling) is evaluated. Typical three-dimensional models are based on the X-ray crystal structure of the NS3 / 4A protease (Figure 1 and Figure 2). Such models can be obtained by methods performed on a computer, methods, or X-ray crystallography. The results obtained can be compared with the data established for the wild-type protease.

The compound can be identified from a combinatorial chemical library or obtained by rational drug design. In exemplary embodiments, the compound is a compound obtained by rational drug design and established based on the structure of VX-950. In other representative embodiments, the identified compound is prepared in the form of a composition comprising the compound and a pharmaceutically acceptable carrier, adjuvant or excipient. Preferably, the composition contains the compound in an amount effective to inhibit the NS3 / 4A serine protease. More preferably, the composition is formulated for administration to a patient. The compositions may also contain an additional agent selected from an immunomodulating agent, an antiviral agent, a second HCV protease inhibitor, another target inhibitor in the HCV life cycle, a cytochrome P-450 inhibitor, or combinations thereof.

Other methods of the invention involving the inhibition of hepatitis C virus NS3 / 4A protease activity include the step of contacting a serine protease with such a compound or composition. In further aspects, methods are provided for treating an HCV infection in a patient, comprising the step of administering to the patient such a compound of the composition.

In further aspects, methods are provided for treating or attenuating an HCV infection in a patient, including determining whether the patient has an HCV infection that is resistant to therapy using the method discussed in the description that enables detection of the described mutations and treating the patient with the composition or therapy aimed at treating drug-resistant HCV infection.

In other further aspects, methods for eliminating or reducing HCV infection of a biological sample or medical or laboratory equipment are discussed, including the step of contacting the biological sample or medical or laboratory equipment with a compound identified as described in the application. In further embodiments, a biological sample or medical or laboratory equipment is infected with a drug-resistant HCV strain, as determined by the determination methods described.

Other features and advantages of the invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples indicating preferred embodiments of the invention are presented for illustration only, as various changes and modifications within the scope of the idea and scope of the invention will become apparent to those skilled in the art from the proposed detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of this description and are included to further illustrate aspects of the present invention. The invention can be better understood by reference to the drawings in conjunction with the detailed description of specific embodiments presented in the description.

Figure 1: X-ray structures of two complexes of HCV NS3 protease — the protease inhibitor (PI) BILN 2061 and VX-950. Two co-complex structures are installed and plotted (VX-950 in blue and BILN 2061 in red). The three ball-and-stick residues (R123, R155 and D168) form salt bridge bonds in the BILN 2061 structure, and not in the VX-950 structure. Removing the negative charge in D168 leads to the loss of the R155 restriction and the subsequent loss of packaging of large P2 BILN 2061 and an increase in the cost of desolvation. R155 is not limited by D168 in the structure of VX-950, and the mutation of D168V does not affect its binding.

Figure 2: A156S mutation causes a loss of binding due to a spatial collision with the VX-950 and not with BILN 2061. The X-ray structures of VX-950 (upper left, blue) or BILN 2061 (lower left, red) with wild-type A156 are brightly lit in yellow . Mutation models A156S (gray color) with VX-950 (upper right, blue) or with BILN 2061 (lower right, red). All permissible torsion angles for the serine side chain in the mutated residue were examined.

Figure 3: A156V mutation causes loss of PIs binding due to spatial collision with either the VX-950 or BILN 2061. A156V mutation models (gray) with VX-950 (left, blue) or BILN 2061 (right, red).

Figure 4: Chemical structures of VX-950 (A) and BILN 2061 (B).

Figure 5: Obtaining replicon-containing HCV cells that are resistant to VX-950. Subgenomic, replicon-containing HCV Con1 cells were serially passaged in the presence of G418 and an increasing concentration of VX-905 (A), as described in the Materials and Methods section. Replicon-containing cells were expanded and fresh PI was added to the medium twice a week. Shaded areas indicate a period of time during which the replicon-containing cells were characterized by slight growth or lack of overall growth, which was accompanied by concomitant massive cell death. The total cellular RNA of replicon-containing cells at various time periods (indicated by extended arrows) was extracted during resistance selection, and the RT-PCR product (PCR-RT), encompassing HCV NS3 serine protease, was sequenced either immediately or after subcloning into the TA vector. IC ₅₀ values for VX-950 relative to Series A or wild-type replicon cells were determined on day 56 in a standard 48-hour study (B).

Figure 6: Preparation of replicon-containing HCV cells that are resistant to BILN 2061. Subgenomic, replicon-containing HCV Con1 cells were serially passaged in the presence of G418 and an increasing concentration of BILN 2061 (A), as described in the Materials and Methods section. Replicon-containing cells were expanded and fresh PI was added to the medium twice a week. Shaded areas indicate a period of time during which the replicon-containing cells were characterized by slight growth or lack of overall growth, which was accompanied by concomitant massive cell death. The total cellular RNA of replicon-containing cells at various time points (indicated by extended arrows) was extracted during resistance selection, and the RT-PCR product (PCR-RT), encompassing HCV NS3 serine protease, was sequenced either immediately or after subcloning into the TA vector. IC ₅₀ values for BILN 2061 relative to B series or wild-type replicon cells were determined on day 59 in a standard 48-hour study (B).

Figure 7: Models of protease complexes: inhibitor. Protein is presented in the form of a picture made on the basis of the secondary structure in light gray color. Inhibitors are shown as a ball-and-stick (VX-950 in purple and BILN 2061 in yellow) with nitrogen colored in blue, oxygen in red, and gray in orange. The side chains of key residues are depicted in the form of rods with different colors: Ala156 (green), Asp168 (orange) and Arg123 (orange). Arg155 BILN 2061 side chain model: the protease is shown in blue, and the Arg155 VX-950 side chain model: protease is colored in orange. The named side chains are depicted as surfaces of dots. The catalytic triad, Ser139, His57 and Asp81 are grayed out. (The figure was created using PyMOL Molecular Graphics Systems, DeLano Scientific LLC, San Carlos, California, U.S.A. Copyright © 1998-2003).

Figure 8: Obtaining double-resistant replicons from VX-950-resistant containing replicon cells. (A) VX-950-resistant, replicon-containing cells were serially passaged in the presence of 0.25 mg / ml G418, 14 μM VX-950 and increasing concentrations of BILN 2061. Replicon-containing cells were propagated and fresh VX-950 and BILN 2061 were added to the medium twice a week, as shown by filled rectangles and triangles, respectively. The total cellular RNA of the replicon-containing cells was extracted on day 32 during resistance selection, and the PCR-RT product encompassing the HCV NS3 serine protease was sequenced either immediately or after subcloning into the TA vector. (B) VX-950 titration versus Series A (VX-950-resistant) (filled rectangles) or Series C (double resistance) (open rectangles) containing religpcon cells for 52 days by VX-950 is presented. HCV RNA levels were determined after 48 hours of incubation with VX-950. (C) Presents titration of BILN 2061 against Series A (VX-950-resistant) (filled triangles) or Series C (double resistance) (open triangles) of replicon cells on day 52 using BILN 2061. The HCV RNA level was determined after 48 hours incubations with BILN 2061.

Figure 9: Obtaining double-resistant replicons from BILN 2061-resistant, containing replicon cells. (A) BILN 2061-resistant, replicon-containing cells were serially passaged in the presence of 0.25 mg / ml G418, 7 or 14 μM VX-950 and increasing concentrations of BILN 2061. The replicon-containing cells were expanded and fresh VX-950 and BILN 2061 were added to Wednesday twice a week, as shown by filled rectangles and triangles, respectively. The total cellular RNA of the replicon-containing cells was extracted on day 32 during resistance selection, and the PCR-RT product encompassing the HCV NS3 serine protease was sequenced either immediately or after subcloning into the TA vector. (B) Presents titration of a VX-950 against a B series (BILN 2061-resistant) (filled rectangles) or a D series (double resistance) (open rectangles) of replicon-containing cells on day 52 using VX-950. HCV RNA levels were determined after 48 hours of incubation with VX-950. (C) Titration of BILN 2061 versus Series B (BILN 2061-resistant) (filled triangles) or Series D (double resistance) (open triangles) of replicon-containing cells on day 52 using BILN 2061 is shown. HCV RNA level was determined after 48-hour incubation with BILN 2061.

Figure 10: Obtaining double-resistant replicons from the "naive" containing replicon cells. Subgenomic HCV replicon-containing cells were serially passaged in the presence of 0.25 mg / ml G418 and increasing concentrations of VX-950 and BILN 2061. Replicon-containing cells were propagated and fresh VX-950 and BILN 2061 were added to the medium twice a week, as shown by filled rectangles and rhombuses respectively. The isolated region indicates the period of time during which the replicon-containing cells are characterized by little or no growth, which is accompanied by concomitant massive cell death. The total cellular RNA of replicon-containing cells at various time periods indicated by open arrows was extracted during resistance selection, and the PCR-RT product encompassing HCV NS3 serine protease was sequenced either immediately or after subcloning into a TA vector.

Figure 11: Scheme of conformation of the side chain of Thr156 depending on the binding of the inhibitor. The thick lines represent the side chain of the Thr156 mutant enzyme and the side chain of the P2 inhibitor or substrate. The same three conformations for the Val156 side chain were also considered. The last (-60 / 180 °) conformations had the lowest energy for any mutation, but both inhibitors were poorly bound.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention has found that HCV strains undergo certain mutations in the presence of certain therapeutic compounds that render HCV strains resistant to the therapeutic potential of such compounds. In particular embodiments, hepatitis C virus NS3 / 4A protease has been mutated into such resistant HCV mutants so that the mutants are resistant to protease inhibitor compounds. The data mentioned can be used to create therapies for the treatment of HCV infections.

In special embodiments, the wild-type amino acid residue 156 NS3 / 4A of HCV (the sequence of which is represented as SEQ ID NO: 2) is found to be sensitive to mutation. Mutation of the aforementioned residue leads to resistance of HCV to therapeutic effects of protease inhibitors. In one embodiment, it is shown that the wild-type codon 156, which encodes alanine in the wild-type NS3 / 4A HCV, is mutated into a codon that encodes the serine at the same corresponding position in the HCV NS3 / 4A polypeptide. In another embodiment, the codon is mutated into a codon that encodes a valine at the same corresponding position in the HCV NS3 / 4A polypeptide. In yet another embodiment, threonine is encoded at the same corresponding position in the NS3 / 4A HCV.

Given the above data, the invention provides HCV DNA encoding the HCV NS3 / 4A protease (or fragment or analog thereof) in which DNA codon 156 encodes serine. In another embodiment of the present invention, HCV DNA encoding an HCV NS3 / 4A protease (or fragment or analog thereof) is provided in which DNA codon 156 encodes a valine. In yet another embodiment, HCV DNA encoding HCV NS3 / 4A protease (or a fragment thereof or an analogue thereof) in which DNA codon 156 encodes threonine is provided.

In further embodiments, it is found that in some embodiments, codon 156 is a code that encodes a valine, serine or threonine at residue 156, which is usually an alanine residue in the native / wild type NS3 / 4A HCV, and there is another mutation in which the codon is at a residue 168 native / wild-type NS3 / 4A HCV, which is usually an aspartic acid residue, is mutated to a residue of valine, alanine, glycine or tyrosine. Although in certain embodiments it is contemplated that the HCV NS3 / 4A mutant protease may contain mutations at positions 156 and 165, it is believed that mutants that contain single mutations are also part of this invention.

Special aspects of the invention include HCV DNA encoding HCV NS3 / 4A protease (or a fragment or analog thereof), in which DNA codon 156 encodes valine or threonine, and codon 168 encodes aspartic acid or glutamic acid. In another embodiment of the present invention, HCV DNA encoding a HCV NS3 / 4A protease (or fragment or analog thereof) is provided in which the DNA codon 168 encodes a valine. In a further embodiment of the present invention, HCV DNA encoding the HCV NS3 / 4A protease (or fragment or analog thereof) is provided, in which DNA codon 168 encodes alanine, glycine or tyrosine.

The numbering system for the DNA of the present invention is in accordance with the sequence of SEQ ID NO.1. DNA according to this invention can be derived from SEQ ID NO.1. DNA can be obtained by solid-phase synthesis or by recombinant methods. In special embodiments, site-directed mutagenesis of the sequence of SEQ ID NO: 2 is used in order to obtain one or the other of the mutants described in the description.

It should be recognized that protein mutations can be complete (that is, all or almost all of the protein is converted into a mutant protein), partial or absent (that is, no or almost no mutation). Therefore, the composition or method of the present invention may include a mixture of wild-type protein and mutant protein.

In accordance with another embodiment of the present invention, an HCV NS3 / 4A protease protein (or fragment or analog thereof) comprising protease amino acid 156 is provided, wherein amino acid 156 is serine.

In another embodiment of the present invention, an HCV NS3 / 4A protease protein (or fragment or analog thereof) comprising protease amino acid 156 is provided, wherein amino acid 156 is valine.

In yet another embodiment of the present invention, an HCV NS3 / 4A protease protein (or fragment or analog thereof) comprising protease amino acid 156 is provided, wherein amino acid 156 is threonine.

In a further embodiment of the present invention, an HCV NS3 / 4A protease protein (or fragment or analogue thereof) comprising amino acid 156 of a protease is provided, wherein amino acid 156 is valine or threonine, and amino acid 168 is aspartic acid or glutamic acid.

In another embodiment of the present invention, an isolated HCV NS3 / 4A protease protein (or fragment or analogue thereof) comprising amino acid 156 of the protease is provided, wherein amino acid 168 is valine.

In another embodiment of the present invention, an HCV NS3 / 4A protease protein (or fragment or analogue thereof) comprising amino acid 156 of a protease is provided, wherein amino acid 168 is alanine, glycine or tyrosine.

DNA and proteins according to this invention can be modified using standard methods. For example, the DNA may contain a modification for attaching the DNA to a solid support. Proteins may contain a covalently attached marker compound.

The DNA or proteins of the invention may be in the form of a computer-readable form, including computer-readable media and / or computer-readable databases, not limited to these forms (see, for example, WO 98/11134).

For some uses, the DNA of the invention may be inserted into a vector. Any suitable vector may fall within the scope of the invention. Suitable vectors are known in the art. In one embodiment, an expression vector is provided. In another embodiment, a viral vector is provided. The vector may be a cloning agent or may additionally contain regulatory sequences, such as a promoter, amplifiers and terminators or polyadenylation signals.

Accordingly, the present invention also provides a vector comprising HCV NS3 / 4A protease DNA (or a fragment or analog thereof) in which:

DNA codon 156 encodes a serine;

DNA codon 156 encodes valine;

DNA codon 156 encodes threonine;

DNA codon 156 encodes valine or threonine, and codon 168 encodes aspartic acid or glutamic acid;

codon 168 DNA encodes valine;

codon 168 DNA encodes alanine;

DNA codon 168 encodes glycine and / or

codon 168 DNA encodes tyrosine.

In another embodiment, an expression vector is provided. In a further embodiment, a viral vector is provided. The vector may be a cloning tool or may additionally contain regulatory sequences, such as a promoter, amplifiers and terminators or polyadenylation signals. These vectors can be used in any suitable host cell. Host cells are known in the art.

Thus, the present invention also provides host cells containing DNA protease NS3 / 4A, in which codon 156 of the DNA encodes serine; DNA codon 156 encodes valine; DNA codon 156 encodes threonine; DNA codon 156 encodes valine or threonine, and codon 168 encodes aspartic acid or glutamic acid; codon 168 DNA encodes valine; codon 168 DNA encodes alanine; a codon of 168 DNA encodes glycine and / or a codon of 168 DNA encodes tyrosine. DNA expression will provide a host cell containing a protease having A156 mutated to serine; A156 with a mutation in valine; A156 with a mutation in threonine; A156 with a mutation in valine or threonine and D168 with a mutation in glutamic acid; D168 mutated to valine; D168 mutated to alanine; D168 with a mutation in glycine and / or D168 with a mutation in tyrosine. Cell lines containing DNA or proteins of the invention are also provided.

The invention also provides an HCV variant comprising DNA according to the invention or a protein according to the invention and compositions comprising DNA and proteins.

The HCV variants, as well as the DNA and / or proteins of the invention, can be used to detect a drug, as well as to monitor appropriate HCV therapies.

Thus, in another embodiment, the invention provides a method for detecting the presence of HCV in a biological sample, comprising determining the presence of DNA according to the invention. Said methods may include the steps of a) preparing (or extracting) the DNA; b) determining the DNA sequence; c) the establishment or assumption whether the codon 156 encodes serine in the DNA, whether the codon 156 encodes a valine, whether the codon 156 encodes a threonine polynucleotide, whether the codon 156 encodes valine or threonine, and whether the codon 168 encodes aspartic acid or glutamic acid, whether the codon 168 encodes valine or whether the codon 168 encodes alanine, glycine, or tyrosine. In some embodiments, a biological sample containing HCV is obtained from a mammal that is infected with HCV. Detecting the presence of such DNA can be used for diagnostic purposes to instruct the practitioner that the individual is an individual in whom HCV infection is likely to be resistant or otherwise insensitive to treatment with protease inhibitors. Given this indication, a professional specialist can modify the method of treatment of a subject having such an infection, for example, in addition to increasing the dose of therapy, using additional methods of treatment using agents to which the HCV strain infecting the subject is not resistant.

The methods of this invention may require the preparation of certain amounts of DNA. As a practitioner must admit, DNA should be obtained and then amplified. Standard methods (e.g., PCR, hybridization) can be used in the practice of this invention. Such methods are well known to those skilled in the art.

The invention also provides methods of treating or preventing HCV infection by continuously monitoring the mutations contemplated by the invention. If a resistant mutant is present in HCV, then the patient can be treated accordingly. Such a method will include: a) sampling (for example, a plasma sample, PMBC, liver cell or other sample) from a patient infected with HCV; and b) evaluating whether the plasma sample contains a nucleic acid encoding an HCV NS3 / 4A protease containing a mutation in codon 156; however, the mutation leads to the replacement of alanine with serine. Similar methods can be performed by substituting 156-alanine with a mutation for serine with other mutations described herein. Accordingly, such methods may include the identification of A156 with a mutation to serine (or another mutation described herein) and another protease mutation. All of these methods will include DNA preparation, DNA amplification and DNA sequence determination.

The present invention also provides methods for evaluating the effectiveness of treatment with HCV-infected NS3 / 4A protease inhibitors. Such methods include: a) sampling (for example, a plasma sample) from a patient infected with HCV; and b) evaluating whether the plasma sample contains a nucleic acid encoding an HCV NS3 / 4A protease having a mutation in codon 156; however, the mutation leads to the replacement of alanine with serine. Similar methods can be performed with other mutations of the present invention.

The methods of this invention are intended to identify resistant mutants in patients who have been administered HCV protease inhibitors. The described method can be practically applied to a patient who is being treated or treated. Mentioned and other diagnostic methods are known in this field (see, for example, US 5631128 and US 6489098).

Thus, in one embodiment, a method is provided for evaluating whether a HCV infected patient has hepatitis C virus protease NS3 / 4A DNA having a mutation in codon 156. The patient may be therapy resistant such as VX-950. Accordingly, the patient can be treated with therapy in which VX-950 substitute is used. In another embodiment, a method is provided for determining whether a HCV infected patient has hepatitis C virus protease NS3 / 4A DNA having a mutation at codon 168.

Some of these mutations result in reduced sensitivity or susceptibility to the VX-950. Similarly, some of the mutations correlate with reduced sensitivity or lead to reduced sensitivity or susceptibility to BILN 2061 (WO 00/599929; US 6608027). Other mutations result in reduced sensitivity or susceptibility to both VX-950 and BILN 2061. Reduced sensitivity or susceptibility to any or both compounds can be evaluated according to this invention. Having certain ideas regarding the characteristics of resistant mutations, a more effective treatment regimen can be developed.

For example, the present invention allows the construction and / or discovery of compounds that are active against the resistant mutants discussed in the description.

Accordingly, the present invention provides a method for evaluating an inhibitor candidate and a potential HCV inhibitor, including:

a) the introduction of a vector containing the DNA according to this invention and an indicator gene encoding an indicator in the host cell;

b) culturing the host cell;

c) quantification of the indicator in the presence of an inhibitor and the absence of an inhibitor.

In the described method, the test compound can be added at any one or more of steps a) to c).

In another embodiment of the present invention, there is provided a method for assaying anti-HCV activity of a compound, comprising:

a) providing a protease according to this invention and a protease substrate;

b) contacting the protease with an inhibitor candidate or potential inhibitor in the presence of a substrate;

c) evaluating or quantifying the inhibition of proteolytic activity of the protease.

In another embodiment of the present invention, there is provided a method for identifying a protease inhibitor according to this invention, including:

a) a study of protease activity in the absence of a compound;

b) a study of protease activity in the presence of a compound;

c) comparison of results a) and results b).

The described method may further include:

d) a study of the activity of wild-type protease in the absence of a compound;

e) testing the activity of wild-type protease in the presence of a compound;

f) a comparison of results d) and results e).

The data obtained by the described methods can then be analyzed, for example, by comparing the results of a) and / or b) and the results of d) and / or e).

Methods are also provided, including:

d) a study of the activity of a second protease NS3 / 4A containing amino acid 168 protease, in which amino acid 168 is valine, alanine, glycine or tyrosine, in the absence of a compound;

e) a study of the activity of the second protease in the presence of a compound;

f) comparing the results of d) and e).

The method may further include:

g) a study of the activity of wild-type protease in the absence of a compound;

h) testing the activity of wild-type protease in the presence of a compound;

i) a comparison of results g) and results h).

In a more specific embodiment, the method includes comparing the results from a) and / or b) and the results d) and / or e) and / or the results from g) and / or h).

After viruses become drug resistant, it is possible that the virus can still mutate and become drug sensitive again. So, this happens when the virus comes into contact with the second drug. Accordingly, the present invention also provides methods for identifying a compound capable of restoring VX-950 activity, wherein the NS3 / 4A protease becomes resistant to VX-950, including:

a) contacting the discussed mutant protease with the compound of interest;

b) testing the ability of VX-950 to inhibit the protease activity of a). Similar methods of restoring BILN 2061 activity against resistant mutants and / or mutants with dual resistance to VX-950 and BILN 2061 are also provided.

Another aspect with respect to drug resistant viruses is that the virus can be treated with another drug. Therefore, methods for identifying compounds that are effective against a drug resistant virus are very useful methods for detecting a drug. The methods described herein can be used in screening methods for a large amount of material. Alternatively, the invention also provides methods for rationally constructing a drug using the HCV NS3 / 4A protease structure disclosed in the description (i.e., that mutation of specific residues of 156 and / or 168 wild-type proteins) as a basis for constructing effective protease inhibitors . More specifically, in the present invention, HCV strains that are resistant to treatment with protease inhibitors, such as VX-950 and BILN 2061, are first identified.

Rational drug design can also be combined with the systematic method of large-scale screening experiments in which targets of possible protease inhibitory drugs are tested with compounds from combinatorial libraries.

Rational drug design is a collective approach that uses information regarding the structure of a drug receptor or one of its natural ligands to identify or create drug candidates. The three-dimensional structure of the protein can be established 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 mutant that contains one, the other, or both mutations of residues 156 or 168 can now be readily determined using standard X-ray crystallography and / or NMR spectroscopy.

Rational drug design can also be combined with the systematic method of large-scale screening experiments in which targets of possible protease inhibitory drugs are tested with compounds from combinatorial libraries. Having the information presented in the description, professional specialists can use computer programs to study databases containing the structures of many different chemical compounds. The computer may select those compounds that are most suitable to interact with the HCV NS3 / 4A protease from drug resistant mutants and test such an identified compound by standard laboratory methods with protease inhibitors, such as the methods described herein.

In some embodiments, it is believed that the structure of VX-950 or BILN 2061 (see figure 4) can be used as the starting structure on the basis of which additional molecules can be constructed. The description shows that mutant proteases are such that the interaction of VX-950 with them is reduced. Structures based on VX-950, which are more suitable and interact with the developed three-dimensional structure, when residue 156 is valine, serine or threonine and / or residue 168 is valine, alanine, glycine or glutamic acid, will be useful new protease inhibitors, which can be used against resistant strains of HCV in which there are mutations in the HCV NS3 / 4A protease. Such compounds may also be effective against wild-type HCV strains in which the HCV NS3 / 4A protease is not mutated. The recommendations of this invention will allow those skilled in the art to concentrate and narrow their studies as much as possible to limit the cost of large-scale screening.

In certain embodiments, the structure of the parent compound has the structure VX-950 (shown below in structure B). Although the VX-950 is an example, any stereoisomer 950 can be used with a mixture of the D and L isomers in the n-propyl side chain specifically included. The following structure A depicts such a diastereoisomer. This is a mixture of compounds of structure B (VX-950) and structure C.

Structure a

Structure B

Structure C

The rational design of drugs can be used to serially change various positions on a given molecule to obtain its derivatives, which can be used as protease inhibitors. The crystal structures of the wild-type HCV NS3 / 4A protease with VX-950 associated with it are shown in Figure 1. The data shown in the figure show that the removal of the negative charge D168 of HCV NS3 / 4A protease leads to the loss of R155 restriction and subsequent loss packaging with large P2 BILN 2061 and an increase in the cost of desolvation. R155 is not limited by D168 to the structure set with VX-950, and the mutation of D168V does not affect its binding. Such binding studies can easily be carried out with derivatives established by rational drug design to identify agents that exhibit binding ability and / or therapeutic efficacy in mutants.

The rational drug design was previously used to identify the release that is used to treat influenza. The design that led to the discovery of relens was developed by selecting the molecules that were most suitable for interacting with neuraminidase, the virus-produced enzyme that is required to release newly formed viruses from infected cells. Numerous modern drugs for treating HCV infections (e.g., ritonivir ™, indinavir ™) have also been identified by rational drug design schemes, in which drugs are designed to interact with a viral protease, an enzyme that breaks down viral proteins and allows them to assemble properly.

Another well-known drug that was created using the ligand-based rational construction method is Viagra ™. The drug mentioned was developed similar to cGMP, a ligand that binds phosphodiesterase.

Given that methods for rational drug design are effective, if only the target structure of the drug is known, it was expected that the discoveries of this invention that reveal the HCV NS3 / 4A protease structures that appear in HCV strains that are resistant to known HCV protease inhibitors in this area can be used for rational drug design to identify drugs suitable for the treatment of HCV.

Accordingly, the present invention also provides a method for identifying a compound effective against the protease according to this invention, including:

a) obtaining a three-dimensional model of the protease;

b) design or selection of a joint;

c) evaluating the ability of a compound to bind to or interact with a protease.

In such methods, the three-dimensional model is based on the X-ray crystal structure of the NS3 / 4A protease (Figure 1 and Figure 2). Known methods for developing models of the crystal structure, for example, computer-based molecular engineering methods (see, for example, US 6162613, WO 98/11134 and / or WO 02/068933). A three-dimensional model can also be obtained by x-ray crystallography of a protein according to this invention. As recognized in the art, a protein can be crystallized in the presence of a lack of a ligand (such as a test compound).

Methods for assessing the ability of a compound to bind to or interact with a protease are known in the art (see, for example, US 6162613, WO 98/11134 and / or WO 02/068933). Evaluation can be done, for example, by molecular construction. Once a compound is selected, it can be tested in the standard studies or studies described herein to establish compounds that act on various HCV proteases.

Thus, if there are recommendations of the present invention, it is possible to conduct screening studies to identify protease inhibitors that are effective against drug-resistant HCV infections. The present invention shows that drug resistance is induced in those HCV strains that have a mutation either at residue 156 or 168 of HCV NS3 / 4A protease. More specifically, it has been shown that mutation A156 to serine, valine or threonine and / or mutation D168 to valine, alanine, glycine or glutamic acid results in drug-resistant HCV.

It is contemplated that compositions that act as HCV mutant NS3 / 4A protease inhibitors that contain one or more of the mutations clearly described above will be useful in therapeutic embodiments for the treatment of HCV. The compounds can be compounds that are designed to mimic the effects of VX-950 or BILN 2061, or are obtained from VX-950 or BILN 2061. In screening studies to identify such compounds, the candidate substance can be tested first for basic in vitro biochemical activity and then tested for its ability to reduce, attenuate, or otherwise therapeutically interfere with HCV infection in an in vivo model of HCV infection. Mutant proteases of the invention have protease activity. Any of the screening studies can be carried out to determine the activity of the mutant HCV NS3 / 4A protease using any conventional test used to establish the activity of wild-type HCV NS3 / 4A protease. In preferred embodiments, the activity of the inhibitors against the mutant HCV NS3 / 4A protease is compared to the activity of the inhibitors against the wild-type HCV NS3 / 4A protease.

The ability of a candidate substance to inhibit a protease is determined by obtaining a sample containing HCV NS3 / 4A protease; and contacting the sample with the candidate substance. HCV protease activity is determined in the presence and absence of a candidate substance. The protease may be an isolated protein, a membrane fraction containing the isolated protein, or it may be in a cell that expresses a protease. Thus, protease activity is usually determined in a sample containing a protease in the absence of a candidate substance. A candidate substance is then added to the same or similar sample composition containing the protease, and protease activity is determined. Any candidate substance that reduces the protease activity of a sample indicates that the candidate substance has the desired inhibitory activity.

In in vivo screening studies, the compound is administered to a model animal over a period of time and at various dosages and the symptoms associated with HCV infection are attenuated. Any improvement in one or more of the symptoms mentioned will indicate that the candidate substance is an effective remedy.

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 wild-type proteases or a mutant species. Such an agent may be a protein or fragment thereof, an inhibitor with a small molecule, or even a nucleic acid molecule. It may turn out that most of the pharmacological compounds used will be compounds that are structurally related to other known HCV protease inhibitors, for example, such as VX-950 or BILN 2061, or other inhibitors discussed in the description. Rational drug design involves not only comparison with known such inhibitors, but predictions regarding the structure of the target molecules of such inhibitors.

On the other hand, one can simply acquire compounds from various commercial sources, libraries of small molecules, which are believed to satisfy the basic criteria for effective drugs, in order to “speed up” the identification of suitable compounds. Screening such libraries, including combinatorially formed libraries (eg, peptide libraries), is a quick and effective way to test a large number of related (and unrelated) compounds for activity. Combinatorial approaches also themselves contribute to the rapid evolution of potential drugs through the creation of compounds of the second, third and fourth generation, formed from active, but in other respects undesirable compounds.

Candidate compounds may include fragments or portions of natural compounds, or may be formulated as active combinations of known compounds that are otherwise inactive. It is believed that compounds isolated from natural sources such as animals, bacteria, fungi, plant sources including leaves and bark, and marine sources can be investigated as candidates for the presence of potentially effective pharmaceuticals. It should be understood that pharmaceuticals to be tested can also be prepared or synthesized from chemical mixtures or compounds made by humans.

"Effective amounts" of a candidate agent under certain circumstances are effective amounts to reproducibly induce a change in the inhibition of HCV NS3 / 4A protease expression or activity, inhibition of HCV production or virulence, suppression of HCV infection, or a decrease in intensity or amelioration of one or more symptoms of HCV infection by compared with the levels of the mentioned parameters in the absence of such a tool. Compounds, upon application of which achieve significant corresponding changes in such parameters, should be used. Significant changes in activity and / or expression will correspond to changes that are changes in activity of at least about 30% -40%, and more preferably, changes of at least 50%, of course, higher are possible quantities.

The dominant VX-950-resistant mutant, A156S, remains sensitive to BILN 2061. To confirm whether the observed mutations in either Ala156 or Asp168 are sufficient for resistance to VX-950 and BILN 2061 to appear, respectively, site-directed mutagenesis was used to introduce each individual mutations at position 156 or 168 in the wild-type NS3 protease domain.

Site-specific mutagenesis is another method used to produce mutant protease proteins used in the methods of the invention. In this method, specific mutagenesis of the basic DNA is used (which encodes the amino acid sequence that is targeted for modification). The method, in addition, provides a quick opportunity to obtain and test sequence variants, taking into account one or more of the above considerations when making one or more changes in the nucleotide sequence in DNA. Site-specific mutagenesis allows mutations to be made by using specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence diversity to form a stable duplex on both sides of the deletion compound that was produced. A primer of approximately 17-25 nucleotides in length with approximately 5-10 residues on both sides of the compound of the sequence that has been altered is generally preferred.

The method typically uses a bacteriophage vector, which exists in both single and double chain forms. Conventional vectors used in site directed mutagenesis include vectors such as phage M13. Said phage vectors are commercially available, and their use is usually well known to those skilled in the art. Double-stranded plasmids are also commonly used in site-directed mutagenesis, which eliminates the stage of gene transfer from phage to plasmid.

In general, site-directed mutagenesis is first carried out by obtaining a single-stranded vector or by melting two strands of a double-stranded vector, which includes in its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer is synthesized that carries the desired mutant sequence. The resulting primer is then annealed with a single-stranded DNA preparation, taking into account the degree of mismatch when choosing hybridization conditions (annealing), and subjected to the action of DNA polymerizing enzymes, such as the maple fragment of E. coli I polymerase I, in order to complete the synthesis of a chain having a mutation. Thus, they form a heteroduplex in which one chain encodes the original unmuted sequence, and the second chain has the desired mutation. The resulting heteroduplex vector is then used to transform suitable cells, such as E. coli cells, and clones are selected that include recombinant vectors carrying the mutant sequence structure.

Of course, the site-directed mutagenesis approach described above is not the only way to obtain potentially suitable mutant protease species, and is essentially not limited to it. Other methods for effecting mutagenesis are also contemplated by the present invention, such as, for example, processing recombinant vectors carrying a gene of mutagenic agents of interest, such as hydroxylamine, to obtain sequence variants.

The kinetic parameters of the FRET substrate for the wild-type NS3 protease domains of genotype 1a and 1b were identical [Table 1A and 1B] under the used research conditions. Although the NS4A peptide cofactor was from HCV genotype 1a, no recognizable differences in kinetic parameters were observed. This is consistent with molecular modeling, suggesting that conservative differences in the central NS4A region between genotypes 1a and 1b do not affect the interaction between the NS4A core peptide and the NS3 protease domain. Ki values for VX-950 and BILN 2061 were determined using wild-type proteases of genotypes 1a and 1b; no statistically significant differences were found between the two wild-type proteases (Table 2).

The kinetic parameters of the FRET substrate for the mutant protease A156S were essentially the same as the parameters of the wild-type protease [Table 1A and 1B]. However, the Ki value for VX-950 was 2.9 μM for the mutant A156S protease, which is 29 times higher than the Ki value against wild-type protease (0.1 μM) (Table 2). The Ki value for BILN 2061 was 112 nM for the A156S mutant, which was 6 times higher than the Ki value against wild-type protease, 19 nM (Table 2).

The level of HCV RNA in replicon-containing cells having A156S substitution was similar to the level of RNA of wild-type replicon cells (data not shown), which is consistent with the similar enzymatic catalytic efficiency of the A156S mutant and wild-type NS3 serine protease. The IC ₅₀ value of VX-950 in relation to replicon-containing A156S cells was 4.65 μM, which is 12 times higher than the IC ₅₀ against wild-type replicon-containing cells (0.40 μM) (Table 3). The differences in IC ₅₀ values for BILN 2061 with respect to A156S (7 nM) and replicon-containing wild-type cells (4 nM) were not significant (Table 3).

The main mutants resistant to BILN 2061, D168V, and D168A remained quite sensitive to the VX-950.

The kinetic parameters of the substrate were not affected by the D168V mutation, and only minor changes (less than 10-fold) for the D168A mutant were revealed, which showed a comparison of the kcat (Kcat) and kcat / Km values for wild-type proteases and two mutant NS3 serine proteases (Table 1A and 1B). Similarly, no significant effect of any substitution according to Asp168 on the Ki VX-950 value was noted (Table 2). However, the replacement of valine or alanine with aspartic acid at position 168 resulted in a mutant NS3 protease that was not inhibited by BILN 2061 concentrations up to 1.2 μM (Table 2). The data obtained indicate that any mutant protease is at least 63 times less sensitive to BILN 2061 compared to the wild-type protease. The exact value of the resistance cannot be established, since BILN 2061 does not dissolve at concentrations above 1.2 μM in the study buffer, which was determined by the optical density at 650 nm. The mutation D168V or D168A was also introduced into the wild-type HCV replicon by site directed mutagenesis and a stable line of replicon-containing cells bearing any substitution was obtained. BILN 2061 had an IC _{50 of} 5.09 μM for D168V replicon-containing cells, which is more than 1300 times higher than for wild-type replicon-containing cells (4 nM) (Table 3). The IC ₅₀ for BILN 2061 was 1.86 μM for the mutant replicon D168A. There was a slight difference in the IC ₅₀ values for VX-950 with respect to D168V and replicon-containing wild-type cells (Table 3).

Accordingly, compounds identified by the methods of the invention are also provided, wherein the compound is an HCV NS3 / 4A protease inhibitor. Such compounds can be obtained, for example, by the rational construction of a drug substance, which was discussed above.

The invention also provides compositions that contain the compounds described above, and their use. Such compositions can be used in pre-treated injectable devices that are administered to a patient to process biological samples, such as blood, before administration to the patient, and for direct administration to the patient. In each case, the composition should be used to inhibit HCV replication and reduce the risk or severity of HCV infection.

In another embodiment of the present invention, there is provided a composition comprising a compound identified in accordance with this invention, or a pharmaceutically acceptable salt thereof. According to a preferred embodiment, the compound identified according to this invention is present in an amount effective to reduce the viral potential in a sample or patient in which said virus encodes a serine protease necessary for the life cycle of the virus and a pharmaceutically acceptable carrier.

If pharmaceutically acceptable salts of the compounds of this invention are used in said compositions, such salts are preferably derived from inorganic or organic acids and bases. Such acid salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, heptoacetanoate, hemoheptosanoate, heptoacetanoate, heptoacetanoate, heptoacetanoate, heptoacetanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2 naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 phenylpropionate, picrate, pivalate, propionate, tuccinate, succinate, Latin and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine salts, N-methyl D-glucamine, and amino acid salts, such as arginine, lysine and so on.

Also, basic nitrogen-containing groups can be quaternized with 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 chlorides, bromides and iodides of decyl, lauryl, myristyl and stearyl, aralkyl halides such as benzyl and phenyl bromides, and others. As a result, soluble or dispersible products in water or oil are obtained.

The compounds used in the compositions and methods of the present invention can also be modified by the addition of suitable functional groups to enhance selective biological properties. Such modifications are known in the art and include modifications that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, improve solubility to allow injection by injection, modify metabolism and change the rate of excretion.

Pharmaceutically acceptable carriers that can be used in the described compositions, but are not limited to, include ion exchangers, alumina, aluminum stearate, lecithin, whey proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate , incomplete glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica zem, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat.

According to a preferred embodiment, the compositions of this invention are prepared for pharmaceutical use by a mammal, preferably a human.

Such pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or through an implanted reservoir. The term “parenteral” as used includes subcutaneous, intravenous, intramuscular, articulation, intraarticular, intrathoracic, intrathecal, pathological focus, intrahepatic and intracranial injection or by infusion. Preferably, the compositions are administered orally or intravenously.

Sterile injectable forms of the compositions of this invention may be an aqueous or oily suspension. These suspensions can be prepared according to methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1.3 butanediol. Suitable excipients and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are generally used as a solvent or suspending medium. For this purpose any light, non-volatile oil is used, including synthetic mono- or diglycerides. In the preparation of injectable forms, fatty acids, such as oleic acid and its glyceride derivatives, which are natural pharmaceutically acceptable oils, such as olive oil or castor oil, are used, especially in their polyoxyethylene versions. Such oil solutions or suspensions may also contain a diluent, long chain alcohol or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as twins, spans, and other emulsifying agents or bioavailability enhancers that are generally used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used to prepare a medicament.

Dose levels of between about 0.01 and 100 mg / kg body weight per day, preferably between about 0.5 and 75 mg / kg body weight per day of the considered protease inhibitor compounds, are used in monotherapy for the prevention and antiviral treatment, especially anti-HCV- mediated disease. Typically, the pharmaceutical compositions of this invention should be administered from about 1 to 5 times a day, or, alternatively, as a continuous infusion. Such an administration can be used as a long-term or emergency treatment. The amount of active ingredient that can be combined with carrier materials to formulate a single dosage form will vary depending on the patient being treated and the particular route of administration. A typical preparation contains from about 5% to 95% of the active compound (w / w). Preferably, such preparations contain from about 20% to 80% of the active compound.

If the compositions of this invention include a combination of a compound identified according to this invention and one or more additional therapeutic or prophylactic agents, then both the compound and the additional agent should be present at dose levels between about 10 and 100%, and more preferably from about 10 up to 80% of the dosage usually administered by a monotherapy regimen.

The pharmaceutical compositions of this invention can be administered orally in any suitable oral dosage form, including capsules, tablets, aqueous suspensions or solutions, not limited to the listed forms. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration of capsules, suitable diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifiers and suspending agents. If necessary, you can also add certain sweeteners, flavoring or coloring agents.

Alternatively, the pharmaceutical compositions of this invention can be used in the form of suppositories for rectal administration. Suppositories can be prepared by mixing the agent with a suitable non-irritating excipient, which is a solid at room temperature but liquid at a rectal temperature and therefore melts in the rectum to release the drug. Such substances include cocoa butter, wax and polyethylene glycols.

The pharmaceutical compositions of this invention can also be administered topically, especially when the treatment target covers areas and organs that are readily available for topical application, including diseases of the eyes, skin, or lower intestine. Suitable topical preparations are readily prepared for each of the areas or organs mentioned.

Topical application for the lower intestine can be effective when using a rectal suppository preparation (see above) or a suitable preparation for an enema. Transdermal patches can also be used topically.

For topical applications, the pharmaceutical compositions may be formulated as a suitable ointment containing an active ingredient suspended or dissolved in one or more carriers. Carriers for topical use of the compounds of this invention, but are not limited to, include mineral oil, liquid paraffin, white petrolatum, polyethylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated as a suitable lotion or cream containing active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters, wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water, but are not limited to these carriers.

For use in ophthalmology, pharmaceutical compositions can be prepared in the form of micronized suspensions in isotonic sterile physiological saline with adjusted pH, or, preferably, in the form of solutions in isotonic sterile physiological saline with adjusted pH, or with or without a preservative, such as benzylalkonium chloride. Alternatively, for use in ophthalmology, pharmaceutical compositions may be formulated as an ointment, such as petrolatum.

The pharmaceutical compositions of this invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to well-known methods of drug preparation technology and can be prepared in the form of solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons and / or other conventional solubilizing or dispersing agents.

Most preferred are pharmaceutical compositions prepared for oral administration.

In another embodiment, the compositions of this invention further comprise another antiviral agent, preferably an anti-HCV agent. Such antiviral agents include, but are not limited to, immunomodulators such as α-, β-, and γ-interferons, pegylated interferon-α compounds, and thymosin; other antiviral agents such as ribavirin, amantadine and telbivudine; other hepatitis C virus protease inhibitors (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase and polymerase inhibitors; ribosome function inhibitors; broad-spectrum viral inhibitors such as IMPDH inhibitors (e.g., compounds from United States Patent 5807876, 6498178, 6344465, 6054472, WO 97/40028, WO 98/40381, WO 00/56331 and mycofinolic acid and its derivatives, including VX-497 , VX-148 and / or VX-944, not limited to the above); 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 were used in the description (with trade names relating to products supplied as applications were registered).

"Peg-Intron" means PEG (PEG) -Intron®, peginterferon alfa-2b, supplied by Schering Corporation, Kenilworth, NJ; "Intron" means intron-A®, interferon alfa-2b, supplied by Schering Corporation, Kenilworth, NJ; “Ribavirin” means ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide available from ICN Pharmaceuticals, Inc., Costa Mesa, CA; described in Merck Index, entry 8365, twelfth edition, also available as rebetol® by Schering Corporation, Kenilworth, NJ, or kopegus® by Hoffmann-La Roche, Nutley, NJ; “Pagasis” means pagasis®, peginterferon alpha 2a, supplied by Hoffmann-La Roche, Nutley, NJ; “Roferon” means roferon®, recombinant interferon alfa-2a, available from Hoffmann-La Roche, Nutley, NJ; “beforor” means beforor®, interferon alfa-2, supplied by Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT; sumiferon®, a purified mixture of natural alpha interferons, such as sumiferon, supplied by Sumitomo, Japan; Wellferon®, interferon alpha n1, supplied by Glaxo-Wellcome LTd., Greit Britain ; Alferon®, a mixture of natural alpha interferons obtained by Interferon Sciences and supplied by Purdue Frederick Co., CT.

As used herein, the term “interferon” refers to a member of the family of highly homologous species of specific proteins that inhibit viral replication and cell proliferation and modulate the immune response, such as interferon alpha, interferon beta and interferon gamma. Merck Index, 5015, twelfth edition. According to one embodiment of the invention, interferon is α-interferon. According to another embodiment, natural alpha interferon 2a is used in the therapeutic combination of the present invention. Alternatively, natural alpha interferon 2b is used in the therapeutic combination of the present invention. In another embodiment, recombinant alpha interferon 2a or 2b is used in the therapeutic combination of the present invention. In yet another embodiment, the interferon is a pegylated alpha interferon 2a or 2b. Interferons suitable for this invention include:

(a) Intron (interferon alfa-2B, Schering Plow),

(b) Peg intron,

(c) Pegasis.

(d) Roferon,

(e) Beaufort,

(f) Sumiferon,

(g) Wellferon,

(h) total alpha interferon available from Amgen, Inc., Newbury Park, CA,

(i) Alferon;

(j) Viraferon®;

(k) Infergen®.

According to practitioners, the protease inhibitor should preferably be administered orally. Interferon is usually not administered orally. However, nothing in the description limits the methods or combinations of this invention for any special dosage forms or dosage regimens. Thus, each component of the combination according to the invention can be administered separately, together or in any combination thereof.

In one embodiment, the protease inhibitor and interferon are administered in separate dosage forms. In yet another embodiment, any additional agent is administered as part of a single dosage form with a protease inhibitor or as a separate drug. Since the invention includes a combination of compounds, the specific amounts of each compound may depend on the specific amounts of each other compound in the combination. As recognized by practitioners, dosages of interferon are usually measured in IU (IU) (for example, from about 4 million IU to 12 million IU).

Thus, agents (either acting as immunomodulators or otherwise) that can be used in combination with a compound of the present invention, without limitation, include interferon alfa-2B (intron A, Schering Plow); rebatron (Schering Plow, interferon alpha 2B + ribavirin); pegylated interferon alfa (Reddy, KR et al. "Efficacy and Safety of Pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2f in noncirrhotic patients with chronic hepatitis C" (Hepatology, 33, pp. 433-438 ( 2001)); total 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)); ribavarin and compounds that increase the development of the response of type 1 T-helper cells (Davis et al. “Future Options for the Management of Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999)); Interferons can reduce the intensity of viral infectious diseases by providing a direct antiviral effect and / or modifying the immune response to the infection. The antiviral effect of interferons is often mediated through inhibition of virus entry or virion decapsidation, viral RNA synthesis, viral protein translocation and / or virus accumulation and release.

Compounds that stimulate interferon synthesis 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), not limited to include double-stranded RNA, alone or in combination with tobramycin, and imiquimod (3M Pharmaceuticals; Sauder, DN “Immunomodulatory and Pharmacologic Properties of Imiquimod” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000)) .

Other non-immunomodulatory and immunomodulatory compounds can be used in combination with the compound of the present invention, without limitation, including the compounds set forth in WO 02/18369, which is incorporated herein by reference (see, for example, page 273, lines 9-22 and from page 274, line 4, up to page 276, line 11, which is included in the description by quotation).

Compounds that stimulate interferon synthesis in cells (Tazulakhova, et al., J. Interferon Cytokine Res., 21 pp. 65-73), without limitation, include double-stranded RNA alone or in combination with tobramycin and imiquimod (3M Pharmaceuticals ) (Sauder, J. Am. Acad. Dermatol., 43 pp. S6-11 (2000)).

Other compounds are known that exhibit or which may exhibit HCV antiviral activity by non-immunomodulating mechanisms, but are not limited to, include ribavarin (ICN Pharmaceuticals), inosine 5'-monophosphate dehydrogenase inhibitors (formula VX-497 described herein), amantadine and rimantadine ( Younossi et al., In Seminars in Liver Disease 19, 95-102 (1999)); LY217896 (U.S. Patent 4,835,168) (Colacino, et al., Antimicrobial Agents & Chemotherapy 34, 2156-2163 (1990)); and methyl ester 9-hydroxyimino-6-methoxy-1,4a-dimethyl 1,2,3,4,4a, 9,10,10a-octahydro-phenanthrene-1-

carboxylic acid; 6-methoxy-1,4a-dimethyl-9- (4-methyl-piperazin-1-ylamino) methyl ester -1,2,3,4,4a, 9,10,10a-octahydro-phenanthrene-1-carbon acid hydrochloride; 1- (2-chloro-phenyl) -3- (2,2-biphenyl-ethyl) -urea (U.S. Patent 6127422).

Preparations, doses and routes of administration for the above molecules are either described in the references cited below or are well known in the art, for example, F.G. Hayden, in 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 in the description. Alternatively, once a compound that exhibits HCV antiviral activity is identified, especially antiviral activity against a drug-resistant HCV strain, a pharmaceutically effective amount of such a compound can be determined using methods that are well known in the art. Mentioned, 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 in the description. Thus, suitable formulations, dose range (h), and regimens for such a compound can be readily established by conventional methods.

The drug combinations of the present invention can be delivered to a cell or cells, or to a human patient, or in separate pharmaceutically suitable preparations, administered simultaneously or sequentially, in preparations containing more than one therapeutic agent, or in the selection of drugs with a single agent or with multiple agents. Regardless of the route of administration, said drug combinations comprise an anti-HCV effective amount of pharmaceutically acceptable drug components.

A large number of other immunomodulators and immunostimulants that can be used in the methods of the present invention are currently available and include: AA-2G; adamantylamide dipeptide; adenosine deaminase, Anzon adjuvant, Alliance; Ribi adjuvants; Vaxcel adjuvants; adjuvax; agelasfin-11; AIDS Therapy (AIDS), Chiron; Algalglucan, SRI; Alganunulin, Anutech; anginlus; anticellular factors, Yeda; anticort; anti-gastrin-17 immunogen, Ap; antigen delivery system, Vac; antigen preparation, IDBC; anti-GnRH immunogen, Aphton; antiherpine; arbidol; azarol; Bay-q-8939; Bay-r-1005; BCH-1393; betafectin; biostim; BL-001; BL-009; bronkostat; cantastym; CDRI-84-246; cefodysime; chemokine inhibitors, ICOS; CMV peptides, City of Hope; CN-5888; cytokine 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; FLT-3 Ligand, Immunex; FR-900483; FR-900494; FR-901235; FTS-Zn; G proteins, Cadus; gladapcin; glutaurin; glycophosphopeptic; GM-2; GM-53; GMDP; growth factor vaccine, EntreM; H-BIG, NABI; H-CIG, NABI; HAB-439; Helicobacter pylori vaccine; herpes-specific immune factor; HIV therapy (HIV), United Biomed; hyperGAM + CF; ImmuMax; Immun BCG; immune therapy, Connective; immunomodulator, Evans; immunomodulators, Novacell; imreg-1; imreg-2; indomun; 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; lycomarasminic acid; LipoTher; LK-409, LK-410; LP-2307; LT (R1926); LW-50020; MAF; Shionogi derivatives of MDP, Merck; met-enkephalin, TNI; methylfuryl butyrolactones; MIMP; myrimostim; mixed bacterial vaccine, Tem, MM-1; moniliastat; MPLA, Ribi; MS-705; murabutide; Marabutide, Vacsyn; muramyl dipeptide derivative; muramyl peptide derivatives, myelopid; -563; NACOS-6; NH-765; NISV, Proteus; NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios; peptidoglycan, Pliva; perton, Advanced Plant; PGM derivative, Pliva; pharmaceutical projects 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, No. 4500, No. 4689, No. 4833, No. 494, No. 5217, No. 530; pidotimod; pimelautide; pinafid; PMD-589; podophyllotoxin, Conpharm; POL-509; poly-ICLC, poly-ICLC, Yamasa Shoyu; polyA-polyU; polysaccharide A; Protein A, Berlux Bioscience; PS34WO; Pseudomonas Mabs, Teijin; psomaglobin; PTL-78419; Pyrexol; pyriferone; retrogen; retropep; RG-003; rhinostat; rifamaxil; RM-06; rollin; romurtide; RU-40555; RU-41821; rubella virus 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; staphage lysate; Stimulon; suppressin; T-150R1; T-LCEF; tabilautide; temurtide; teradigm-HBV; teradigm-HBV; teradigm-HSV; THF, Pharm &Upjohn; THF, Yeda; thymalfasin; thymus hormone fractions; thymocartin; thymolymphotropin; thymopentin; thymopentin analogues; thymopentin, Peptech; thymosin fraction 5, Alpha; thymostimulin; thymotrinan; TMD-232: TO-115; transfer factor, Viragen; tuftsin, Selavo; yubenimeks; Ulsastat; ANGG-; CD-4 +, collage +; COLSF +; COM +; 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 +; RHA-SYN-; SY-CW-; TH-A-I +; TH-5 +; THF +; UN.

Representative nucleoside and nucleotide compounds used in this invention, without limitation, include: (+) - cis-5-fluoro-1- [2- (hydroxy-methyl)] - [1,3-oxathiolan-5-yl ] cytosine; (-) - 2'-deoxy-3'-thiocytidine-5'-triphosphate (3TC); (-) - cis-5-fluoro-1- [2 (hydroxymethyl) - [I, 3-oxathiolan-5-yl] cytosine (FTC); (-) 2 ', 3', dideoxy-3'-thiacytidine [(-) - 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-1,2,4-triazole-3-carboxamide; 2 ', 3'-dideoxy-3'-fluoro-5-methyl-dexocytidine (FddMeCyt); 2 ', 3'-dideoxy-3'-chloro-5-methyl-dexocytidine (ClddMeCyt); 2 ', 3'-dideoxy-3'-amino-5-methyl-dexocytidine (AddMeCyt); 2 ', 3'-dideoxy-3'-fluoro-5-methyl-cytidine (FddMeCyt); 2 ', 3'-dideoxy-3'-chloro-5-methyl-cytidine (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'-deoxy-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-dexocytidine (AddMeCyt); 2-amino-1,9 - [(2-hydroxymethyl-1- (hydroxymethyl) ethoxy] methyl] -6H-purin-6-one (ganciclovir); 2- [2- (2-amino-9H-purin-9yl ) ethyl) -1,3-propanediyl diacetate (famciclovir); 2-amino-1,9-dihydro-9 - [(2-hydroxy-ethoxy) methyl] 6H-purin-6-one (acyclovir); 9- (4-hydroxy-3-hydroxymethyl-but-1-yl) guanine (penciclovir); 9- (4-hydroxy-3-hydroxymethyl-but-1-yl) -6-deoxy-guanine diacetate (famciclovir); 3'-azido-3'-deoxythymidine (AZT); 3'-chloro-5-methyl-dexocytidine (ClddMeCyt); 9- (2-phosphonyl-methoxyethyl) -2 ', 6'-diaminopurin-2', 3'-dideoxyriboside; 9- (2-phosphonylmethoxyethyl) adenine (PMEA); acyclovir triphosphate (ACVTP); D-carbocyclic-2'-deoxyguanosine (CdG); dideoxy-cytidine; dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine (ddl); E-5- (2-bromovinyl) -2'-deoxyuridine triphosphate; fluoro-arabinofuranosyl-ioduracil; 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-purine-6-amine-5'-monophosphate monohydrate (Ara-AMP); 2-deoxy-3'-thia-5-fluorocytidine; 2 ', 3'-dideoxy-guanosine; and 2', 3'-dideoxy-guanosine.

The synthesis methods for producing nucleosides and nucleotides used in this 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 Nucleotides 3, 421-535 (1994); Ann. Reports in Med. Chena, Academic Press; and Exp. Opin. Invest. Drugs 4, 95-115 (1995).

The chemical reactions described in the references cited above are usually considered from the point of view of their broadest application to obtain the compounds of this invention. Sometimes the described reactions are not suitable for each compound among the compounds described in the description. Compounds for which the described reactions are not suitable are readily established by those skilled in the art. In all such cases or reactions, it is possible to carry out successfully using conventional modifications known to those skilled in the art, for example, appropriate protection of the interfering groups, substitution with alternative conventional reagents, usual modification of the reaction conditions and the like, or other reactions described herein, or in other respects, suitable should be used in the preparation of the corresponding compounds of this invention. For all preparative methods, all starting materials are known or are easily obtained from raw materials.

While nucleoside analogs are usually used as antiviral agents as they are, nucleotides (nucleoside phosphates) should sometimes be converted to nucleosides in order to facilitate their transport across cell membranes. An example of a chemically modified nucleotide capable of penetrating into cells is S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine (HPMPC, Gilead Science). The nucleoside and nucleotide compounds used in this invention, which are acids, can form salts. Examples include alkali metal or alkaline earth metal salts such as sodium, potassium, calcium or magnesium, or organic base salts or basic quaternary ammonium salts.

The skilled person may also prefer to administer a cytochrome P450 monooxygenase inhibitor. Such inhibitors can be used to increase concentrations in the liver and / or increase blood levels of compounds that are inhibited by cytochrome P450.

If an embodiment of the present invention encompasses a CYP inhibitor, any CYP inhibitor that improves the pharmacokinetics of the corresponding NS3 / 4A protease can be used in the method of the present invention. Mentioned CYP inhibitors, but are not limited to, include ritonavir (WO 94/14436), ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporine, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, flufinvrethine, indolefinvin, fluloquiretin, flufonvetin, indolefinvin, amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944 and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporine and clomethiazole. For preferred dosage forms of ritonavir, see United States Patent 6037157 and documents cited in the description: United States Patent 5484801, United States Patent Application 08/402690 and International Applications WO 95/07696 and WO 95/09614.

Methods for assessing the ability of a compound to inhibit the activity of cytochrome P450 monooxygenase are known (see US 6037157 and Yun, et al. Drug Metabolism & Disposition, vol. 21, pp. 403-407 (1993)).

Immunomodulators, immunostimulants and other agents used in the combination therapy methods of the present invention can be administered in amounts lower than those accepted in the art. For example, interferon alfa is typically administered to a person to treat HCV infectious diseases in an amount of about 1 × 10 ⁶ units / subject three times a week to 10 × 10 ⁶ units / subject three times a week (Simon et al., Hepatology 25: 445- 448 (1997)). In the methods and compositions of this invention, said dose may range from about 0.1 × 10 ⁶ units / subject three times a week to 7.5 × 10 ⁶ units / subject three times a week; more preferably from about 0.5 × 10 ⁶ units / subject three times a week to 5 × 10 ⁶ units / subject three times a week; most preferably from about 1 × 10 ⁶ units / subject three times a week to 3 × 10 ⁶ units / subject three times a week. Due to the increased antiviral against hepatitis C virus, the effectiveness of immunomodulators, immunostimulants or other anti-HCV agents in the presence of HCV serine protease inhibitors of the present invention, reduced amounts of said immunomodulators / immunostimulants can be used in the treatment methods and compositions described herein. Similarly, due to the increased antiviral against hepatitis C virus efficacy of these HCV serine protease inhibitors in the presence of immunomodulators and immunostimulants, reduced amounts of these HCV serine protease inhibitors can be used in the methods and compositions described herein. Such reduced amounts can be established by routine monitoring of hepatitis C virus titers in infected patients who are being treated. This can be done, for example, by monitoring HCV RNA in patient serum using slot blotting, dot blotting, or PCR-RT, or by detecting surface and other HCV antigens. Patients can be similarly monitored during treatment with combination therapy using the HCV serine protease inhibitors described herein and other compounds exhibiting anti-HCV activity, for example, nucleoside and / or nucleotide antiviral agents, to determine the lowest effective doses of each compound. used in combination.

In the combination therapy methods discussed herein, nucleoside or nucleotide antiviral compounds or mixtures thereof can be administered to humans in an amount in the range of about 0.1 mg / subject / day to about 500 mg / subject / day; preferably from about 10 mg / subject / day to 300 mg / subject / day; more preferably from about 25 mg / subject / day to 200 mg / subject / day; even more preferably from about 50 mg / subject / day to 150 mg / subject / day, and most preferably in the range of about 1 mg / subject / day to 50 mg / subject / day.

Doses of the compounds can be administered to the patient in a single dose or in divided doses. In the latter case, the drug standard composition may contain such quantities of submultiple doses that make up the daily dose. Multiple daily doses may also increase the total daily dose when required by the subject to whom the drug is prescribed.

The treatment regimen for a patient suffering from an HCV-induced infectious disease, compounds and / or compositions of this invention is selected in accordance with a number of factors, including the patient’s age, weight, gender, diet and general condition, severity of infection, route of administration, pharmacological characteristics, such such as activity, efficacy, pharmacokinetic and toxicological profiles of the individual compounds used, and whether the drug delivery system is used. The administration of the drug combinations described herein should usually be continued for a period of several weeks to several months or years until the titers of the virus have reached appropriate levels, indicating that the infection is controlled or eliminated. Patients who are treated with the drug combinations described herein are usually monitored continuously by detecting hepatitis RNA in patient serum using slot blotting, dot blotting, or PCR-RT, or by detecting hepatitis C virus antigens, such as surface antigens, in serum, to establish the effectiveness of therapy. A constant analysis of the data obtained by the above methods allows you to change the scheme during therapy in order to enter the optimal amounts of each component in combination, and in order to also establish the duration of treatment. Thus, the treatment / drug administration schedule can be rationally changed during therapy in order to administer the smallest amounts of each of the antiviral compounds used in combination, which together show satisfactory efficacy against the hepatitis C virus, and so that the use of such antiviral compounds in combination continued only as long as necessary to successfully treat an infectious disease.

The present invention encompasses the use of the HCV serine protease inhibitors discussed herein in various combinations with the above and similar types of compounds exhibiting anti-HCV activity to treat or prevent infectious diseases caused by HCV in patients, especially those patients who have infectious HCV diseases that show resistance to treatment with VX-950 and other standard protease inhibitors. For example, one or more HCV serine protease inhibitors can be used in combination with: one or more interferons or interferon derivatives exhibiting anti-HCV activity; one or more non-interferon compounds exhibiting anti-HCV activity; or one or more interferons or derivatives of interferon exhibiting anti-HCV activity, and one or more non-interferon compounds exhibiting anti-HCV activity. When used in combination to treat or prevent HCV infection in humans, any of the HCV serine protease inhibitors described herein and any of the above compounds exhibiting anti-HCV activity may be present in a pharmaceutically or anti-HCV effective amount. Due to their additive and synergistic effects, when used in the combinations described above, each can also be present in a subclinical pharmaceutically effective or anti-HCV effective amount, that is, in an amount that, when used alone, provides reduced pharmaceutical efficacy in completely inhibiting or reducing accumulation HCV virions and / or in weakening or decreasing the intensity of conditions or symptoms associated with HCV infection or pathogenesis in patients compared to such HCV serine protease inhibitors and compounds exhibiting anti-HCV activity when used in pharmaceutically effective amounts. In addition, the present invention includes the use of combinations of HCV serine protease inhibitors and anti-HCV activity compounds as discussed above to treat or prevent HCV infectious diseases in which one or more of the above inhibitors or compounds are present in a pharmaceutically effective amount, and the other (s) is present (s) in a subclinical pharmaceutically effective or anti-HCV effective amount (s) due to their additive or synergistic effect. Used in the description, the term "additive effect" describes the combined effect of two (or more) pharmaceutically active agents, which is equal to the sum of the actions of each agent produced separately. A synergistic effect is an action in which the combined effect of two (or more) pharmaceutically active agents is greater than the sum of the effects of each agent produced separately.

When an improvement in the patient's condition is achieved, a maintenance dose of a compound, composition or combination of the present invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced as a function of symptoms to a level at which an improved condition is maintained, if the symptoms are stopped to the desired level, treatment should be discontinued. However, patients may require intermittent treatment on a long-term basis for any relapse of the symptoms of the disease.

It should also be understood that the specific dosage and treatment regimen for an individual patient will depend on a number of factors, including the activity of the particular compound used, age, body weight, general condition, gender, diet, time of administration, excretion rate, drug combination and evaluation the attending physician and the severity of the disease that is being treated. The amount of active ingredients also depends on the individual compound described and the presence or absence and 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 virus encoded serine protease, which is necessary for the life cycle of the virus, by administering to the patient a pharmaceutically acceptable composition of the present invention. Preferably, the methods of this invention are used to treat a patient suffering from HCV infection. Such treatment can completely eliminate the viral infection or reduce its severity. More preferably, the patient is a human.

In an alternative embodiment, the methods of the invention further include the step of administering to said patient an antiviral agent, preferably an anti-HCV agent. Such antiviral agents, without limitation, include immunomodulators such as α-, β- and γ-interferons, pegylated derivatives of α-interferon compounds and thymosin; other antiviral agents such as ribavirin and amantadine; other hepatitis C virus protease inhibitors (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase and polymerase inhibitors; ribosome function inhibitors; broad-spectrum viral inhibitors such as IMPDH inhibitors (e.g., VX-497 and other IMPDH inhibitors described in United States Patent 5807876, mycophenolic acid and its derivatives), or combinations of any of the above inhibitors.

Said additional agent may be administered to said patient as part of a single-use dosage form containing both a compound of the invention and an additional antiviral agent. Alternatively, the additional agent can be administered separately from the compound of the present invention, as part of a unit dosage form, wherein said additional agent is administered before, together with the composition, or after the composition containing the compound of the present invention.

In yet another embodiment, the invention provides a method of pretreating a biological substance for administration to a patient, comprising the step of contacting said biological substance with a pharmaceutically acceptable composition comprising a compound of the invention. Such biological substances, without limitation, include blood and its components, such as plasma, platelets, subpopulations of blood cells and the like; organs such as kidney, liver, heart, lung, and so on; sperm and eggs; the bone marrow and its components and other fluids that must be infused into the patient, such as saline, dextrose and so on.

According to another embodiment, the invention provides methods for processing materials that could potentially be in contact with a virus characterized by a virus encoded serine protease necessary for its life cycle. This method includes the step of contacting said material with a compound of the invention. Such materials include, but are not limited to, surgical instruments and clothing; laboratory tools and clothing; equipment and materials for blood sampling and devices introduced into the body, such as shunts and stents, and so on.

In another embodiment, the compounds of this invention can be used as laboratory agents to facilitate the isolation of a virus-encoded serine protease. This method includes the steps of ensuring the attachment of the compounds of this invention to a solid base; contacting said solid base with a sample containing a viral serine protease under conditions that cause said protease to bind to said solid base, and eluting said serine protease with said solid base. Preferably, the viral serine protease isolated by the described method is HCV NS3 / NS4A protease. More specifically, it is a HCV NS3 / NS4A mutant protease that is resistant to the treatment of VX-905 and / or BILN 2061, which are described in the description. Examples of such proteases include those described herein, which have mutant residues at positions 156 and / or 168 of the protein SEQ ID NO: 2.

Used in the description, unless otherwise indicated, the term "contains" and its variations mean the inclusion of an established element, and not an exception to any other element.

Conventional methods that are known to those skilled in the art can be used in the practice of this invention. Such methods can be found in published documents. For example, standard recombinant DNA and molecular cloning methods are well known in the art. See, for example, F.M. Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 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 cited in US Pat. Nos. 6,617,156 and 6,617,130, all of which are incorporated herein by reference.

Examples

For a more complete understanding of the present invention, the following preparative and testing examples are provided. The following examples are included to demonstrate some preferred embodiments of the invention. Specialists in this field should be borne in mind that the methods described in the examples, which follow the presented methods developed in the invention, are effective in the practical application of the invention and, therefore, are believed to be the preferred methods for their practical application. However, specialists in this field, in the light of this discovery, it should be borne in mind that many changes can be made to special embodiments that are described, and still get a similar or similar result without deviating from the idea and scope of the invention.

Example 1: Obtaining plasmids

The DNA fragment encoding the residues of Ala ¹ -Ser ¹⁸¹ protease NS3 HCV (GenBank CAB46913) was obtained by PCR from a replicon-containing plasmid Con1 HCV, I ₃₇₇ neo / NS3-3 '/ wt. (renamed pBR322-HCV-Neo in this study) [V. Lohmann et al., Science, 285, pp. 110-113 (1999)] and inserted into pBEV11 (S. Chamber, et al., Personal communication) for expression in HCV proteins with a C-terminal hexahistidine tag in E. coli. Resistant mutations against PI NS3-4A HCV were introduced into this construct through PCR-based site-directed mutagenesis. To obtain a HCV replicon containing PI-resistant mutations, 1.2-kb (thousand base pairs; kbp) Hind III / BstX I fragment derived from HCV replicon Con1 was subcloned into a TA-cloning vector, pCR2. 1 (Invitrogen). PI-resistant mutations in the NS3 serine protease domain were introduced into the pCR2 vector containing the Hind III / BstX I fragment of HCV by PCR, and the BsrG I / BstX I fragment, 579 base pairs containing the mutated residue, was subcloned back into the Con1 plasmid second a generation having three adaptive mutations, pBR322-HCV-Neo-mADE (see below). All constructs were confirmed by sequencing.

Example 2: Preparation of Replicon-Containing HCV Cells

The subgenomic replicon-containing plasmid Con1, pBR322-HCV-Neo [Lohmann et al., Science, 285, pp. 110-113 (1999)] were digested with Sca I (New England Biolabs). A full-size subgenomic replicon containing HCV RNA was obtained from a linearized DNA matrix using a T7 Mega-script kit (Ambion) and treated with DNase to remove the template DNA. After reading, RNA transcripts were introduced by electroporation into Huh-7 cells and stable lines of HCV replicon cells were selected from 0.25 or 1 mg per ml of G418 (Geneticin) into the minimum Dulbecco's modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Stable replicon-containing HCV cells were maintained in DMEM, 10% FBS and 0.25 mg per ml G418.

During the preparation of subgenomic stable cell lines containing a replicon, several different types of adaptive mutations were identified. One type had three substitutions in non-structural HCV proteins that were introduced into the original pBR322-HCV-Neo plasmid by site directed mutagenesis to obtain a second generation subgenomic replicon-containing plasmid, pBR322-HCV-Neo-mADE. When T7 RNA transcripts, after reading from Sca plasmid I-linearized pBR322-HCV-Neo-mADE by electroporation, were introduced into Huh7 cells, stable colonies of replicon-containing cells were formed with much greater efficiency than the original Con1 R1 replicon RNA replicon. Resistant mutations identified in the described study were introduced into the replicon-containing plasmid pBR322-HCV-Neo-mADE using site-directed mutagenesis. Stable cell lines containing replicon were obtained using T7 transcripts derived from either pBR322-HCV-Neo-mADE or types with resistant mutations.

Example 3: Study Protocol Containing HCV Replicon Cells

Cells containing hepatitis C virus replicon (HCV) were maintained in DMEM containing 10% fetal bovine serum (FBS), 0.25 mg per ml G418 with appropriate additives (medium A).

On day 1, a monolayer of replicon-containing cells was treated with trypsin: EDTA, removed, and then medium A was diluted to a final concentration of 100,000 cells per ml wt (wild type), 10,000 cells in 100 μl were placed in each well of a 96-well tissue culture plate and cultured overnight in a tissue culture incubator at 37 ° C.

On day 2, the compounds (in 100% DMSO) were sterile diluted in DMEM containing 2% FBS, 0.5% DMSO with appropriate additives (medium B). The final concentration of DMSO was maintained at 0.5% in the entire dilution series.

The medium from the monolayer of replicon-containing cells was removed, and then medium B containing various concentrations of compounds was added. Medium B without any compound was added to other wells as controls containing no compound.

Cells were incubated with compound or 0.5% DMSO in medium B for 48 hours in a tissue culture incubator at 37 ° C. At the end of the 48-hour incubation, the medium was removed, and the monolayer of replicon-containing cells was washed once with PBS and stored at -80 ° C until RNA extraction.

Culture plates with treated monolayers of replicon-containing cells were thawed, and a specified amount of another virus containing RNA, such as bovine viral diarrhea virus (BVDV), was added to the cells in each well. Reagents for RNA extraction (such as reagents from RNase kits) were immediately added to the cells to avoid RNA degradation. Total RNA was extracted according to the manufacturer's instructions with the modification in order to improve extraction efficiency and consistency. Finally, total cellular RNA, including HCV replicon RNA, was eluted and stored at -80 ° C until further processing.

The method of quantitative determination of Tagman real-time PCR-RT was carried out with two series of specific primers and a probe. One series is for HCV and the other is for BVDV. Total RNA extracts from treated replicon-containing HCV cells were added to PCR reaction mixtures to quantify both HCV and BVDV RNA in the same PCR well. An experimental “failure” was noted and deemed unsuccessful based on the level of BVDV RNA in each well. The HCV RNA level in each well was calculated according to the mileage of the standard curve for the same PCR plate. The percentage of inhibition or decrease in HCV RNA level due to treatment with the compound was calculated by taking DMSO or control without compound as 0% inhibition. The cytotoxicity of the compounds was determined using a cell viability analysis based on the mitochondrial enzyme, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). IC ₅₀ values (concentration at which a 50% inhibition of HCV RNA level was observed) and CC ₅₀ (concentration at which a 50% decrease in cell viability was observed) were calculated from the titration curve of any given compound using a curve with four parameters (SoftMax Pro).

Example 4: Selection of PI-resistant replicon-containing HCV cells

Subgenomic, replicon-containing stable HCV Con1 cells were serially passaged in the presence of 0.25 mg per ml G418 and gradually 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). VX-950 concentrations ranged from 3.5 μM (or

10 × IC ₅₀ ) in a 48-hour study (see above) to 28 μM (80 × IC ₅₀ ), and the final concentration was 12.5 μM (12500 × IC ₅₀ ). During the selection, the replicon-containing cells multiplied twice a week when they reached 70-90% confluence. Fresh PI HCV was added every 3-4 days, regardless of whether the cell culture was shared or not.

Example 5: Identification of PI-Resistant HCV Mutants

During the selection of PI-resistant replicon-containing HCV cells, cell pellets were collected each time the cell culture was digested. Total cellular RNA was extracted using RNase mini prep. set (Qiagen). A cDNA fragment of 1.7 thousand base pairs (kbp) containing the HCV NS3 serine protease region was amplified with a pair of HCV-specific oligonucleotides (5'-CCTTCTATCGCCTTCTTG-3 '(SEQ ID NO: 3) and 5'-CTTGATGGTCTCGATGG-3 '(SEQ ID NO: 4)) using a one-step PCR-RT titanium kit (Roche Applied Science). The amplified products were purified using a QIA-fast PCR purification kit (Qiagen). To control the appearance of PI-related HCV mutations in the HCV NS3 serine protease domain during selection, purified 1.7 kb sequences were determined. PCR-RT-processed PI products taken at some different time periods of cultivation. To determine the frequency of occurrence of PI-resistant mutations, 1.7 kb HCV RNA PCR products of replicon resistant VX-950 or BILN 2061 cells were ligated into a TA-cloning vector (Invitrogen). Numerous individual bacterial colonies were isolated for each time point, and the region encoding the HCV NS3 protease purified plasmid DNA was sequenced.

Example 6: Expression and Purification of the HCV NS3 Serine Protease Domain

Each expression construct for the HCV NS3 serine protease domain containing a wild-type sequence or resistant mutations (A156S, D168V or D168A) was transformed into E. coli BL21 / DE3 pLysS cells (Stratagene). Fresh transformed cells grew at 37 ° C in BHI medium (Difco Laboratories) supplemented with 100 μg per ml of carbenicillin and 35 μg per ml of chloramphenicol to an optical density of 0.75 at 600 nM. Induction by 1 mM IPTG was carried out for four hours at 24 ° C. Cell masses were collected by centrifugation and the vial was frozen at -80 ° C until protein purification. All purification steps were carried out at 4 ° C. For each NS3 HCV protease, 100 g of cell mass was lysed in 1.5 L of buffer A {50 mM HEPES (pH 8.0), 300 mM NaCl, 0.1% n-octyl-β-D-glucopyranoside, 5 mM β-mercaptoethanol, 10% (v / v) glycerol} and stirred for 30 minutes Lysates were homogenized using a microfluidizer (Microfluidics, Newton, MA), followed by ultracentrifugation at 54000g for 45 minutes. Imidazole was added to the supernatants to a final concentration of 5 mM together with 2 ml of Ni-NTA resin, previously equilibrated with buffer A containing 5 mM imidazole. The mixture was rocked for three hours and washed with 20 column volumes of buffer A plus 5 mM imidazole. The mixture was rocked for three hours and washed with 20 column volumes of buffer A plus 5 mM imidazole. HCV NS3 proteins were eluted into buffer A containing 300 mM imidazole. The eluates were concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, previously equilibrated with buffer A. The appropriate fractions of the purified HCV proteins were combined and stored at -80 ° C.

Example 7 Enzymatic Investigation of the HCV NS3 Serine Protease Domain

A. Protocol for the study of the enzyme by HPLC

HPLC-Microbore method for separation of 5AB substrate and products

Substrate:

NH2-Glu-Asp-Val-Val- (alpha) Abu-Cys-Ser-Met-Ser-Tyr-COOH

A stock solution of 20 mM 5AB (or the concentration of your choice) was prepared in DMSO wt./0.2 M DTT. The solution was stored in aliquots at -20 ° C.

Buffer: 50 mM HEPES, pH 7.8; 20% glycerin; 100 mM NaCl

The total sample volume was 100 μl

X1 μl Study Concentration Buffer 86.5 see above 5 mM KK4A 0.5 25 μm 1M DTT 0.5 5 mm DMSO or inhibitor 2.5 2.5% v / v 50 μM tNS3 0.05 25 nM 250 μM 5AB (initiates) twenty 25 μm

Buffer, KK4A, DTT and tNS3 were mixed, each 78 μl was dispensed into the wells of a 96-well plate. The plate was incubated at 30 ° C for ~ 5-10 minutes.

2.5 μl of the appropriate concentration of the test compound was dissolved in DMSO (DMSO only for control) and added to each well. The mixture was incubated at room temperature for 15 minutes.

The reaction was initiated by adding 20 μl of 250 μM 5AB substrate (25 μM concentration is equivalent to or slightly lower than Km for 5AB). The reaction mixture was incubated for 20 minutes at 30 ° C, and then the reaction was completed by adding 25 μl of 10% TFA. Aliquots of 120 μl of the final product of the final reaction were transferred to HPLC ampoules. The SMSY product was separated from the substrate and KK4A in the following way:

Microbore-separation method:

Hardware: Agilent 1100

Apparatus for degassing G1322A

Double pump G1312A

Auto Dispenser G1313A

Thermostatic column chamber G1316A

G1315A Diode Array Detector

Speaker:

Phenomenex Jupiter; 5 microns C18; 300 angstroms; 150 × 2 mm; P / O OOF-4053-BO

Column type thermostat: 40 ° C

Injection volume: 100 μl

Solvent A = water, pure for HPLC, + 0.1% TFA

Solvent B = acetonitrile, pure for HPLC, + 0.1% TFA

Time (min) % B Speed (ml / min) Maximum pressure 0 5 0.2 400 12 60 0.2 400 13 one hundred 0.2 400 16 one hundred 0.2 400 17 5 0.2 400 Retention time: 17 min. Time after holding: 10 min.

B. Protocol research enzyme using FRET

Enzymatic activity was determined using a modification of the method described by Taliani et al., [Taliani et al., Anal. Biochem. 240, pp. 60-67 (1996)]. An internally quenched fluorogenic peptide (FRET substrate), Ac-DED (EDANS) EEαAbuψ [COO] ASK (DABCYL) -NH2, was purchased from AnaSpec Incorporated (San Jose, CA). The study was conducted in a continuous manner in the format of a 96-well microtiter plate. The buffer was composed of 50 mM HEPES (pH 7.8), 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 μM KK4A peptide (KKGSVVIVGRIVLSGK; SEQ ID NO: 5). The KK4A peptide is the central region of the NS4A cofactor of genotype 1a with lysine residues introduced to improve solubility [Landro et al. Biochemistry, 36, pp. 9340-9348 (1997)]. The reaction was initiated by the addition of a FRET substrate after 10 minutes of preincubation of the components of the buffer with 2 nM NS3 protease at room temperature. The reaction was constantly monitored at 30 ° C for 20 minutes using a Molecular Devices fmax fluorimetric plate reader. Filters for excitation and radiation wavelengths were 355 nm and 495 nm, respectively. To determine the kinetic parameters of the substrate, the concentration of the FRET peptide was varied from 0.5 to 0.7 μM. No intermolecular quenching was observed in this region. The kinetic parameters of the substrate, Km and Vmax, were determined by establishing data according to the Michaelis-Menten equation. Inhibition constants (Ki) were determined by titration of the enzyme activity using the method described above, except that the compound dissolved in DMSO (not more than 2% v / v DMSO, one solvent was used as a control) was added to the components of the buffer and the enzyme after the initial 10-minute pre-incubation, as described above. The resulting mixture was incubated for an additional 15 minutes at room temperature prior to incubation with a FRET substrate for the next 20 minutes at 30 ° C. Seven to nine concentrations of the compound were tested, and the results corresponded to the integrated form of the Morrison equation to inhibit strong binding [J.F. Morrison, Biochem. Biophys. Acta, 185 pp. 269-286 (1969)]. All data on the substrate and the inhibitor was established using non-linear regression Marquardt-Levenberg (Marquardt-Levenberg) with software GraphPad Prism.

Example 8: Obtaining resistance to VX-950 in replicon-containing HCV cells

VX-950 (Figure 4A, chemical structure) is a clinical candidate for a treatment for hepatitis C. VX-950 is a reversible, covalent HCV NS3 / 4A serine protease inhibitor. Although the inhibitor competes with the peptide substrate in the active center, it exhibits clearly non-competitive inhibition as a result of its tightly binding properties and time-dependent inhibition mechanism (C. Gates and YP. Luong). Incubation of subgenomic, replicon-containing Con1 HCV cells with VX-950 led to a concentration-dependent drop in HCV RNA level, which was determined by real-time PCR-RT (Figure 5B). The IC ₅₀ value for the VX-950 was 354 nM in a 48-hour study.

To identify VX-950 resistant mutations, subgenomic, replicon-containing Con1 cells were serially passaged (i.e., subcultures were obtained) in the presence of 0.25 mg per ml G418 and gradually increasing concentrations of VX-950 (series A) (Figure 5A, selection curve) . The initial concentration of VX-950 was 3.5 μM, or 10-fold IC ₅₀ , and the highest concentration was 28 μM, or 80 times higher than IC ₅₀ . Replicon-containing cells were separated or replenished every 3 or 4 days and fresh VX-950 was added. Since VX-950 inhibits the activity of HCV serine NS3 protease and therefore blocks the replication of HCV RNA, the equilibrium state of HCV proteins and neomycin transferase protein gradually decreased and, in the end, became undetectable in the presence of a high concentration of VX-950. Cells with low or no protein content of neomycin transferase proliferated at a gradually decreasing rate and, in the end, died in the presence of G418. Only HCV RNA with mutations that are resistant to VX-950 could replicate in the presence of a high concentration of VX-950 and support the growth of replicon-containing cells holding them. The replicon-containing cells in series A grew normally during the first 10 days in the presence of 3.5 μM VX-950. After 10 days, Series A cells grew much more slowly, and mass cell death was observed between 10 and 17 days (Figure 5A, selection curve). Normal growth did not resume until 21 days. It was found that the IC ₅₀ for VX-950 relative to series A replicon-containing cells ranged from 8.1 to 12.0 μM, which is 23-34 times higher than the IC ₅₀ (354 nM) for wild-type replicon-containing cells (Figure 5B, curve IC ₅₀ ).

Total cellular RNA from Series A cells was extracted on day 7, 21, and 56 and subjected to PCR-RT to amplify the coding region of the NS3 serine protease domain. The PCR-RT product was sequenced in bulk to identify the position (s) of possible mutations that may be responsible for the observed decrease in sensitivity to VX-950. The nucleotide and amino acid sequences of wild-type HCV protease from the original replicon-containing cells are shown in SEQ ID NO: 1 and SEQ ID NO: 1. No mutation associated with VX-950 was observed in the serine protease domain of NS3 replicon series A cells on day 7, when compared to wild-type replicon containing Con1 cells in the absence of VX-950.

On days 21 and 56 in series A, Ala156 substitutions were found in the protease domain, suggesting that mutations at residue 156 may be crucial for reduced sensitivity to VX-950. No mutation was detected in any of the four proteolytic sites in the region of the non-structural HCV protein, which is cleaved by the NS3 / 4A serine protease. To establish the dimensions of identity and frequency of substitutions, the PCR-OT product is 1.7 kb. On day 7 or 98, the replicon-containing cells of series A were subcloned into a TA vector and numerous clones were sequenced for both samples. All clones obtained from 7-day old samples contained wild-type Ala156. In a 98-day sample of Series A replicon-containing cells that were cultured in the presence of 28 μM VX-950 for 63 days, 79% or 60 of the 76 clones had alanine substitution for serine (A156S).

In addition, VX-950-resistant cells were selected at a constant concentration of VX-950 and G418. In this case, numerous colonies of resistant cells were detected after a long cultivation period with VX-950 and G418. NS3 serine protease sequences were determined from these resistant colonies and similar mutations were found in the amino acid 156 of HCV serine protease.

Example 9: Obtaining resistance to BILN 2061 in replicon-containing HCV cells

Another HCV NS3 / 4A protease inhibitor, BILN 2061 (Figure 4B, chemical structure) (WO 00/599929; US 6608027), has been shown to be effective in patients with hepatitis C (Lamarre et al., Nature Medicine, 2003). Replicon-containing HCV cells resistant to BILN 2061 (series B) were selected in the same manner as for VX-950. Again, subgenomic, replicon-containing wild-type HCV Con1 cells were serially passaged in the presence of 0.25 mg per ml of G418 and a gradually increasing concentration of BILN 2061 (Figure 6A, selection curve). Replicon-containing B cells grew normally during the first 7 days in the presence of 80 nM BILN 2061, or 80 times higher IC ₅₀ . However, after 7 days, the proliferation of B cells significantly slowed down, and there was a massive cell death between 7 and 17 days. As before, normal growth did not resume until 21 days. The IC ₅₀ value for BILN 2061 ranged from 1.0 to 1.8 μM against series B cells on day 59, which was 1000 to 1800 times higher than the IC ₅₀ (1 nM) for wild type replicon-containing cells (Figure 6B, IC ₅₀ curve).

No BILN 2061-related mutation was observed in the NS3 serine protease domain on day 7. By day 24, a series of substitutions were identified for amino acid 168 of NS3 protein, which suggests that substitutions at residue 168 may explain resistance to BILN 2061. No mutation was observed in four regions of the non-structural HCV protein region that was cleaved by the NS3 / 4A serine protease. To determine the frequency of substitutions by residue 168 NS3, the NS3 serine protease of replicon series B cells on day 98, which were cultured in the presence of 3.2 μM BILN 2061, was sequenced. 60 out of 94 clones or 64% had an Asp168 substitution for Val (D168V), and 23 clones or 24% had an Asp168 mutation for Ala (D168A).

In addition, BILN 2061-resistant cells were selected at a constant concentration of BILN 2061 and G418. In this case, numerous colonies of resistant cells were detected after an extended cultivation period with BILN 2061 and G418. HCV NS3 serine protease sequences were determined from these resistant colonies and similar mutations were found for the amino acid 168 HCV serine protease.

Example 10: Selection of replicon-containing cells resistant to both VX-950 and BILN 2061

A. Obtaining cross-resistant HCV replicons from VX-950-resistant cells

Several breeding schemes have been used to identify resistant mutations that are cross-resistant to both the VX-950 and BILN 2061. First, a line of replicon-containing cells resistant to VX-950 [series A of C. Lin et al., J. Biol. Chem. 279, pp. 17508-17514 (2004)], serially passaged in the presence of 0.25 mg / ml G418, 14 μM VX-950 and gradually increasing concentrations of BILN 2061 (series C) (Figure 8A). For BILN 2061, the initial concentration was 40 nM and the final concentration was 6.4 μM. Replicon-containing cells were separated or replenished every 3 or 4 days and fresh VX-950 and BILN 2061 were added. Since PIV HCV inhibits the activity of NS3 / 4A serine protease and therefore blocks the replication of HCV RNA, the equilibrium state of HCV proteins and neomycin protein β-transferase gradually decreased and, in the end, became undetectable in the presence of a high concentration of PI HCV (data not shown). Cells with a low protein content of neomycin transferase proliferated at a gradually decreasing rate and, in the end, died in the presence of G418. It is believed that replicon-containing cells with a major VX-950-resistant mutation, A156S, died in the presence of increasing concentrations of BILN 2061, as they were shown to be sensitive to inhibition of BILN 2061 [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)]. Only HCV RNA with mutations that are cross-resistant to both VX-950 and BILN 2061 could replicate in the presence of high concentrations of both HCV PIs and support the growth of the replicon-containing cells holding them. However, the replicon-containing cells in series C grew normally during the entire selection process, which lasted for 56 days. It was found that the IC ₅₀ for BILN 2061 in relation to series C replicon cells on day 52 was ~ 3 μM, which is 300 times higher than the IC ₅₀ for series A replicon cells (VX-950-resistant) (~ 10 nM ) (Figure 8B). Since VX-950, 30 μM, did not result in a more than 50% decrease in HCV RNA in replicon C series cells by day 52, the exact IC ₅₀ values could not be determined, indicating that replicon C series cells were 52 day remained resistant to VX-950 (Figure 8C).

Total cell RNA from Series C cells on day 32, which were cultured in the presence of 14 μM VX-950 and 0.32 μM BILN 2061, was extracted and subjected to PCR-RT to amplify the coding region of the NS3 serine protease domain. The PCR-RT product was sequenced in bulk to identify the position (s) of possible mutations that may be responsible for the observed decrease in sensitivity to both PIs of HCV. In addition, Ala156 substitutions were found in the protease domain, suggesting that mutations at residue 156 may be crucial for reduced sensitivity to both PIs. These data are somewhat unexpected, as it is established that the main VX-950-resistant mutation 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-structural HCV protein region that are cleaved by the NS3 / 4A serine protease. In order to establish the identity and frequency of substitutions, the PCR-RT product was 1.7 kb. On day 32, replicon-containing C cells were subcloned into a TA vector and 10 individual colonies were sequenced. 6 clones had Ala156 substitution for Thr (A156T), and 3 clones had Ala156 substitution for Val (A156V). The 10th clone retained the A156S mutation.

B. Obtaining cross-resistant HCV replicons from BILN 2061-resistant cells

A second selection scheme included growing BILN 2061-resistant, replicon-containing cells in the presence of both BILN 2061 and VX-950. In this case, a line of replicon-containing cells resistant to BILN 2061 [series B in C. Lin et al., J. Biol. Chem. 279, pp. 17508-17514 (2004)], serially passaged in the presence of 0.25 mg / ml G418 and gradually increasing concentrations of VX-950 and BILN 2061 (series D) (Figure 9A). For 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. Replicon-containing cells with major BILN 2061-resistant mutations, D168V or D168A, were thought to die in the presence of high concentrations of VX-950, as they were shown to be sensitive to inhibition of VX-950 [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)]. In addition, only HCV RNA with mutations that are cross-resistant to both VX-950 and BILN 2061 could replicate in the presence of high concentrations of both HCV PIs and support the growth of the replicon-containing cells holding them. However, D-series replicon-containing cells grew normally during most of the selection process, which also continued for 56 days. Since 30 μM VX-950 did not result in a more than 50% reduction in HCV RNA in replicon-containing D-series cells by day 52, the exact IC ₅₀ values for VX-950 could not be determined, but they should be at least more than 100 times higher than IC ₅₀ (~ 0.3 μM) against replicon-containing B-series cells (BILN 2061-resistant) (Figure 9B). It was found that the IC ₅₀ values for BILN 2061 relative to D series replicon cells by day 52 were ~ 4 μM, indicating that D series replicon cells by day 52 remained resistant to BILN 2061 (Figure 9C).

Total cell RNA from D series cells on day 32, which were also cultured in the presence of 14 μM VX-950 and 0.32 μM BILN 2061, was extracted and subjected to PCR-RT to amplify the coding region of the NS3 serine protease domain. The PCR-RT product was sequenced in bulk to identify the position (s) of possible mutations that may be responsible for the observed decrease in sensitivity to both PIs of HCV. In addition, Ala156 substitutions were established in the protease domain, which confirms that mutations at residue 156 may be crucial for reduced sensitivity to both PIs. No mutation was found in any of the four proteolytic sites in the non-structural HCV protein region that are cleaved by the NS3 / 4A serine protease. In order to establish the identity and frequency of substitutions, the PCR-RT product was 1.7 kb. on day 32, replicon-containing cells of series A were subcloned into a TA vector and 14 individual colonies were sequenced. 12 clones had the A156V substitution, and 1 clone had the A156T mutation. Clone 14 had two mutations, A156S and D168V.

C. Obtaining cross-resistant HCV replicons from naive replicon cells

In preliminary studies of mutations that are resistant to one PI HCV, or VX-950, or BILN 2061, cell growth slowed down for several days, during which mass cell death was observed [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)], which indicates the appearance of resistant mutant replicon-containing cells and the concomitant death of non-resistant replicons. However, no such cell death or deceleration of cell growth was observed in the selection of the cross-resistant replicons of series C or D described above. It is possible that cross-resistant mutations, A156T and A156V, can already be in VX-950-resistant (series A) or BILN 2061-resistant (series B) cells containing replicon in the form of a minor population. If so, the two selection schemes described may provide a tendency to mutate A156T or A156V within other potential cross-resistant mutations. Thus, the third selection scheme can be carried out using naive replicon-containing HCV cells that are sensitive to any inhibitor.

Subgenomic, replicon-containing Con1 cells derived from pBR322-HCV-Neo-mADE [C. Lin et al. J. Biol. Chem. 279, pp. 17508-17514 (2004)], serially passaged in the presence of 0.25 mg / ml G418 and gradually increasing concentrations of VX-950 and BILN 2061 (series E) (Figure 10). The initial concentration of VX-950 was 3.5 μM, and the highest concentration was 14 μM. For BILN 2061, the initial concentration was 80 nM and the final concentration was 3.2 μM. The replicon-containing cells were separated or replenished every 3 or 4 days and fresh VX-950 and BILN 2061 were added. The E-series replicon cells grew normally during the first 10 days in the presence of 3.5 μM VX-950 and 160 nM BILN 2061. After For 10 days, the E-series cells grew much more slowly, and mass cell death was observed between 10 and 21 days (Figure 10). Normal growth did not recover until 21 days. Total cell RNA from C series cells was extracted on days 10, 21, and 48 and subjected to PCR-RT to amplify the coding region of the NS3 serine protease domain. No mutation associated with PI HCV was observed in the NS3 serine protease domain of the E series replicon cells on day 10 when they were compared with replicon containing Con1 cells cultured in the absence of both HCV PIs. In order to establish the identity and frequency of substitutions, the PCR-RT product was 1.7 kb. E series replicon-containing cells on day 21 or day 48 were subcloned into a TA vector and numerous clones were sequenced for both samples. In a 21-day sample of replicon-containing E-series cells that were cultured in the presence of 3.5 μM VX-950 and 0.32 μM BILN 2061 for 14 days, 65% or 30 of the 46 clones had Ala156 substitution on Thr (A156T), whereas another substitution of Ala156 with Val (A156V) was found in 35% or 16 of 46 clones. As for the 48-day sample of E-series cells that were cultured in the presence of 14 μM VX-950 and 1.6 μM BILN 2061 for 14 days, 80% or 35 of 44 clones had A156T substitution, while A156V substitution was found in 20% or in 9 out of 44 clones. In any case, no other mutations were found in the NS3 serine protease domain in more than 10% TA plasmid clones, demonstrating that A156T and A156V are the only mutations that include cross-resistance to both VX-950 and BILN 2061.

Example 11: Demonstration and confirmation of resistant mutations of amino acid 156 or 168 in an enzymatic study of replicon cells

To confirm whether the observed mutations in either Ala156 or Asp168 are sufficient to induce resistance to VX-950 or BILN 2061, respectively, a site-directed mutagenesis method was used to introduce each individual mutation at position 156 or 168 into the wild-type NS3 protease domain type.

Site-directed mutagenesis is another method suitable for producing mutant protease proteins used in the methods of the invention. In this method, specific mutagenesis of the main DNA is used (which encodes the amino acid sequence that is targeted for modification). The method also provides the ability to quickly obtain and test sequence variants, taking into account one or more of the above considerations, by introducing one or more changes in the nucleotide sequence in the DNA. Site-directed mutagenesis allows mutants to be obtained by using specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence variability to form a stable duplex on both sides of the compound of the deletion. A primer of approximately 17 to 25 nucleotides with a length of approximately 5-10 residues on both sides of the variable sequence compound is generally preferred.

The method usually uses a bacteriophage vector, which exists in both single-stranded and double-stranded forms. Typical vectors suitable for site-directed mutagenesis include vectors such as M13 phage. Said phage vectors are commercially available, and their use is usually well known to those skilled in the art. Double-stranded plasmids are also commonly used in the site-directed mutagenesis method, which eliminates the step of transferring the gene of interest from the phage to the plasmid.

In general, site-directed mutagenesis was first carried out by obtaining a single-stranded vector or by melting two strands of a double-stranded vector, which enclosed in its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer synthesizing the desired mutant sequence was synthesized. The resulting primer was then annealed to obtain single-stranded DNA, taking into account the degree of mismatch when choosing hybridization conditions (annealing), and was subjected to the action of enzymes that polymerize DNA, such as the maple fragment of E. coli polymerase I, in order to complete the synthesis of the chain carrying the mutation. Thus, a heteroduplex was obtained in which one chain encodes the original unmuted sequence, and the second chain has the desired mutation. The resulting heteroduplex vector was then used to transform suitable cells, such as E. coli cells, and clones that included recombinant vectors carrying the mutant sequence structure were selected.

Of course, the site-directed mutagenesis approach described above is not the only way to obtain potentially suitable types of mutant protease and, in essence, is not limited to it. Other methods for effecting mutagenesis have also been contemplated by the present invention, for example, such as processing recombinant vectors carrying a gene of mutagenic agents of interest, such as hydroxylamine, to obtain sequence variants.

A. The dominant VX-950-resistant mutant, A156S, remains sensitive to BILN 2061

To confirm whether the observed Ala156 substitution by Ser is sufficient to achieve resistance to VX-950 rather than BILN 2061, site directed mutagenesis was used to replace Ala156 by Ser in the wild-type NS3 protease domain.

The kinetic parameters of the FRET substrate for the wild-type NS3 protease domains from genotypes 1a and 1b were identical (table 1) under the used research conditions. Although the NS4A peptide cofactor was from HCV genotype 1a, no recognizable differences in kinetic parameters were observed. This is consistent with molecular modeling, suggesting that conservative variations in the central NS4A region between genotypes 1a and 1b do not affect the interaction between the NS4A core peptide and the NS3 protease domain. Ki values for VX-950 and BILN 2061 were determined using wild-type proteases of genotypes 1a and 1b, and no statistically significant differences were found between the two wild-type proteases (Table 1).

The kinetic parameters of the FRET substrate for the mutant A156S protease were essentially the same as the parameters of the wild-type protease (Table 1). However, the Ki value for VX-950 was 2.9 μM for the mutant A156S protease, which is 29 times higher than the Ki value for the wild-type protease (0.1 μM) (Table 2). The Ki value for BILN 2061 was 112 nM for the A156S mutant, which was 6 times higher than the Ki value against wild-type protease, 19 nM (Table 2).

The level of HCV RNA in replicon-containing cells having A156S substitution was similar to the level of RNA of wild-type replicon cells (data not shown), which is consistent with the similar enzymatic catalytic efficiency of the A156S mutant and wild-type NS3 serine protease. The IC ₅₀ value for VX-950 for replicon-containing A156S cells was 4.65 μM, which is 12 times higher than the IC ₅₀ value for wild-type replicon cells (0.40 μM) (Table 3). The differences in IC ₅₀ values for BILN 2061 in relation to A156S (7 nM) and replicon-containing wild-type cells (4 nM) were not significant (Table 3).

B. The major BILN 2061-resistant mutants, D168V and D168A, remain completely sensitive to the VX-950

To confirm whether the observed replacement of Asp168 by Val or Ala is sufficient to achieve resistance to BILN 2061 rather than VX-950, site-directed mutagenesis was used to replace Asp168 by Val or Ala in the wild-type NS3 protease domain.

The kinetic parameters of the substrate were not affected by the D168V mutation, and only minor changes (less than 10 times) for the D168A mutant were established, which was established by comparing the kcat and kcat / Km values for the wild-type protease and two mutant NS3 serine proteases (Table 1 ) Similarly, no significant effect of any substitution according to Asp168 on the Ki VX-950 value was noted (Table 2). However, the substitution of valine or alanine with aspartic acid at position 168 resulted in a mutant NS3 protease that was not inhibited at BILN 2061 concentrations up to 1.2 μM (Table 2). The data obtained indicate that any mutant protease is at least 63 times less sensitive to BILN 2061 compared to the wild-type protease. The exact value of the resistance cannot be established, since BILN 2061 did not dissolve at concentrations above 1.2 μM in the study buffer, which was determined by the optical density at 650 nm. The mutation D168V or D168A was also introduced into the wild-type HCV replicon by site directed mutagenesis and a stable line of replicon-containing cells bearing any substitution was obtained. The IC ₅₀ value for BILN 2061 was 5.09 μM relative to replicon-containing D168V cells, which is more than 1300 times higher than relative to wild-type replicon-containing cells (4 nM) (Table 3). The IC ₅₀ value for BILN 2061 was 1.86 μM relative to the mutant replicon D168A. There was a slight difference in the IC ₅₀ values for VX-950 with respect to D168V and replicon-containing wild-type cells (Table 3).

Table 1 summarizes the enzymatic properties of the wild-type serine protease or the mutant NS3 HCV serine protease domains. Five domain proteins of HCV NS3 serine proteases, including wild-type (wt) proteases of genotype 1a and 1b and three mutants (A156S, D168V and D168A) of genotype 1b, were expressed and purified as described in the Materials and Methods section. The kcat and Km values for these proteases were determined using the KK-NS4A core peptide and FRET substrate.

Table 1
Enzymatic characterization of wild-type or mutant serine proteases NS3 HCV Protease HCV Km (μm) Katk ( ^sec-1 ) Katk / Km
(M ^-1 sec ^-1 ) Wild type (1a) 1.3 1,2 8.97 × 10 ⁵ Wild type (1b) 1,1 1,0 9.47 × 10 ⁵ A156S (1b) 0.9 0.6 6.64 × 10 ⁵ D168V (1b) 1,2 0.6 4.98 × 10 ⁵ D168A (1b) 2.0 0.3 1.50 × 10 ⁵

Table 2 presents confirmation of resistance in an enzymatic study. Ki values for VX-950 and BILN 2061 were determined for five purified domains of HCV NS3 serine proteases, including wild-type (wt) proteases from genotype 1a or 1b, as well as three mutants (A156S, D168V and D168A) of genotype 1b, using KK-NS4A peptide and FRET substrate. The solubility of BILN 2061 in the reaction buffer was limited at concentrations above 1.2 μM. No inhibition of the mutant NS3 protease was observed, neither D168V nor D168A, in the presence of 1.2 μM BILN 2061.

table 2
Ki values for VX-950 and BILN 2061 for wild-type and mutant NS3 proteases Ki (μm) Mutant BILN 2061 VX-950 Wild type (1a) 0.006 0,047 Wild type (1b) 0.019 0,100 A156S 0,112 2.9 D168V > 1.2 0,043 D168A > 1.2 0.150

Table 3 shows the confirmation of resistance in HCV replicons. Four subgenomic stable lines of replicon-containing HCV cells including wild type (wt) and three mutants, A156S, D168V and D168A, were obtained using T7 RNA transcripts after reading from the corresponding highly efficient replicon-containing Con1 plasmids. IC ₅₀ values for VX-950 and BILN were determined against four lines of replicon-containing HCV cells in a standard 48-hour study.

Table 3
IC ₅₀ values for VX-950 and BILN 2061 for replicon-containing HCV cells with wild-type NS3 protease and mutant NS3 proteases Replicon IC ₅₀ (μM) Mutant BILN 2061 VX-950 Wild type 0.004 0.402 A156S 0.007 5.09 D168V 4.65 0.163 D168A 1.86 0.193

C. Mutations A156T and A156V are cross-resistant to both VX-950 and BILN 2061

To confirm whether mutations in Ala156 are sufficient to achieve cross-resistance to both VX-950 and BILN 2061, site-directed mutagenesis was used to replace Ala156 or Val or Thr in the wild-type NS3 protease domain.

The catalytic efficiency (kcat / Km) of the A156T or A156V mutant protease with respect to the FRET substrate was approximately 5-7 times lower than the catalytic efficiency of the wild-type protease (Table 4). The Ki value for VX-950 was 9.9 μM or 33 μM for the mutant protease A156T or A156V, respectively, which is 99 or 330 times higher than the Ki value for wild-type protease (0.1 μM), respectively (Table 5) . No mutant protease was inhibited at a BILN concentration of 2061 up to 1.2 μM (Table 5). The data obtained indicate that each mutant protease was at least 63 times less sensitive to BILN 2061 when compared with wild-type protease. The exact value of the resistance cannot be determined, since BILN 2061 did not dissolve at concentrations above 1.2 μM in the study buffer, which was determined by the optical density at 650 nm (data not shown).

Table 4
Enzymatic properties of wild-type or mutant domains of HCV NS3 serine proteases Protease HCV Km (μm) Kkat (sec ^-1 ) Kkat / Km (M ^-1 sec ^-1 ) Wild type 1,1 1,0 9.47 × 10 ⁵ A156T 1.3 0.2 1.36 × 10 ⁵ A156V 2.6 0.6 2.34 × 10 ⁵

Table 4, summary: Three domain proteins of serine proteases NS3 of HCV Con1 strain Con1, including wild-type proteases and two mutants, A156T and A156V, were expressed and purified. The kcat and Km values of these proteases were determined using the KK-NS4A core peptide and FRET substrate, and the average values of two independent studies were presented.

Table 5
Confirmation of resistance in an enzymatic study Mutant Ki (μm) BILN 2061 VX-950 Wild type 0.019 0,100 A156T _{(n = 2)} > 1.2 9.9 A156V _{(n = 1)} > 1.2 33

Table 5, summary: Ki values for VX-950 and BILN 2061 were determined for five purified domains of HCV NS3 serine proteases, including wild-type protease, as well as two mutants, A156T and A156V, using KK-NS4A peptide and FRET substrate. The solubility of BILN 2061 in the reaction buffer is limited at concentrations above 1.2 μM. No inhibition of any mutant protease NS3, A156T or A156V was observed in the presence of 1.2 μM BILN 2061.

The level of HCV RNA in replicon-containing cells having A156T or A156V substitution was lower than the level of RNA containing wild-type replicon cells (data not shown), which is consistent with the lower enzymatic catalytic efficiency of two mutants compared to the enzymatic catalytic efficiency of NS3 serine proteases wild type. No decrease in HCV replicon RNA at a concentration of VX-950 up to 30 μM was observed in any line of mutant cells containing replicon, which indicates at least a 75-fold decrease in sensitivity that occurs with any mutation (Table 6). The IC ₅₀ value for BILN 2061 for any replicon-containing A156T cells was 1.09 μM, which is about 272 times higher than the IC ₅₀ for wild-type replicon cells (4 nM). For mutant replicons, A156V BILN 2061 was characterized by an IC ₅₀ value of 5.76 μM, indicating a more than 1400-fold decrease in sensitivity achieved with the A156V mutation (Table 6).

Table 6
Confirmation of resistance in HCV replicons Mutant Replicon IC ₅₀ (μM) BILN 2061 VX-950 Wild type 0.004 0.402 A156T 1.09 > 30 A156V 5.76 > 30

Table 6, summary: three subgenomic stable cell lines containing replicon, HCV, including the wild type and two mutants, A156T and A156V, were obtained using T7 RNA transcripts after reading from the corresponding highly efficient replicon-containing Con1 plasmids. IC ₅₀ values for VX-950 and BILN 2061 were determined for three lines of replicon-containing HCV cells in a standard 48-hour study and presented averages of two independent studies.

Example 12: Modeling - I

VX-950 and BILN 2061 were modeled in the active center of the NS3 serine protease domain using the full length HCV NS3 protein structure published by Yao et al. [Yao., Et al., Structure Fold DES., 7, pp. 1353-1363 (1999)] (sod PDB: 1CU1). The coordinates of the protease domain of segment A in this structure showed that the C-terminal strand of the NS3 protein binds in the substrate-binding portion of the protease. The terminal carboxyl group of this filament is located near the active center residues His57, Asp81 and Ser139 so that it forms hydrogen bonds with the side chains of His57 and Ser139, as well as with the amides of the backbone of residues 137 and 139, which form an oxyanion hole (“hole”). Additionally, the last six residues (from 626 to 631) of the NS3 protein form an extended antiparallel β-strand along the edge of the E2 strand of the protease β-rod and create twelve backbone-backbone hydrogen bonds. A product-based inhibitor like BILN 2061 is believed to bind the NS3 protease in a similar manner. Therefore, the described crystal structure was used to create a model of inhibitor protease co-complexes. The BILN 2061 molecule was inserted into QUANTA molecular modeling software (Accelrys Inc., San Diego, California, USA) and manually docked to the active site so that its carboxyl group was coated with the C-terminal NS3 carboxylate of the full-length NS3 protein. Then, the inhibitor molecule was rotated so that it created all of the following hydrogen bonds of the backbone: NH PI with Arg155 carbonyl, P3 carbonyl with NH Ala157 and NH P3 with Ala157 carbonyl. The binding method under consideration brought a large group of P2 BILN 2061 into direct collision with the Arg155 side chain. To avoid a collision, the Arg155 side chain was modeled into an extended conformation, which was prompted by a description of the crystal structure of the NS3 protease complex with an inhibitor, which is analogous to BILN 2061 [Y.S. Tsantrizos, Angew. Chem. Int. Ed. Engl. 42, pp. 1356-1360 (2003)]. The energy of the inhibitor was minimized in two stages. In the first stage, only the inhibitor and side chain atoms of Arg155, Asp168, and Arg123 proteases were allowed to move during energy minimization during 1000 operations. In the second stage, all atoms of the side chains of the active center were allowed to move along the inhibitor during 1000 additional operations. The resulting model structure closely mimics the published structure of the analogue of BILN 2061 [Y.S. Tsantrizos, Angew. Chem. Int. Ed. Engl. 42, pp. 1356-1360 (2003)].

A similar procedure was adapted for modeling VX-950 in the active center of the protease. The inhibitor was modeled as a covalent adduct with si-surface attachment of the Ser139 side chain to the ketocarbonyl inhibitor. Said binding method has been described for similar ketoamide inhibitors [Perni et al., Bioorg. Med. Chem. Lett. 14, in press (2004)] and ketoacid inhibitors [Di Marco et al., J. Biol. Chem., 275, pp. 7152-7257 (2000)]. The main chain of the inhibitor was covered with residues 626-631 of the C-terminal strand of NS3 so that it formed all of the following hydrogen bonds of the backbone: NH P1 with Arg155 carbonyl, carbonyl P3 with NH Ala157, NH P3 with Ala157 carbonyl and P4 covering carbonyl with NH Cys159. In the described binding method, the P2 group of the VX-950 was placed in a P2 pocket without any need to move the Arg155 side chain. The tert-butyl and cyclohexyl groups were placed in pockets S3 and S4, respectively. For matching, the same two-step energy minimization protocol used for the BILN 2061 model was used. The two co-complex models described were used to predict the effect of Ala156 and Asp168 mutations on the binding of protease inhibitors.

The Asp168 side chain was exposed to solvent. The side chain of valine mutant D168V can take three canonical conformations with χ ₁ = 60 °, -60 ° or 180 °. All three Val168 side chain orientations were modeled. The interaction energy of the mutant enzyme D168V and the inhibitor was minimized, allowing the inhibitor and Val168 atoms to move while fixing the positions of all other atoms of the protein molecule. In all cases, the Val168 side chain did not cause any steric collision with the atoms of the inhibitor. Ala156 serine mutation was modeled as follows. The Ala156 side chain is in van der Waals contact with the P2 group of both inhibitors (Figure 9). The side chain of the serine of the A156S mutant was modeled in three canonical conformations with χ ₁ = 60 °, -60 °, or 180 °, and the energy was minimized by keeping the resting conformation of the fixed protein. These models were used to study the effect of this mutation on inhibitor binding. The -60 ° conformation was found to have the lowest energy when it forms a hydrogen bond with the neighboring Arg155 carbonyl, but it causes the maximum number of adverse contacts with both inhibitors. The 60 ° and 180 ° conformations are energetically equivalent, but the 60 ° conformation is characterized by fewer adverse contacts and was used in research.

Ala156 was located on the E2 strand in the NS3 protease structure ^. 4A HCV [RA Love et al., Cell 87, pp. 331-342 (1996)]. Several atoms of the core of the aforementioned filament (mainly Arg155 carbonyl and both nitrogen of the main chain and carbon Ala157) formed hydrogen bonds with the atoms of the backbone of substrates or substrate-like inhibitors. In the structural model of the invention of the VX-950: NS3 co-complex (Figure 9), three hydrogen bonds are formed between NH P1 and Arg155 carbonyl, P3 carbonyl and NH Ala157 and NH P3 and Ala157 carbonyl. Some hydrogen bonds were also formed in the BILN 2061 co-complex model. The Ala156 side chain is in van der Waals contact with the P2 group of the mentioned inhibitors. In the mutant model A156S of the invention, the terminal oxygen of Ser156 is too close to the cyclohexyl group P4 of VX-950 and it is also close to the group of terminal cyclopentyl BILN 2061. Since the cyclopentyl group BILN 2061 is at the flexible end of the inhibitor, it can be moved away from this unfavorable contact without losing most of the binding. A similar movement of the cyclohexyl P4 group of VX-950 causes destabilization of the interactions between the inhibitor and the S4 and S5 protease subsites. Therefore, a greater loss of binding was suggested for VX-950 than for BILN 2061, with the mutant protease A156S.

Asp168 was located in the F2 strand of the NS3 protease structure and involved in the salt-bridging interactions with the side chains of Arg123 and Arg155 (Figure 9) [R.A. Love et al., Cell 87, pp. 331-342 (1996)]. It is also part of the S4 binding pocket. The aliphatic part of this side chain is in van der Waals contact with the terminal group of cyclopentyl BILN 2061, which was not believed to be affected by the D168V mutation, since the valine side chain does not cause any steric collision with the inhibitor at this position. However, the aforementioned D168V substitution results in a loss of the salt-bridging interaction with the Arg155 side chain on the adjacent E-2 strand (Figure 1), which creates numerous contacts with the large BILN 2061 group P2 in the model described here. The Arg155 conformation (Figure 9, blue) on the model of the BILN 2061 complex: wild-type NS3 protease is not energetically more favorable in the D168V mutant for two reasons. First, it is impossible to stay close to the skeletons of the E-2 thread in the absence of interaction between Arg155 and Asp168. The second, uncompensated and solvent-exposed positive charge of the Arg155 side chain will tend to a more solvate shell, as was observed in the crystalline structures of apo-protease and two protease complexes: an inhibitor, which are available in Protein Data Bank (code: 1DY8 and 1DY9) [S. Di Marco et al., J. Biol. Chem., 275, pp. 7152-7157 (2000)]. The described conformation of Arg155 is in direct collision with the quinoline group P2 of BILN 2061 and destabilizes its binding. Therefore, substitution of Asp168 with any amino acid except glutamine will disrupt the salt-bridge interaction with Arg155 and lead to a decrease in BILN 2061 binding. On the other hand, the Arg155 conformation in two published crystal structures of the NS3 protease complex: the inhibitor is similar to the conformation in the model of the invention of the VX-950 complex : protease (orange in Figure 9). In addition, the said Arg155 conformation stabilizes the binding of VX-950, since it allows the maximum number of van der Waals contacts between the Arg155 side chain and the inhibitor. Therefore, it is believed that the VX-950 is not affected by substitution according to Asp168 compared to BILN 2061.

Example 13: Modeling - II

Modeling VX-950 and BILN 2061 in the active center of the NS3 serine protease domain using the crystal structure of HCV NS3 full-length protein [N. Yao et al., Structure Fold Des. 7, pp. 1353-1363 (1999)] (Protein Data Base code: 1CU1)) has been described previously [C. Lin et al., J. Biol. Chem. 279, 17508-17514 (2004)]. The Ala156 side chain on the β-strand E2 of HCV NS3 / 4A protease separates the pockets S4 and S2 of the active site of the enzyme and is in van der Waals contact with the P2 group of the two inhibitors (Figure 7). The Val or Thr substitution of Ala156 extends to the side chain with two additional (methyl or hydroxyl) groups in a compact space between the wild-type enzyme and the inhibitors. The side chain Val156 or Thr156 was modeled in all three possible canonical configurations with χ ₁ = 60 °, -60 ° or 180 °, following the procedure outlined earlier for modeling the A156S mutation [C. Lin et al., J. Biol. Chem. 279, 17508-17514 (2004)]. For side chain conformations, energy was minimized by holding the resting conformation of a fixed protein. Inhibitors, VX-950 and BILN 2061, docked at the active centers of these mutant enzymes to determine the effect of mutations on the binding of inhibitors.

Of the three possible conformations of the Ser side chain at position 156 (Figure 11), the conformation χ ₁ = 60 ° had the least number of adverse contacts with VX-950 and BILN 2061. The other two conformations (with χ ₁ = 180 ° and -60 °) numerous adverse contacts with both inhibitors in either the P2 side chain or the P3 carbonyl group. In the A156T or A156V mutation, an additional group at the Cβ atom of the side chain was forced to occupy one of the two positions under consideration with χ ₁ = 180 ° and -60 °, which created adverse interactions with inhibitors. Three possible Thr conformations are shown schematically in Figure 11. In all cases, when an additional group interacts with the atoms of the core of the inhibitor and / or enzyme, repulsion occurs. When minimizing energy, it was found that the –60 / 180 ° conformation is characterized by the least repulsion during the interaction, and the main reason for the repulsion is a close collision between the terminal methyl or hydroxyl group of the mutant side chain and the carbonyl group of P3 inhibitors. Therefore, mutations A156T and A156V are resistant to both inhibitors.

All of the compositions and / or methods discussed and claimed in the description, can be made and implemented without undue experimentation in the light of this discovery. Although the compositions and methods of the present invention are described (in the form of preferred embodiments, those skilled in the art will appreciate that changes can be made to the compositions and / or methods and to the step or sequence of steps of the method described in the description without departing from the concept, idea and More specifically, it will be apparent that certain agents that are related both chemically and physiologically may be replaced by the agents described in the specification, if the same or similar effects are achieved. the results. It is believed that all such similar substitutions and modifications apparent to those skilled in the art, correspond ideas and scope of the inventive concept as defined by the appended claims.

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Claims (71)

1. An isolated HCV polynucleotide containing a nucleotide sequence encoding an HCV NS3 / 4A protease, or a biologically active fragment thereof, in which a codon that corresponds to codon 156 of a wild-type polynucleotide is mutated so that it does not encode alanine, and optionally, a codon, which corresponds to codon 168 of the wild-type polynucleotide, is mutated so that it does not encode aspartic acid. 2. The selected HCV polynucleotide according to claim 1, wherein the polynucleotide encodes an HCV NS3 / 4A protease, or a biologically active fragment thereof, that are resistant to at least one protease inhibitor. 3. The selected HCV polynucleotide according to claim 1, wherein the codon of said polynucleotide, which corresponds to codon 156 of the wild-type polynucleotide, encodes a series. 4. The selected HCV polynucleotide according to claim 1, wherein the codon of said polynucleotide, which corresponds to codon 156 of the wild-type polynucleotide, encodes a valine. 5. The selected HCV polynucleotide according to claim 1, wherein the codon of said polynucleotide, which corresponds to codon 156 of the wild-type polynucleotide, encodes threonine. 6. The selected HCV polynucleotide according to claim 1, wherein the codon that matches the codon 168 of the wild-type polynucleotide is mutated so that it does not encode aspartic acid. 7. The selected HCV polynucleotide according to claim 1, wherein the code for said polynucleotide, which corresponds to codon 156 of wild-type polynucleotide, encodes valine or threonine, and the codon of said polynucleotide, which corresponds to codon 168 of wild-type polynucleotide, encodes aspartic acid or glutamic acid. 8. The selected HCV polynucleotide according to claim 6, wherein the codon of said polynucleotide, which corresponds to codon 168 of the wild-type polynucleotide, encodes a valine. 9. The selected HCV polynucleotide according to claim 6, in which the codon 168 of the polynucleotide encodes alanine, glycine or tyrosine. 10. The selected HCV polynucleotide according to claim 1, where the polynucleotide contains the sequence of SEQ ID NO: 1, provided that codon 156 of SEQ ID NO: 1 is mutated so that it does not encode alanine, and, optionally, codon 168 of SEQ ID NO : 1 is mutated in such a way that it does not encode aspartic acid. 11. An isolated polynucleotide containing a nucleotide sequence that encodes a biologically active fragment of HCV NS3 / 4A protease, in which a codon that encodes amino acid 156 of the wild-type HCV NS3 / 4A protease is mutated so that it does not encode alanine, and optionally, a codon which encodes the amino acid 168 of the wild-type HCV NS3 / 4A protease, is mutated so that it does not encode aspartic acid. 12. The selected polynucleotide according to claim 11, wherein the polynucleotide encodes a biologically active fragment of HCV NS3 / 4A protease that is resistant to at least one protease inhibitor. 13. The selected polynucleotide according to claim 11, in which codon 156 encodes the remainder of valine, threonine or serine. 14. The isolated polynucleotide of claim 11, wherein codon 156 encodes valine or threonine. 15. The selected polynucleotide according to claim 11, wherein the codon that corresponds to the codon 168 of the wild-type polynucleotide is mutated so that it does not encode aspartic acid. 16. The selected polynucleotide according to claim 15, wherein the polynucleotide comprises a codon that corresponds to codon 168 of the HCV NS3 / 4A protease domain, in which codon 168 encodes valine. 17. The selected polynucleotide according to claim 15, wherein the polynucleotide comprises a codon that corresponds to codon 168 of the HCV NS3 / 4A protease domain, where codon 168 encodes alanine, glycine or tyrosine. 18. The selected HCV polynucleotide according to claim 11, wherein the polynucleotide contains the sequence of SEQ ID NO: 1, provided that codon 156 of SEQ ID NO: 1 is mutated so that it does not encode alanine, and, optionally, codon 168 of SEQ ID NO: 1 is mutated in such a way that it does not code for aspartic acid. 19. An isolated HCV NS3 / 4A protease protein or biologically active fragment thereof comprising a sequence in which the amino acid residue that corresponds to amino acid 156 of the wild-type HCV NS3 / 4A protease is not an alanine residue, and, optionally, the amino acid residue that corresponds to the amino acid 168 wild-type HCV NS3 / 4A protease, is not aspartic acid. 20. The isolated HCV NS3 / 4A protease protein or its biologically active fragment according to claim 19, wherein the HCV NS3 / 4A protease protein or its biologically active fragment is resistant to at least one protease inhibitor. 21. The isolated HCV NS3 / 4A protease protein or fragment thereof according to claim 19, wherein the amino acid that corresponds to wild-type protease amino acid 156 is valine, threonine or serine. 22. The isolated HCV NS3 / 4A protease protein or fragment thereof according to claim 19, comprising an amino acid sequence in which the amino acid corresponding to amino acid 168 of the wild-type HCV NS3 / 4A protease is not aspartic acid. 23. The isolated HCV NS3 / 4A protease protein or fragment of claim 19, wherein the amino acid that corresponds to protease amino acid 156 is serine, valine or threonine, and amino acid 168 is aspartic acid or glutamic acid. 24. The isolated HCV NS3 / 4A protease protein or fragment thereof according to claim 22, wherein the amino acid that corresponds to amino acid 168 of the wild-type protease is valine. 25. The isolated HCV NS3 / 4A protease protein or fragment thereof according to claim 22, wherein the amino acid that corresponds to amino acid 168 of the wild-type protease is alanine, glycine or tyrosine. 26. The selected HCV NS3 / 4A protease protein according to claim 19, wherein the HCV NS3 / 4A protease protein contains the sequence SEQ ID NO: 2, provided that amino acid 156 of SEQ ID NO: 2 is not alanine, and optionally amino acid 168 SEQ ID NO: 2 is not aspartic acid. 27. An expression vector containing a polynucleotide according to any one of claims 1 to 18. 28. A host cell containing a polynucleotide according to any one of claims 1 to 18, to obtain a protein of HCV NS3 / 4A protease or its fragment encoded by the specified polynucleotide. 29. The cell line containing the polynucleotide according to any one of claims 1 to 18, to obtain a protein of the HCV NS3 / 4A protease or its fragment encoded by the specified polynucleotide. 30. A cell line transformed or transfected with a vector according to claim 27, to obtain a HCV NS3 / 4A protease protein or a fragment thereof encoded by a vector polynucleotide. 31. The host cell, transformed or transfected with the vector according to item 27, to obtain the protein of the HCV NS3 / 4A protease or its fragment encoded by the polynucleotide of the vector. 32. The selected HCV mutant containing the polynucleotide according to any one of claims 1 to 18. 33. The selected HCV mutant containing the protein according to any one of claims 19-26. 34. Composition for detecting a drug against HCV and / or for monitoring appropriate HCV therapies, which contains a polynucleotide according to any one of claims 1 to 18. 35. Composition for detecting a drug against HCV and / or for monitoring appropriate HCV therapies, which contains a polynucleotide according to any one of claims 1 to 5 or 9-13 and a polynucleotide according to any one of claims 7, 8, 14 or 16. 36. Composition for detecting a drug against HCV and / or for monitoring appropriate HCV therapies, which contains the protein according to any one of claims 19-26. 37. A method for determining the presence of HCV drug-resistant HCV in a biological sample, comprising determining the presence of a polynucleotide according to any one of claims 1 to 18 in said biological sample, unless the mutation is A156S (codon of said polynucleotide , which corresponds to codon 156 of the wild-type polynucleotide, encodes serine), and the drug is BILN 2061. 38. The method according to clause 37, including:
a) obtaining the specified polynucleotide from the specified sample;
b) determining the polynucleotide sequence;
c) determining whether codons of said polynucleotide corresponding to codon 156 and optionally codon 168 of wild-type HCV protease polynucleotide are codons of the non-wild type in that codon 156 does not encode alanine and codon 168 does not encode aspartic acid . 39. The method according to clause 37, wherein said wild-type codons encode a series of valine or threonine in a residue that corresponds to residue 156 of wild-type HCV NS3 / 4A protease and / or encode glutamic acid, valine, alanine, glycine or tyrosine in a residue that corresponds to residue 168 of the wild-type HCV NS3 / 4A protease. 40. A method for determining whether an HCV-mediated infection in a patient is resistant to an anti-HCV drug, including:
a) collecting a biological sample from a patient infected with HCV; and
b) evaluating whether the sample contains nucleic acid encoding a mutant HCV NS3 / 4A protease, where the nucleic acid contains a mutation in a codon corresponding to the codon 156 of wild-type HCV NS3 / 4A protease polynucleotide, wherein the presence of said mutant HCV NS3 / 4A protease indicates that said patient has a drug-resistant HCV infection, unless the mutation is A156S (the codon of said polynucleotide, which corresponds to codon 156 of the wild-type polynucleotide, encodes serine), and the drug vennoe agent is BILN 2061. 41. The method of claim 40, wherein the nucleic acid contains a mutation in a codon corresponding to codon 168 of wild-type HCV NS3 / 4A protease polynucleotide, wherein the presence of a mutant NS3 / 4A HCV protease containing an amino acid that is not alanine is at amino acid position 156 of NS3 Wild type / 4A HCV and an amino acid that is not aspartic acid at amino acid position 168 of the NS3 / 4A wild type HCV indicates that the patient has a drug-resistant HCV infection. 42. The method of claim 40 or 41, wherein said mutant HCV NS3 / 4A protease comprises serine, valine or threonine at amino acid position 156 of the wild-type HCV NS3 / 4A protease. 43. The method according to paragraph 41, where the mutant HCV NS3 / 4A protease contains valine, alanine, glycine or tyrosine at amino acid position 168 of the wild-type HCV NS3 / 4A protease. 44. The method of claim 40, wherein the nucleic acid encoding a mutant HCV NS3 / 4A protease present in said biological sample comprises a second mutation in a codon corresponding to codon 168 of wild-type HCV NS3 / 4A protease polynucleotide; where the second mutation leads to the replacement of aspartic acid at amino acid position 168 with glutamic acid. 45. The method of claim 40, wherein the nucleic acid encoding an HCV NS3 / 4A protease present in said biological sample comprises a second mutation in codon 168; where the second mutation leads to the replacement of aspartic acid with alanine, glycine, valine or tyrosine. 46. A method for evaluating whether an HCV-infected patient has a reduced sensitivity or susceptibility to VX-950, comprising determining whether the indicated patient has hepatitis C virus protease NS3 / 4A DNA having a mutation in the codon that encodes amino acid 156 of NS3 / protease Wild-type hepatitis C virus 4A. 47. A method for evaluating whether an HCV-infected patient has a reduced sensitivity or susceptibility to a protease inhibitor, comprising determining whether the patient has hepatitis C virus protease NS3 / 4A DNA having a mutation in the codon that encodes amino acid 156 of the NS3 / 4A protease wild-type hepatitis C virus, unless the mutation is A156S (the codon of the indicated polynucleotide, which corresponds to the codon 156 of the wild-type polynucleotide, encodes serine), and the drug is oh BILN 2061. 48. The method according to any one of claims 46 and 47, wherein the mutation correlates with or leads to reduced sensitivity or susceptibility to BILN 2061. 49. The method according to any one of claims 46 and 47, wherein the mutation correlates with or results in reduced sensitivity or susceptibility to VX-950 and BILN 2061. 50. A method for examining a candidate for an inhibitor or potential inhibitor of HCV, including:
a) introducing a vector containing the polynucleotide according to any one of claims 1 to 18, and an indicator gene encoding an indicator into the host cell;
b) culturing the host cell; and
c) quantification of the indicator in the presence of an inhibitor and in the absence of an inhibitor. 51. A method of researching a candidate for an inhibitor or potential inhibitor of HCV in relation to anti-HCV activity, comprising:
a) providing a protease according to any one of claims 19 to 26 and a protease substrate;
b) contacting the protease with an inhibitor candidate or potential inhibitor in the presence of a substrate; and
c) evaluating or quantifying the inhibition of proteolytic activity of the protease. 52. A method for identifying a compound effective as a protease inhibitor according to any one of claims 19-26, including:
a) a study of the activity of the protease according to any one of claims 19 to 26 in the absence of a compound;
b) a study of protease activity in the presence of a compound;
c) a comparison of results a) and results b). 53. The method according to paragraph 52, which includes:
d) a study of the activity of wild-type protease in the absence of a compound;
e) testing the activity of wild-type protease in the presence of a compound;
f) a comparison of results d) and results e). 54. The method according to item 53, which includes comparing the results from a) and / or b) and the results d) and / or e). 55. The method according to paragraph 52, which includes:
d) testing, in the absence of a compound, the activity of the second NS3 / 4A protease, wherein said second protease contains an amino acid that corresponds to a residue of a wild-type protease 168, wherein said amino acid is mutated to valine, alanine, glycine or tyrosine;
e) a study of the activity of the second protease in the presence of a compound; and
f) comparing the results of d) and e). 56. The method according to clause 55, which includes:
g) a study of the activity of wild-type protease in the absence of a compound;
h) testing the activity of wild-type protease in the presence of a compound;
i) a comparison of results g) and results h). 57. The method according to p. 56, which includes comparing the results from a) and / or b) and the results of d) and / or e); and / or results from g) and / or h). 58. A method for identifying a compound capable of restoring VX-950 activity, wherein the NS3 / 4A protease becomes resistant to VX-950, including:
a) contacting the protease according to any one of claims 19 to 26 with the compound;
b) testing the ability of VX-950 to inhibit the protease activity of a). 59. A method for identifying a compound effective as a protease inhibitor according to any one of claims 19-26, which includes:
a) obtaining a three-dimensional model of the protease according to any one of paragraphs.19-26;
b) design or selection of a joint;
c) evaluating the ability of a compound to bind or interact with a protease. 60. The method according to § 59, where the three-dimensional model is based on the x-ray crystal structure of the NS3 / 4A protease (Figure 1 and Figure 2). 61. The method of claim 60, wherein the model is obtained by methods performed on a computer. 62. The method according to § 59, where the three-dimensional model is obtained by x-ray crystallography of a protein according to any one of paragraphs.19-26. 63. The method according to any one of paragraphs 59-62, where the assessment is carried out by molecular modeling. 64. The method according to any one of claims 59-62, wherein the compound is contacted with a wild-type protease. 65. The method according to any one of claims 59-62, wherein the compound is contacted with a protein according to any one of claims 19-25. 66. The method according to any one of claims 59-62, wherein said compound is a compound identified from a combinatorial chemical library. 67. The method according to any one of paragraphs 59-62, where the specified compound is a compound obtained by the method of rational construction of a medicinal product. 68. The method according to any one of claims 59-62, wherein said compound is a compound obtained by rational drug design and obtained from the structure of VX-950.
Priority on points: 10/27/2003 according to claims 1-65, 67; 04/13/2004 according to claims 66, 68; 11/26/2003 according to claims 1-68.

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