HCV NS3-NS4 Protease Resistance Mutants

Abstract

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

1 . An isolated HCV polynucleotide encoding a HCV NS3/4A protease or a biologically active analog thereof wherein the codon that corresponds to codon 156 of the wild-type polynucleotide and/or the codon that corresponds to codon 168 of the wild-type polynucleotide is mutated such that it does not encode an alanine at 156 and/or aspartic acid at 168. 
     
     
         2 . The isolated HCV polynucleotide of  claim 1 , wherein the codon of said polynucleotide that corresponds to codon 156 of the wild-type polynucleotide encodes a serine. 
     
     
         3 . The isolated HCV polynucleotide of  claim 1 , wherein the codon of said polynucleotide that corresponds to codon 156 of the wild-type polynucleotide encodes a valine. 
     
     
         4 . The isolated HCV polynucleotide of  claim 1  wherein the codon of said polynucleotide that corresponds to codon 156 of the wild-type polynucleotide encodes a threonine. 
     
     
         5 . The isolated HCV polynucleotide of  claim 1 , wherein the codon of said polynucleotide that corresponds to codon 156 of the wild-type polynucleotide encodes a valine or a threonine and the codon of said polynucleotide that corresponds to codon 168 of the wild-type polynucleotide encodes an aspartic acid or glutamic acid. 
     
     
         6 . The isolated HCV polynucleotide of  claim 1 , wherein the codon of said polynucleotide that corresponds to codon 168 of the wild-type polynucleotide encodes a valine. 
     
     
         7 . The isolated HCV polynucleotide of  claim 1 , wherein codon 168 of the polynucleotide encodes an alanine, a glycine, or a tyrosine. 
     
     
         8 . The isolated HCV polynucleotide of  claim 1 , wherein wild-type HCV polynucleotide has a sequence of SEQ ID NO:1. 
     
     
         9 . A polynucleotide that encodes a biologically active fragment of an HCV NS3/4A protease, the fragment of said polynucleotide is characterized in that it encodes an HCV NS3/4A protease domain that comprises a codon that corresponds to codon 156 of the wild-type polynucleotide and/or the codon that corresponds to codon 168 of the wild-type polynucleotide, wherein said codon that corresponds to codon 156 and/or said codon that corresponds to codon 168 is mutated such that it does not encode an alanine at 156 and/or aspartic acid at 168. 
     
     
         10 . The polynucleotide of  claim 9 , wherein said polynucleotide is characterized in that it comprises codon 156 of the HCV NS3/4A protease domain wherein the codon 156 encodes a valine, threonine or serine residue instead of an alanine residue. 
     
     
         11 . The polynucleotide of  claim 9 , wherein said polynucleotide is characterized in that it comprises codons that correspond to codons 156 and 168 of the HCV NS3/4A protease domain wherein the codon 156 encodes a valine or threonine instead of an alanine and codon 168 encodes an aspartic acid or glutamic acid. 
     
     
         12 . The polynucleotide of  claim 9 , the polynucleotide characterized in that it comprises a codon that corresponds to codon 168 of the HCV NS3/4A protease domain wherein the codon 168 encodes a valine. 
     
     
         13 . The polynucleotide of  claim 9 , wherein the polynucleotide is characterized in that it comprises a codon that corresponds to codon 168 of the HCV NS3/4A protease domain wherein the codon 168 encodes an alanine, glycine, or tyrosine. 
     
     
         14 . The isolated HCV polynucleotide of  claim 9 , wherein wild-type HCV polynucleotide has a sequence of SEQ ID NO:1. 
     
     
         15 . An isolated HCV NS3/4A protease protein or biologically active fragment or biologically active analog thereof comprising a sequence in which the amino acid residue which corresponds to amino acid 156 of wild-type HCV NS3/4A protease is not an alanine residue and/or amino acid residue which corresponds to amino acid 168 HCV NS3/4A protease is not an aspartic acid residue. 
     
     
         16 . The isolated HCV NS3/4A protease protein or fragment or analog of  claim 15 , wherein the amino acid that corresponds to amino acid 156 of the wild-type protease is valine, threonine or serine. 
     
     
         17 . The isolated HCV NS3/4A protease protein or fragment or analog of  claim 15 , wherein the amino acid that corresponds to amino acid 156 of the protease is serine, valine or threonine and amino acid 168 is aspartic acid or glutamic acid. 
     
     
         18 . The isolated HCV NS3/4A protease protein or fragment or analog of  claim 15 , wherein the amino acid that corresponds to amino acid 168 of the wild-type protease is valine. 
     
     
         19 . The isolated HCV NS3/4A protease protein or fragment or analog of  claim 15 , wherein the amino acid that corresponds to amino acid 168 of the wild-type protease is alanine, glycine, or tyrosine. 
     
     
         20 . The isolated HCV NS3/4A protease protein of  claim 15 , wherein wild-type HCV NS3/4A protease has a sequence of SEQ ID NO:2. 
     
     
         21 . A vector comprising the polynucleotide according to any one of  claims 1 - 14 . 
     
     
         22 . A host cell or cell line comprising the polynucleotide according to any one of  claims 1 - 14 . 
     
     
         23 . A host cell transformed or transfected with a vector of  claim 21 . 
     
     
         24 . A host cell or cell line comprising the protein according to any one of  claims 15 - 21 . 
     
     
         25 . An isolated HCV variant comprising a polynucleotide according to any one of  claims 1 - 14 . 
     
     
         26 . An isolated HCV variant comprising a protein according to any of  claims 15 - 23 . 
     
     
         27 . A composition comprising the polynucleotide according to any one of  claims 1 - 14 . 
     
     
         28 . A composition comprising the polynucleotide according to any one of  claims 1 - 4  or  7 - 10  and the polynucleotide according to any one of  claim 5 ,  6   11  or  12 . 
     
     
         29 . A composition comprising a protein according to any one of  claims 15 - 21 . 
     
     
         30 . A composition comprising a protein according to any one of  claims 15 - 18  and a protein according to  claim 19  or  claim 23 . 
     
     
         31 . A method for detecting the presence of drug-resistant HCV in a biological sample comprising detecting the presence of a polynucleotide according to any one of  claims 1 - 14  in said biological sample. 
     
     
         32 . The method according to  claim 31  comprising:
 a) obtaining said polynucleotide from said sample; 
 b) determining the sequence of the polynucleotide; 
 c) determining whether in said polynucleotide, codon 156 and/or codon 168 are codons that are a non-wild-type codons in that said codons do not encode aspartic acid. 
 
     
     
         33 . The method of  claim 31 , wherein said non-wild-type codons encode a serine, valine, or a threonine at a residue that corresponds to residue 156 of the wild-type HCV NS3/4A protease and/or encode a glutamic acid, valine, alanine, glycine, or tyrosine at a residue that corresponds to residue 168 of the wild-type HCV NS3/4A protease. 
     
     
         34 . The method according to  claim 33  comprising determining or inferring whether, in the polynucleotide,
 a) codon 156 encodes a serine, codon 156 encodes a valine, codon 156 encodes a threonine, codon 156 encodes a valine or a threonine and codon 156 encodes an aspartic acid or glutamic acid; and 
 b) codon 168 encodes a valine or codon 168 encodes an alanine, a glycine, or a tyrosine. 
 
     
     
         35 . A method for determining whether an HCV infection in a patient is drug-resistant comprising:
 a) collecting a biological sample from the HCV infected patient; and   b) evaluating whether the plasma sample contains nucleic acid encoding a mutant HCV NS3/4A protease, wherein the presence of said mutant HCV NS3/4A protease is indicative of said patient having a drug-resistant HCV infection.   
     
     
         36 . The method of  claim 35 , wherein said nucleic acid encodes a mutant HCV NS3/4A protease having a mutation at an amino acid that corresponds to residue 156 and/or residue 168 of said HCV NS3/4A, wherein the presence of an amino acid that is not alanine at a residue that corresponds to residue 156 of wild-type HCV NS3/4A, and/or an amino acid that is not aspartic acid at residue that corresponds to residue 168 of wild-type HCV NS3/4A, is indicative that said patient has a drug-resistant HCV infection. 
     
     
         37 . The method of  claim 35 , wherein said mutation comprises the presence of a serine, valine or threonine at the amino acid residue that corresponds to residue 156 of wild-type HCV NS3/4A protease. 
     
     
         38 . The method according to any of  claim 35 ,  36  or  37  wherein the HCV NS3/4A protease comprises the presence of an aspartic acid, valine, alanine, glycine, or tyrosine at the amino acid residue that corresponds to residue 168 of wild-type HCV NS3/4A protease. 
     
     
         39 . The method of according to any of  claim 35 ,  36  or  37 , wherein the nucleic acid encoding HCV NS3/4A protease present in said biological sample comprises a mutation at codon 156; wherein the mutation results in a substitution of alanine with serine, valine or threonine or and said mutation comprises a substation of aspartic acid at residue 168 with glutamic acid. 
     
     
         40 . The method according to any one of  claims 35 - 39 , wherein the nucleic acid encoding HCV NS3/4A protease present in said biological sample comprises a mutation at codon 168; wherein the mutation results in a substitution of aspartic acid with alanine, glycine, valine, or tyrosine. 
     
     
         41 . A method for evaluating whether a HCV-infected patient has a decreased sensitivity or susceptibility to VX-950 comprising evaluating whether said patient has a Hepatitis C virus NS3/4A protease DNA having a mutation at the codon that encodes residue 156 of wild-type Hepatitis C virus NS3/4A protease. 
     
     
         42 . A method for evaluating whether a HCV-infected patient has a decreased sensitivity or susceptibility to a protease inhibitor comprising evaluating whether said patient has a Hepatitis C virus NS3/4A protease DNA having a mutation at the codon that encodes residue 156 of wild-type Hepatitis C virus NS3/4A protease. 
     
     
         43 . The method according to any one of  claims 41 - 42 , wherein the mutation correlates with or results in decreased sensitivity or susceptibility to BILN 2061. 
     
     
         44 . The method according to any one of  claims 41 - 42 , wherein the mutation correlates with or results in decreased sensitivity or susceptibility to VX-950 and to BILN 2061. 
     
     
         45 . A method for evaluating a candidate or potential HCV inhibitor comprising:
 a) introducing a vector comprising a polynucleotide according to any one of  claims 1 - 14  and an indicator gene encoding an indicator into a host cell;   b) culturing the host cell; and   c) measuring the indicator in the presence of inhibitor and in the absence of inhibitor.   
     
     
         46 . A method for assaying compounds for activity against HCV comprising:
 a) providing a protease according to any one of  claims 15 - 23  and a protease substrate;   b) contacting the protease with a candidate or potential inhibitor in the presence of the substrate; and   c) evaluating or measuring the inhibition of proteolytic activity of the protease.   
     
     
         47 . A method for identifying a compound as an inhibitor of a protease according to any one  claims 15 - 23  comprising:
 a) assaying the activity of the protease in the absence of the compound; 
 b) assaying the activity of the protease in the presence of compound; 
 c) comparing the results of a) and the results of b). 
 
     
     
         48 . The method according to  claim 47  comprising:
 d) assaying the activity of a wild-type protease in the absence of the compound; 
 e) assaying the activity of the wild-type protease in the presence of compound; 
 f) comparing the results of d) and the results of e). 
 
     
     
         49 . The method according to  claim 48  comprising comparing the results from a) and/or b) and the results of d) and/or e). 
     
     
         50 . The method according to  claim 47  comprising 
       d) assaying, in the absence of the compound, the activity of a second NS3/4A protease wherein said second protease comprises an amino acid that corresponds to residue 168 of the wild-type protease, wherein said amino acid is mutated to a valine, alanine, glycine, or tyrosine;
 e) assaying the activity of the second protease in the presence of compound; and 
 f) comparing the results of d) and e). 
 
     
     
         51 . The method according to  claim 50  comprising:
 g) assaying the activity of a wild-type protease in the absence of the compound; 
 h) assaying the activity of the wild-type protease in the presence of compound; 
 i) comparing the results of g) and the results of h). 
 
     
     
         52 . The method according to  claim 51  comprising comparing the results from a) and/or b) and the results of d) and/or e); and/or the results from g) and/or h). 
     
     
         53 . A method for identifying a compound able to rescue the activity of VX-950, wherein a NS3/4A protease has become resistant to VX-950 comprising:
 a) contacting a protease according to any one  claims 15 - 23  with the compound;   b) assaying the ability of VX-950 to inhibit the activity of the protease of a).   
     
     
         54 . A method for identifying a compound effective against a protease according to any one of  claims 15 - 23 , comprising:
 a) obtaining a three dimensional model of the protease;   b) designing or selecting a compound;   c) evaluating the ability of the compound to bind to or interact with the protease.   
     
     
         55 . The method according to  claim 54 , wherein the three dimensional model is based on the x-ray crystal structure ( FIG. 1  and  FIG. 2 ) of NS3/4A protease. 
     
     
         56 . The method according to  claim 55 , wherein the model obtained by computer-implemented methods. 
     
     
         57 . The method according to  claim 54 , wherein the three dimensional model is obtained by x-ray crystallography of the protein according to any one of  claims 15 - 21 . 
     
     
         58 . The method according to any one of  claims 54 - 57 , wherein the evaluating is by molecular modeling. 
     
     
         59 . The method according to any one of  claims 54 - 58 , wherein the compound is contacted with a wild-type protease. 
     
     
         60 . The method according to any one of  claims 54 - 59 , wherein the compound is contacted with a protein according to any one of  claims 15 - 19 . 
     
     
         61 . The method according to any one of  claims 54 - 60 , wherein the compound is contacted with a protein according to any one of  claims 20 - 21 . 
     
     
         62 . The method of any of  claims 54 - 61 , wherein said compound is a compound identified from a combinatorial chemical library. 
     
     
         63 . The method of any of  claims 54 - 62 , wherein said compound is a compound prepared through rational drug design. 
     
     
         64 . The method of any of  claims 54 - 62 , wherein said compound is a compound prepared through rational drug design and derived from the structure of VX-950. 
     
     
         65 . A compound identified according to any one of  claims 56 - 61 . 
     
     
         66 . A composition comprising the compound according to  claim 65 ; and a pharmaceutically acceptable carrier, adjuvant or vehicle. 
     
     
         67 . The composition according to  claim 66  wherein the compound is in an amount effective to inhibit NS3/4A serine protease. 
     
     
         68 . The composition according to  claim 67 , wherein said composition is formulated for administration to a patient. 
     
     
         69 . The composition according to  claim 68 , wherein said composition comprises an additional agent selected from an immunomodulatory agent; an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; a cytochrome P-450 inhibitor; or combinations thereof. 
     
     
         70 . The composition according to  claim 69 , wherein said immunomodulatory agent α, β- or γ interferon or thymosin; the antiviral agent is ribavirin, amantadine, or thymosin; or the inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease. 
     
     
         71 . The composition according to  claim 70 , wherein said cytochrome P-450 inhibitor is ritonavir. 
     
     
         72 . A method of inhibiting the activity of a Hepatitis C NS3/4A protease comprising the step of contacting said serine protease with a compound according to  claim 65 . 
     
     
         73 . A method of treating an HCV infection in a patient comprising the step of administering to said patient a compound according to  claim 65 . 
     
     
         74 . A method of treating or reducing an HCV infection in a patient comprising:
 a) determining whether said patient has an HCV infection that is resistant to therapy using a method of any of  claims 35 - 44 ;   b) treating said patient with a composition or therapy directed at the treatment of drug-resistant HCV.   
     
     
         75 . The method according to  claim 73  or  74 , comprising the additional step of administering to said patient an additional agent selected from an immunomodulatory agent; an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; or combinations thereof; wherein said additional agent is administered to said patient as part of a single or as a separate dosage form. 
     
     
         76 . The method according to  claim 75 , wherein said immunomodulatory agent α, β- or γ interferon; or thymosin; said antiviral agent is ribavirin or amantadine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease. 
     
     
         77 . A method of eliminating or reducing HCV contamination of a biological sample or medical or laboratory equipment, comprising the step of contacting said biological sample or medical or laboratory equipment with a compound according to  claim 65 . 
     
     
         78 . The method of  claim 77  wherein said biological sample or medical or laboratory equipment is contaminated with a drug-resistant strain of HCV as determined according to a method of  claims 31 - 34 . 
     
     
         79 . The method according to  claim 77 , wherein said sample or equipment is selected from blood; other body fluids, biological tissue, a surgical instrument, a surgical garment, a laboratory instrument, a laboratory garment, a blood or other body fluid collection apparatus; a blood or other bodily fluid storage material.