WO2001002601A2 - Cell-based assay systems for examining hcv ns3 protease activity - Google Patents

Cell-based assay systems for examining hcv ns3 protease activity Download PDF

Info

Publication number
WO2001002601A2
WO2001002601A2 PCT/US2000/018590 US0018590W WO0102601A2 WO 2001002601 A2 WO2001002601 A2 WO 2001002601A2 US 0018590 W US0018590 W US 0018590W WO 0102601 A2 WO0102601 A2 WO 0102601A2
Authority
WO
WIPO (PCT)
Prior art keywords
protease
hcv
activity
cell
cdna
Prior art date
Application number
PCT/US2000/018590
Other languages
French (fr)
Other versions
WO2001002601A3 (en
Inventor
Anu Bansal
Claudio Pasquinelli
Original Assignee
Du Pont Pharmaceuticals Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Du Pont Pharmaceuticals Company filed Critical Du Pont Pharmaceuticals Company
Priority to AU59204/00A priority Critical patent/AU5920400A/en
Publication of WO2001002601A2 publication Critical patent/WO2001002601A2/en
Publication of WO2001002601A3 publication Critical patent/WO2001002601A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention is directed to cell-based plasmid expression systems for analyzing the activity of hepatitis C virus NS3 protease upon its viral protein substrates, and hence, for the analysis and discovery of compounds having a potential for inhibiting viral replication.
  • HCV Hepatitis C virus
  • HCV is a positive-stranded RNA virus having a genome 9.6 kb long comprised of a single, uninterrupted open reading frame encoding a polyprotein of about 3000-3011 amino acids; this polyprotein is a precursor to the individual HCV proteins necessary for replication, packaging and infectivity.
  • the structural region of the polyprotein precursor (including the C, El, E2 and p7 proteins) is processed by host cell signal peptidases.
  • the nonstructural region of the precursor including the NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins
  • HCV NS3 protease is thus an essential enzyme for HCV replication and viral infection (see, e. g., Kolykhalov et al. (J. Virol. 74, 2046-2051 (2000)); compounds which inhibit NS3 protease activity would therefore be useful as anti-HCV agents.
  • Several studies have been reported detailing the biochemical features of the NS3 protease in the cell free assay systems (Bartenschlager et al . (J Virol. 67, 3835-3844 (1993)); Baftenschlager et al . (J Virol. 68, 5045-5055 (1994)), (J Virol. 69, 7519-7528 (1995)); Failla, et al . (J.
  • This invention provides such an identification means, through its assays involving transfection of mammalian cells with DNA encoding both HCV NS3 protease activity and an HCV polyprotein substrate including an NS3 cleavage site, thus allowing for assessment of NS3 activity in the cells, as well as the inhibition of such activity through the use of particular compounds .
  • This invention provides a method of assessing the protease activity of HCV NS3 protein in mammalian cells, said method comprising the step of first transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequences encoding the NS3 protease cleavage activity and an HCV NS3 protease substrate polyprotein sequence including an NS3 cleavage site, wherein the 5' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon; said mammalian cells are incubated for a period of time effective for translation of the cDNA, and then lysed.
  • the cell lysate is analyzed for the presence of mature, i.e., processed, HCV NS3 protease substrate, the presence of such processed substrate in said cell lysate being indicative of the presence of NS3 protease activity in the cell.
  • Results of the assessment are useful, for example, in determining the ability of a compound to inhibit HCV NS3 protease cleavage activity. This is accomplished by conducting the assay in the presence and absence of the compound, wherein a higher level of NS3 protease activity in the absence of the compound than is exhibited by the transfected cells in its presence is indicative of the activity of the compound in inhibiting NS3 protease activity. Moreover, the results are useful for demonstrating the expression of HCV nonstructural proteins in cell-based expression systems.
  • HCV NS3 protein herein is expressed as either (1) constitutive or (2) inducible.
  • NS3 protein is expressed as (1) the protease catalytic domain, (2) full- length NS3 protein comprised of both the protease and helicase catalytic domains, (3) full-length NS3 protein followed by NS4A-NS4B polyprotein, (4) protease catalytic domain or full-length catalytic domain coexpressed with another NS3 protease substrate (e.g., NS5A-NS5B polyprotein) , (5) full-length NS3 protein followed by NS4A-NS5B polyprotein, or (6) mutant NS3 protease.
  • NS3 protease substrate e.g., NS5A-NS5B polyprotein
  • FIGURE 1 Gel Electrophoresis Immunoblot Examining
  • FIGURE 2 Gel Electrophoresis Immunoblot Assessing Activity of Protease-Defective Mutant NS3 Protein on NS4B Processing - as measured using
  • HepG2 cells transfected with pCXN2/NS3-4B or p CXN2 /NS3m (mutant ) -4B DNA see Figure 3).
  • FIGURE 3 Schematic Representations of NS3 Expression Plasmids and NS Substrate Polyproteins .
  • Bold arrows indicate NS3 protease cleavage sites on substrate polyproteins;
  • NS3* NS3 protease catalytic domain;
  • 5A*-5B* recombinant pCDNA3 expression plasmid containing C-terminal NS5A through N-terminal NS5B sequence (including the NS5A-5B cleavage site);
  • NS3 full-length NS3 protease;
  • NS3/4A/4B full-length polyprotein comprising HCV sequence from the NS3 N-terminus to the NS4B C-terminus (including the NS3-NS4A and NS4A-NS4B cleavage sites) .
  • FIGURE 4 Gel Electrophoresis Immunoblot Examining Protein Processing by NS3 Catalytic Domain - as measured using HepG2 cells co-transfected with DNA's corresponding to both the NS3 protease domain and a polyprotein encoding NS5A and NS5B.
  • A Schematic representations of full-length NS3- NS5B domain (top) and NS3 catalytic domain and recombinant ("r") NS5A/5B (bottom).
  • B - I Untransfected cells; II: NS3 catalytic domain + rNS5A/NS5B; III: inhibitor; IV: rNS5A/NS5B; V: NS5A (truncated) .
  • FIGURE 5 Gel Electrophoresis Immunoblot Examining Cleavage of Recombinant NS5A/NS5B by Wild-Type (Full-Length) NS3 Protein - as measured using HepG2 cells co-transfected with pCXN2/NS3 and pCDNA3/NS5A5B plasmid DNA, and then incubated in the presence and absence of an NS3 -specific inhibitor.
  • A Schematic representations of full-length NS3-NS5B domain (top) and NS3 and rNS5A/NS5B (bottom).
  • B - I No inhibitor; II: inhibitor concentration; III: untransfected cells; IV: NS5A (truncated) .
  • FIGURE 6 Gel Electrophoresis Immunoblot Examining Cleavage of HCV Polyprotein by Wild-Type NS3 Protein - as measured by cotransfection of HepG2 cells with pCDNA3 /NS5A5B and pCXN2/NS3-4B plasmid DNA in the absence and presence of NS3- specific inhibitor.
  • A Schematic representations of full-length NS3-NS5B domain (top) and NS3-NS4B full length (bottom left) and rNS5A/NS5B (bottom right).
  • B - I Untransfected cells; II: no inhibitor; III: inhibitor concentration; IV: NS4B (full length); V: NS5A (truncated) .
  • FIGURE 7. Gel Electrophoresis Immunoblot Examining
  • FIGURE 8 Gel Electrophoresis Immunoblot Examining Ecdysone-Induced Cleavage of NS3-NS4B
  • This invention provides a method of assessing the protease activity of hepatitis C virus ( "HCV” ) non- structural 3 (“NS3”) protein, said method comprising the steps of: (a) transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequence encoding the NS3 protease cleavage activity and an HCV nucleic acid sequence encoding an NS3 protease substrate, containing an NS3 cleavage site - the 5 ' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon; (b) incubating said mammalian cells for a period of time effective for translation of the cDNA of step (a) ; (c) lysing said incubated mammalian cells of step (b) ; and, (d) detecting the presence of cleaved HCV NS3 protease substrate in
  • HCV being a positive-stranded RNA virus
  • a cDNA copy of said RNA is made for transfections herein according to procedures well known in, and readily practiced by, the art.
  • Transfecting means any process generally accepted in the art for introducing nucleic acid sequences into cells such that the sequences are capable of being translated into proteins; these include, for example and without limitation, transfection by encapsulation of the nucleic acids in liposomes as well as by electroporation.
  • Transfection of mammalian cells herein is by introduction into the cells of a plasmid comprising a cDNA which comprises from 5 ' to 3 ' in operable linkage a methionine codon, the NS3 protease activity-encoding sequence, the NS3 substrate-encoding sequence and a termination codon, or by cotransfection of a plasmid comprising a cDNA which comprises from 5 ' to 3 ' in operable linkage a methionine codon, the NS3 protease- activity encoding sequence and a termination codon and a plasmid comprising a cDNA which comprises from 5' to 3 ' in operable linkage a methionine codon the NS3 substrate- encoding sequence and a termination codon.
  • “Operable linkage” of a codon, such as a methionine codon or a termination codon to another DNA sequence is well known in the art, and is taken herein, to mean linkage of the codon to the additional DNA sequence such that the codon functions for its intended purposes during transcription and translation of that sequence.
  • “Mammalian cells” transfected are any such cells into which nucleic acids can be introduced and then subsequently translated; most preferably herein the mammalian cell transfected is a human hepatocarcinoma cell such as HepG2 , although other cell types, e.g., the human transformed embryonic kidney 293 cell line, and human hepatocarcinoma cell line , Huh- 7, also yield similar results or are also useful.
  • Transfected cells are incubated according to step (b) of this invention's method for a period of time effective for translation of the transfected cDNA; such incubation is under conditions most suitable for growth of cells of a particular type, these being either known to those of ordinary skill in the art or readily determined by them without undue experimentation.
  • An "effective period of time, " and that generally preferred herein, for incubation is about 20 hours, although this period may be longer or shorter as needed.
  • the cells After incubation for the effective period, the cells are lysed so as to release their protein contents intact so that the presence of HCV proteins in the lysates can be detected; such lysis is under conditions suitable for subsequent detection of specific proteins in the lysate and is by methods well known to ordinarily skilled artisans (see, e.g., Example 1 hereinbelow) . Protein detection is also by means, and using techniques, well known to, and readily practiced by, the artisans.
  • NS3 protease cleaves its HCV polyprotein precursor substrate into individual viral proteins at specific cleavage sites on the precursor.
  • transfection into mammalian cells, as provided herein, of NS3 protease- encoding and substrate-encoding sequences allows for the assessment of protease activity on the substrate in vivo; such activity assessment, in turn, allows for examination of its inhibition by particular compounds of interest inside cells.
  • this invention further provides a method of assessing the activity of a compound in inhibiting the activity of HCV NS3 protease, said method comprising conducting the assay provided herein in the presence, and absence, of the compound whose inhibitory activity is being assessed.
  • Production of HCV proteins from NS3 polyprotein substrates by NS3 protease in the absence and presence of the particular compound is compared, a decrease in such production in the presence of the compound over that exhibited in its absence being indicative of inhibition of NS3 activity by the compound.
  • NS3 protease activity in this system is assessed on almost all of its natural cleavage sites, and in a biologically relevant context.
  • the anti-NS3 protease activity of potential compounds on various NS3 cleavage sites is assessed inside cells, giving a direct suggestion of their potency in whole virus, and hence its replication.
  • this invention also provides a means of treating a mammal afflicted with a hepatitis C virus infection, said method comprising the steps of administering to the mammal a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound found, by the assay and screening means provided herein, to be NS3 activity inhibitors.
  • compositions suitable for parenteral administration include various aqueous media such as aqueous dextrose and saline solutions; glycol solutions are also useful carriers, and preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • suitable stabilizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or in combination, are suitable stabilizing agents; also used are citric acid and its salts, and EDTA.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • compositions can be administered orally in solid dosage forms, such as capsules, tablets and powders; or in liquid forms such as elixirs, syrups, and/or suspensions .
  • Gelatin capsules can be used to contain the active ingredient and a suitable carrier such as but not limited to lactose, starch, magnesium stearate, stearic acid, or cellulose derivatives. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of time. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste, or used to protect the active ingredients from the atmosphere, or to allow selective disintegration of the tablet in the gastrointestinal tract.
  • “Therapeutically effective amounts” of a particular compound are any amounts of the compound effective to treat the infected mammal. Such amounts, typically from about 1 to 1000 mg per kg of the body weight of the mammal, are readily determined by ordinarily skilled artisans given the teachings of this invention, e.g., by dose-ranging trials.
  • This invention still further provides a method of expressing HCV nonstructural proteins in a cell-based expression system, said method comprising the step of first transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequences encoding the NS3 protease cleavage activity and an HCV NS3 protease substrate including an NS3 cleavage site, wherein the 5' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon.
  • 2xlO ⁇ HepG2 or 293 cells were electroporated with 2»g of pCXN2/NS3-NS4B plasmid DNA, the electroporated cells were immediately diluted with cell culture medium such as Dulbecco-modified MEM (DMEM) supplemented with 10% fetal calf serum and penicillin-streptomycin mix (Gibco BRL) and plated into 6 well plates, which were incubated in a standard C0 2 incubator at 37 degrees Celsius for 20-24 hrs in the presence or absence of the compounds to be tested. Synthesis of the NS3 protease and its substrates begin within hours after DNA introduction into the cells; compounds were therefore added to the cell culture medium within few minutes after the transfection.
  • DMEM Dulbecco-modified MEM
  • Gibco BRL penicillin-streptomycin mix
  • Incubated cells were harvested by scraping, centrifugation and then aspiration of the cell culture media.
  • the cells were lysed with commercially available lysis buffer (GIBCO BRL) .
  • the protein in the cell lysates were separated by standard SDS polyacrylamide gel electrophoresis, and transferred from gel onto nitrocellulose membranes, which were subsequently used for immunoblot analysis by methods described in Sambrook et al . (Molecular Cloning: A laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al . (Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).
  • HCV-infected human patient serum containing antibodies against several HCV proteins including NS4B and NS5 proteins, was used as the source of primary antibody; HRP conjugated goat antihuman antibody was used as the secondary antibody for immunobloting.
  • Results demonstrate the presence of NS4B protein in transfected, but not untransfected cells; NS4B protein is also produced in cells exposed to the non-NS3 specific inhibitors A and C, and its substantial absence in cells exposed to increasing concentrations of the NS3 -specific Inhibitor B and to increasing concentrations of Peflabloc®, a commercially available inhibitor of chymotrypsin-like serine protease enzymes .
  • HepG2 cells were transfected with either pCXN2/NS3-4B or pCXN2/NS3m-4B DNA plasmid DNA (where "m” represents an active site, non-functional mutation of the NS3 protease) in the manner described in Example 1.
  • Results demonstrate expression of NS4B in cells transfected with pCXN2/NS3-4B, but not in either untransfected cells or in cells transfected with pCXN2/NS3m-4B; thus demonstrating that the NS4B released in these cells is through the action of NS3 protease, and not due to the activity of a nonspecific host cellular protease.
  • Example 3 Gel Electrophoresis Immunoblot Examining Protein Processing by NS3 Catalytic Domain HepG2 cells were, according to the procedures described in Example 1 hereinabove, co-transfected with a plasmid encoding the NS3 protease catalytic domain
  • NS3* a plasmid encoding recombinant
  • r a plasmid encoding recombinant
  • HepG2 cells were co-transfected with a pCXN2/NS3 and pcDNA3/NS5A-5B plasmids incubated for twenty hours, lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 5, demonstrate production of NS5A protein
  • HepG2 cells were co-transfected with pCXN2/NS3-4B and pcDNA3/NS5A-5B, incubated for twenty hours incubated in the absence and presence of NS3-specific inhibitor, lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 6, demonstrate production of both NS4B and NS5A (truncated) proteins with no inhibitor present, but not in untransfected cells or at increasing inhibitor concentrations .

Abstract

Assays for identifying levels of hepatitis C virus NS3 protease activity are provided herein, and involve the transfection into mammalian cells of cDNA corresponding to HCV nucleic acid sequences encoding both the viral NS3 protease activity and an NS3 polyprotein precursor substrate including an NS3 cleavage site. The presence of mature, processed substrate in lysates obtained from the transfected cells is indicative of the level of activity of the NS3 protease in the cells. These assays can thus also be used as a screen to examine the activity of a particular compound in inhibiting NS3 protease activity, by conducting the assays in the absence and presence of the compound. The degree to which NS3 protease activity is expressed in the former assays over the latter is indicative of the compound's activity in inhibiting the NS3 protein substrate cleavage, and thus also, of inhibiting HCV replication.

Description

CEL -BASED ASSAY SYSTEMS FOR EXAMINING HCV NS3 PROTEASE ACTIVITY
Field of the Invention
This invention is directed to cell-based plasmid expression systems for analyzing the activity of hepatitis C virus NS3 protease upon its viral protein substrates, and hence, for the analysis and discovery of compounds having a potential for inhibiting viral replication.
Background of the Invention
Hepatitis C virus ("HCV") is a parenterally transmitted, hepatotropic virus that causes acute and chronic hepatitis, as well as hepatocellular carcinoma; approximately 2% of the world's population is afflicted with HCV infections, many of which remain chronic. No vaccine for HCV is currently available, present treatment generally being limited to interferon monotherapy, or the combination of alpha-interferon with the nucleoside analog ribavirin (Bartenschlager and Lohman (J. Gen. Virol. 81, 1631-1648 (2000)); Choo et al . (Science 244, 358- 362(1989)); Kolykhalov et al . (Science 277, 570-574, (1997)); Neuman et al . (Science 282, 103-107 (1998)); Saito et al . (Proc. Natl . Acad. Sci . USA 87, 6547-6549 (1990)); Tong et al . (Lancet 345, 1058-1059 (1995))).
HCV is a positive-stranded RNA virus having a genome 9.6 kb long comprised of a single, uninterrupted open reading frame encoding a polyprotein of about 3000-3011 amino acids; this polyprotein is a precursor to the individual HCV proteins necessary for replication, packaging and infectivity. The structural region of the polyprotein precursor (including the C, El, E2 and p7 proteins) is processed by host cell signal peptidases. The nonstructural region of the precursor (including the NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) is processed between NS2 and NS3 by NS2-3 protease, while processing in the NS3-NS5B region of the polyprotein is accomplished by
NS3 protease activity (Bartenschlager et al . (J. Virol.
68, 5045-5055 (1994)); Eckart et al . (BBRC 192, 399-406 (1993)); Grakoui et al . (J. Virol. 67, 2832-2843 (1993));
Lin et al . (J. Virol. 68, 5063-5073 (1994)); Lin et al .
(J. Virol. 68, 8147-8157 (1994))).
HCV NS3 protease is thus an essential enzyme for HCV replication and viral infection (see, e. g., Kolykhalov et al. (J. Virol. 74, 2046-2051 (2000)); compounds which inhibit NS3 protease activity would therefore be useful as anti-HCV agents. Several studies have been reported detailing the biochemical features of the NS3 protease in the cell free assay systems (Bartenschlager et al . (J Virol. 67, 3835-3844 (1993)); Baftenschlager et al . (J Virol. 68, 5045-5055 (1994)), (J Virol. 69, 7519-7528 (1995)); Failla, et al . (J. Virol. 68, 3753-3760 (1994)) Gallinari et al . (J. Virol. 72, 6758-6769 (1998)); Grakoui et al. (J. Virol. 67, 2832-2843 (1993)); Lin et al . (J Virol. 68, 8147-8157 (1994)); Lin and Rice (Proc. Natl Acad. Sci. (USA) 92, 7622-7626 (1995)); Shimizu et al . (J Virol. 70, 127-130 (1996)). Additionally, three dimensional structural features of this enzyme have also been described both by x-ray crystallography as well as by NMR studies, utilizing NS3 protease domain alone, full- length NS3 (includes both protease and RNA helicase domains), NS3 tethered with its cofactor NS4A (Kim et al . (Cell 87, 343-355 (1996)); Love et al . (Cell 87, 331-342 (1996)); Yan et al . (Prot. Sci. 7, 837-847 (1998))). Compared to all this detailed knowledge about physical and biochemical properties of the NS 3 protein, very little is known about the characteristics of NS3 protease and its substrate proteins in a cellular system. Several studies have been made into cellular localization and activity of NS3 , but results are inconsistent among different HCV strains, and cell types (Harada et al . (J. Gen. Virol. 76,
1215-1221 (1995)); Ishido et al . (Biochem. Biophys . Res. Comm. 230, 431-436 (1997)); Koch and Bartenschlager (Virol. 237, 78-88 (1997)); Muramatsu et al . (J. Virol. 71, 4954-4961 (1997)); Sakamuro et al . (J. Virol. 69, 3893-3896 (1995)); Wolk et al . (J. Virol. 74, 2293-2304 (2000)); Sho i et al . (Virol. 254, 315-323 (1999))).
Lack of an efficient cell-culture system and a convenient small animal model, as well as the low titers of infectious virus typically found in samples obtained from afflicted subjects has hampered progress in the discovery and development of anti HCV chemotherapy. This has also affected our understanding of the molecular mechanism of HCV replication and the functioning of HCV proteins, including NS3 (see, e.g., Bartenschlager and Lohman, 2000) . Rather, most of the studies have been limited to in-vitro analysis of HCV proteins.
Accordingly, there is a need for the identification of compounds able to inhibit viral replication and hence, to treat HCV infections, including by inhibiting HCV NS3 protease activity. This invention provides such an identification means, through its assays involving transfection of mammalian cells with DNA encoding both HCV NS3 protease activity and an HCV polyprotein substrate including an NS3 cleavage site, thus allowing for assessment of NS3 activity in the cells, as well as the inhibition of such activity through the use of particular compounds .
Summary of the Invention
This invention provides a method of assessing the protease activity of HCV NS3 protein in mammalian cells, said method comprising the step of first transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequences encoding the NS3 protease cleavage activity and an HCV NS3 protease substrate polyprotein sequence including an NS3 cleavage site, wherein the 5' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon; said mammalian cells are incubated for a period of time effective for translation of the cDNA, and then lysed. Subsequently, the cell lysate is analyzed for the presence of mature, i.e., processed, HCV NS3 protease substrate, the presence of such processed substrate in said cell lysate being indicative of the presence of NS3 protease activity in the cell.
Results of the assessment are useful, for example, in determining the ability of a compound to inhibit HCV NS3 protease cleavage activity. This is accomplished by conducting the assay in the presence and absence of the compound, wherein a higher level of NS3 protease activity in the absence of the compound than is exhibited by the transfected cells in its presence is indicative of the activity of the compound in inhibiting NS3 protease activity. Moreover, the results are useful for demonstrating the expression of HCV nonstructural proteins in cell-based expression systems.
Expression of HCV NS3 protein herein is expressed as either (1) constitutive or (2) inducible. NS3 protein is expressed as (1) the protease catalytic domain, (2) full- length NS3 protein comprised of both the protease and helicase catalytic domains, (3) full-length NS3 protein followed by NS4A-NS4B polyprotein, (4) protease catalytic domain or full-length catalytic domain coexpressed with another NS3 protease substrate (e.g., NS5A-NS5B polyprotein) , (5) full-length NS3 protein followed by NS4A-NS5B polyprotein, or (6) mutant NS3 protease. Brief Description of the Drawings
FIGURE 1. Gel Electrophoresis Immunoblot Examining
Inhibition of HCV N3-NS4B Polyprotein Region
Processing - as measured using inhibitors A, B and C on HepG2 cells, human hepatocarcinoma cell line, transfected with the plasmid pCXN2/NS3-4B
(see Figure 3) . Bold arrow: NS4B protein; I: no inhibitor present; II: untransfected cells; III: cells incubated in the presence of inhibitor A; IV: inhibitor B; V: inhibitor C; VI: Peflabloc™
(A: 125 »M; B: 500 »M) ) .
FIGURE 2. Gel Electrophoresis Immunoblot Assessing Activity of Protease-Defective Mutant NS3 Protein on NS4B Processing - as measured using
HepG2 cells transfected with pCXN2/NS3-4B or p CXN2 /NS3m (mutant ) -4B DNA (see Figure 3). Bold arrow: NS4B protein; I: wild-type NS3 protein (pCXN2/NS3-4B) ; II: mutant NS3 protein (pCXN2/NS3m-4B) .
FIGURE 3. Schematic Representations of NS3 Expression Plasmids and NS Substrate Polyproteins . Bold arrows indicate NS3 protease cleavage sites on substrate polyproteins; "NS3*": NS3 protease catalytic domain; "5A*-5B*": recombinant pCDNA3 expression plasmid containing C-terminal NS5A through N-terminal NS5B sequence (including the NS5A-5B cleavage site); "NS3" : full-length NS3 protease; "NS3/4A/4B": full-length polyprotein comprising HCV sequence from the NS3 N-terminus to the NS4B C-terminus (including the NS3-NS4A and NS4A-NS4B cleavage sites) .
FIGURE 4. Gel Electrophoresis Immunoblot Examining Protein Processing by NS3 Catalytic Domain - as measured using HepG2 cells co-transfected with DNA's corresponding to both the NS3 protease domain and a polyprotein encoding NS5A and NS5B. A: Schematic representations of full-length NS3- NS5B domain (top) and NS3 catalytic domain and recombinant ("r") NS5A/5B (bottom). B - I: Untransfected cells; II: NS3 catalytic domain + rNS5A/NS5B; III: inhibitor; IV: rNS5A/NS5B; V: NS5A (truncated) .
FIGURE 5. Gel Electrophoresis Immunoblot Examining Cleavage of Recombinant NS5A/NS5B by Wild-Type (Full-Length) NS3 Protein - as measured using HepG2 cells co-transfected with pCXN2/NS3 and pCDNA3/NS5A5B plasmid DNA, and then incubated in the presence and absence of an NS3 -specific inhibitor. A: Schematic representations of full-length NS3-NS5B domain (top) and NS3 and rNS5A/NS5B (bottom). B - I : No inhibitor; II: inhibitor concentration; III: untransfected cells; IV: NS5A (truncated) .
FIGURE 6. Gel Electrophoresis Immunoblot Examining Cleavage of HCV Polyprotein by Wild-Type NS3 Protein - as measured by cotransfection of HepG2 cells with pCDNA3 /NS5A5B and pCXN2/NS3-4B plasmid DNA in the absence and presence of NS3- specific inhibitor. A: Schematic representations of full-length NS3-NS5B domain (top) and NS3-NS4B full length (bottom left) and rNS5A/NS5B (bottom right). B - I: Untransfected cells; II: no inhibitor; III: inhibitor concentration; IV: NS4B (full length); V: NS5A (truncated) . FIGURE 7. Gel Electrophoresis Immunoblot Examining
Tetracycline-Induced Cleavage of HCV NS3 /NS4B Polyprotein - as measured using HepG2 cells co- transfected with pTRE/NS3-4B and ptet/ON plasmid DNA in the presence (A) and absence of doxycyclin. I: NS3/NS4A polyprotein; II: NS4B protein.
FIGURE 8. Gel Electrophoresis Immunoblot Examining Ecdysone-Induced Cleavage of NS3-NS4B
Polyprotein - as measured using HepG2 cells co- transfected with pIND (Spl) /NS3-NS4B and pVGRXR plasmid DNA incubated in the presence of ecdysone (I: Ponasterone®; I: 15 »M; II: 5«M; III: 0 -M) . II (arrow): NS4B protein.
Detailed Description of the Invention
This invention provides a method of assessing the protease activity of hepatitis C virus ( "HCV" ) non- structural 3 ("NS3") protein, said method comprising the steps of: (a) transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequence encoding the NS3 protease cleavage activity and an HCV nucleic acid sequence encoding an NS3 protease substrate, containing an NS3 cleavage site - the 5 ' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon; (b) incubating said mammalian cells for a period of time effective for translation of the cDNA of step (a) ; (c) lysing said incubated mammalian cells of step (b) ; and, (d) detecting the presence of cleaved HCV NS3 protease substrate in the cell lysate of step (c) , the presence of such substrate in said cell lysate being indicative of the presence of NS3 protease activity in the cell. HCV being a positive-stranded RNA virus, a cDNA copy of said RNA is made for transfections herein according to procedures well known in, and readily practiced by, the art. "Transfecting" as used herein means any process generally accepted in the art for introducing nucleic acid sequences into cells such that the sequences are capable of being translated into proteins; these include, for example and without limitation, transfection by encapsulation of the nucleic acids in liposomes as well as by electroporation. Transfection of mammalian cells herein is by introduction into the cells of a plasmid comprising a cDNA which comprises from 5 ' to 3 ' in operable linkage a methionine codon, the NS3 protease activity-encoding sequence, the NS3 substrate-encoding sequence and a termination codon, or by cotransfection of a plasmid comprising a cDNA which comprises from 5 ' to 3 ' in operable linkage a methionine codon, the NS3 protease- activity encoding sequence and a termination codon and a plasmid comprising a cDNA which comprises from 5' to 3 ' in operable linkage a methionine codon the NS3 substrate- encoding sequence and a termination codon. "Operable linkage" of a codon, such as a methionine codon or a termination codon to another DNA sequence is well known in the art, and is taken herein, to mean linkage of the codon to the additional DNA sequence such that the codon functions for its intended purposes during transcription and translation of that sequence. "Mammalian cells" transfected are any such cells into which nucleic acids can be introduced and then subsequently translated; most preferably herein the mammalian cell transfected is a human hepatocarcinoma cell such as HepG2 , although other cell types, e.g., the human transformed embryonic kidney 293 cell line, and human hepatocarcinoma cell line , Huh- 7, also yield similar results or are also useful. Transfected cells are incubated according to step (b) of this invention's method for a period of time effective for translation of the transfected cDNA; such incubation is under conditions most suitable for growth of cells of a particular type, these being either known to those of ordinary skill in the art or readily determined by them without undue experimentation. An "effective period of time, " and that generally preferred herein, for incubation is about 20 hours, although this period may be longer or shorter as needed. After incubation for the effective period, the cells are lysed so as to release their protein contents intact so that the presence of HCV proteins in the lysates can be detected; such lysis is under conditions suitable for subsequent detection of specific proteins in the lysate and is by methods well known to ordinarily skilled artisans (see, e.g., Example 1 hereinbelow) . Protein detection is also by means, and using techniques, well known to, and readily practiced by, the artisans.
NS3 protease cleaves its HCV polyprotein precursor substrate into individual viral proteins at specific cleavage sites on the precursor. Thus, transfection into mammalian cells, as provided herein, of NS3 protease- encoding and substrate-encoding sequences allows for the assessment of protease activity on the substrate in vivo; such activity assessment, in turn, allows for examination of its inhibition by particular compounds of interest inside cells.
Thus, this invention further provides a method of assessing the activity of a compound in inhibiting the activity of HCV NS3 protease, said method comprising conducting the assay provided herein in the presence, and absence, of the compound whose inhibitory activity is being assessed. Production of HCV proteins from NS3 polyprotein substrates by NS3 protease in the absence and presence of the particular compound is compared, a decrease in such production in the presence of the compound over that exhibited in its absence being indicative of inhibition of NS3 activity by the compound. NS3 protease activity in this system is assessed on almost all of its natural cleavage sites, and in a biologically relevant context. The anti-NS3 protease activity of potential compounds on various NS3 cleavage sites is assessed inside cells, giving a direct suggestion of their potency in whole virus, and hence its replication.
Since NS3 -mediated protein cleavage is necessary for HCV replication, compounds discovered by such a screening method to be NS3 inhibitors are also useful as HCV replication inhibitors. Thus, this invention also provides a means of treating a mammal afflicted with a hepatitis C virus infection, said method comprising the steps of administering to the mammal a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound found, by the assay and screening means provided herein, to be NS3 activity inhibitors.
Administration is, for example, by various parenteral means. Pharmaceutical compositions suitable for parenteral administration include various aqueous media such as aqueous dextrose and saline solutions; glycol solutions are also useful carriers, and preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents, such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or in combination, are suitable stabilizing agents; also used are citric acid and its salts, and EDTA. In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
Alternatively, compositions can be administered orally in solid dosage forms, such as capsules, tablets and powders; or in liquid forms such as elixirs, syrups, and/or suspensions . Gelatin capsules can be used to contain the active ingredient and a suitable carrier such as but not limited to lactose, starch, magnesium stearate, stearic acid, or cellulose derivatives. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of time. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste, or used to protect the active ingredients from the atmosphere, or to allow selective disintegration of the tablet in the gastrointestinal tract.
"Therapeutically effective amounts" of a particular compound are any amounts of the compound effective to treat the infected mammal. Such amounts, typically from about 1 to 1000 mg per kg of the body weight of the mammal, are readily determined by ordinarily skilled artisans given the teachings of this invention, e.g., by dose-ranging trials.
This invention still further provides a method of expressing HCV nonstructural proteins in a cell-based expression system, said method comprising the step of first transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequences encoding the NS3 protease cleavage activity and an HCV NS3 protease substrate including an NS3 cleavage site, wherein the 5' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon. The cells incubated for a period of time effective for translation of the transfected cDNA, and cell lysates are then collected - the presence of cleaved HCV NS3 protease substrate in the lysates demonstrates that NS3 protease had been expressed in the transfected.
The examples provided hereinbelow further describe the invention provided herein. However, those of ordinary skill in the art will readily understand that said examples are merely illustrative of the invention, as defined in the claims following immediately thereafter.
Examples Example 1
Gel Electrophoresis Immunoblot Examining Inhibition of HCV N3-NS4B Polyprotein Region Processing
2xlOδ HepG2 or 293 cells were electroporated with 2»g of pCXN2/NS3-NS4B plasmid DNA, the electroporated cells were immediately diluted with cell culture medium such as Dulbecco-modified MEM (DMEM) supplemented with 10% fetal calf serum and penicillin-streptomycin mix (Gibco BRL) and plated into 6 well plates, which were incubated in a standard C02 incubator at 37 degrees Celsius for 20-24 hrs in the presence or absence of the compounds to be tested. Synthesis of the NS3 protease and its substrates begin within hours after DNA introduction into the cells; compounds were therefore added to the cell culture medium within few minutes after the transfection. Incubated cells were harvested by scraping, centrifugation and then aspiration of the cell culture media. The cells were lysed with commercially available lysis buffer (GIBCO BRL) . The protein in the cell lysates were separated by standard SDS polyacrylamide gel electrophoresis, and transferred from gel onto nitrocellulose membranes, which were subsequently used for immunoblot analysis by methods described in Sambrook et al . (Molecular Cloning: A laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al . (Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). HCV-infected human patient serum, containing antibodies against several HCV proteins including NS4B and NS5 proteins, was used as the source of primary antibody; HRP conjugated goat antihuman antibody was used as the secondary antibody for immunobloting.
Results, presented in Figure 1 herein, demonstrate the presence of NS4B protein in transfected, but not untransfected cells; NS4B protein is also produced in cells exposed to the non-NS3 specific inhibitors A and C, and its substantial absence in cells exposed to increasing concentrations of the NS3 -specific Inhibitor B and to increasing concentrations of Peflabloc®, a commercially available inhibitor of chymotrypsin-like serine protease enzymes .
Example 2 Gel Electrophoresis Immunoblot Assessing Activity of Mutant NS3 Protein on NS4B Processing
HepG2 cells were transfected with either pCXN2/NS3-4B or pCXN2/NS3m-4B DNA plasmid DNA (where "m" represents an active site, non-functional mutation of the NS3 protease) in the manner described in Example 1. Results (presented in Figure 2) demonstrate expression of NS4B in cells transfected with pCXN2/NS3-4B, but not in either untransfected cells or in cells transfected with pCXN2/NS3m-4B; thus demonstrating that the NS4B released in these cells is through the action of NS3 protease, and not due to the activity of a nonspecific host cellular protease.
Example 3 Gel Electrophoresis Immunoblot Examining Protein Processing by NS3 Catalytic Domain HepG2 cells were, according to the procedures described in Example 1 hereinabove, co-transfected with a plasmid encoding the NS3 protease catalytic domain
("NS3*") and a plasmid encoding recombinant ("r") NS5A/5B DNA, incubated for twenty hours, lysed and analyzed by the procedures described in Example 1 hereinabove. Results, presented in Figure 4, demonstrate processing of rNS5A/NS5B, and production therefrom of truncated NS5A, in transfected, but not in untransfected cells, as well as decreased processing at increasing inhibitor concentrations .
Example 4
Gel Electrophoresis Immunoblot Examining Cleavage of Recombinant NS5A/5B by Wild-Type (Full-Length) NS3 Protein
HepG2 cells were co-transfected with a pCXN2/NS3 and pcDNA3/NS5A-5B plasmids incubated for twenty hours, lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 5, demonstrate production of NS5A protein
(truncated) with no NS3 protease inhibitor present, but not in untransfected cells, as well as decreasing production at increasing inhibitor concentrations.
Example 5
Gel Electrophoresis Immunoblot Examining Cleavage of HCV Polyprotein by Wild-Type NS3 Protein
HepG2 cells were co-transfected with pCXN2/NS3-4B and pcDNA3/NS5A-5B, incubated for twenty hours incubated in the absence and presence of NS3-specific inhibitor, lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 6, demonstrate production of both NS4B and NS5A (truncated) proteins with no inhibitor present, but not in untransfected cells or at increasing inhibitor concentrations . Example 6
Gel Electrophoresis Immunoblot Examining Tetracvcline- Induced Cleavage of HCV NS3/NS4B Polyprotein HepG2 cells were co-transfected with pTRE/NS3-4B and ptet/ON, incubated for twenty hours, in the absence ("-") and presence ("+") of doxycyclin (an activator of the gene expression in this plasmid system) , lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 7, demonstrate decreasing production of 4B protein at decreasing doxycyclin concentrations and the absence of production in the absence of doxycyclin. In the absence of Doxycyclin there is no expression of NS3-NS4B polyprotein, hence, there is no NS3 protease activity. These results clearly establish that the band on the immunoblots corresponding to NS4B is NS4B protein, and is produced as a result of NS3 protease activity, as it is only seen in the cells treated with Doxycyclin, but not in the untreated cells.
Example 7
Ecdysone®-Induced Expression of NS3-4B Polyprotein in HepG2 Cells HepG2 cells were co-transfected with pIND (Spl) /NS3-4B and pVGRXR plasmids, incubated for twenty hours, lysed and analyzed by immunoblotting according to the procedures described in Example 1 hereinabove. Results, presented in Figure 8, demonstrate decreasing production of NS4B protein in the presence of ecydsone (Ponasterone®, an activator of gene expression in this system) , and the substantial absence of production in its absence.

Claims

What is claimed is:
1. A method of assessing the protease activity of hepatitis C virus ("HCV") non-structural 3 ("NS3") protein, said method comprising the steps of:
(a) transfecting a mammalian cell with cDNA corresponding to the HCV nucleic acid sequences encoding:
(i) the NS3 protease cleavage activity; and, (ii) an HCV NS3 protease substrate including an
NS3 cleavage site, wherein the 5 ' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon;
(b) incubating said mammalian cells for a period of time effective for translation of the cDNA of step (a);
(c) lysing said incubated mammalian cells of step (b) ; and,
(d) detecting the presence of cleaved HCV NS3 protease substrate in the cell lysate of step (c) , the presence of such substrate in said cell lysate being indicative of the presence of NS3 protease activity in the cell, wherein the transfection of step (a) is by a plasmid comprising a cDNA which comprises from 5' to 3 ' in operable linkage a methionine codon, the NS3 protease activity-encoding sequence, the NS3 substrate- encoding sequence and a termination codon, or wherein the transfection of step (a) is a cotransfection of:
(i) a plasmid comprising a cDNA which comprises from 5 ' to 3' in operable linkage a methionine codon, the NS3 protease-activity encoding sequence and a termination codon; and, (ii) a plasmid comprising a cDNA which comprises from 5 ' to 3 ' in operable linkage a methionine codon the NS3 substrate-encoding sequence and a termination codon.
2. The method of claim 1, wherein the mammalian cell is a human cell.
3. A method of assessing the activity of a compound in inhibiting HCV NS3 protease cleavage activity, said method comprising the steps of :
(1) conducting the assay of claim 1, wherein step (a) of the assay is done in the absence of the compound;
(2) conducting the assay of claim 1, wherein step (a) of the assay is done in the presence of the compound; and,
(3) comparing the levels of HCV NS3 protease cleavage activity in the assay of step (1) of this method with the level of HCV NS3 protease cleavage activity in the assay of step (2) of this method, wherein a higher level of said NS3 protease activity of the absence of the compound is indicative of the activity of the compound in inhibiting such protease activity
4. A method of treating a mammal afflicted with hepatitis C, said method comprising the steps of administering to the mammal a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound assessed by the method of claim 4 to inhibit HCV NS3 substrate-cleavage activity.
5. A method of expressing HCV nonstructural proteins in a cell-based expression system, said method comprising: (a) transfecting a cell with cDNA corresponding to the HCV nucleic acid sequences encoding:
(i) the NS3 protease cleavage activity; and, (ii) an HCV NS3 protease substrate including an NS3 cleavage site, wherein the 5 ' end of said cDNA is operably linked to a methionine codon and the 3 ' end of said cDNA is operably linked to a termination codon; (b) incubating said cell for a period of time effective for translation of the cDNA of step (a) ;
(c) lysing said transfected cell of step (b) ; and,
(d) detecting the presence of cleaved HCV NS3 protease substrate in the cell lysate of step (c) , the presence of such substrate in said cell lysate being indicative of the presence of NS3 protease activity in the cell, wherein the cell is a mammalian cell.
PCT/US2000/018590 1999-07-07 2000-07-07 Cell-based assay systems for examining hcv ns3 protease activity WO2001002601A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU59204/00A AU5920400A (en) 1999-07-07 2000-07-07 Cell-based assay systems for examining hcv ns3 protease activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14256199P 1999-07-07 1999-07-07
US60/142,561 1999-07-07

Publications (2)

Publication Number Publication Date
WO2001002601A2 true WO2001002601A2 (en) 2001-01-11
WO2001002601A3 WO2001002601A3 (en) 2001-07-26

Family

ID=22500325

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2000/018655 WO2001002424A2 (en) 1999-07-07 2000-07-07 Peptide boronic acid inhibitors of hepatitis c virus protease
PCT/US2000/018590 WO2001002601A2 (en) 1999-07-07 2000-07-07 Cell-based assay systems for examining hcv ns3 protease activity

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2000/018655 WO2001002424A2 (en) 1999-07-07 2000-07-07 Peptide boronic acid inhibitors of hepatitis c virus protease

Country Status (4)

Country Link
EP (1) EP1196436A2 (en)
AU (2) AU5920400A (en)
CA (1) CA2376965A1 (en)
WO (2) WO2001002424A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005024051A1 (en) * 2003-06-23 2005-03-17 Vanderbilt University Cell-based assay for identifying peptidase inhibitors

Families Citing this family (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002230763A1 (en) * 2000-12-13 2008-01-03 Bristol-Myers Squibb Pharma Company Inhibitors of hepatitis c virus ns3 protease
US6653295B2 (en) 2000-12-13 2003-11-25 Bristol-Myers Squibb Company Inhibitors of hepatitis C virus NS3 protease
US6586615B1 (en) 2001-01-10 2003-07-01 Bristol-Myers Squibb Company α-aminoboronic acids prepared by novel synthetic methods
EP3078667B1 (en) 2001-01-25 2018-11-21 The United States of America, represented by the Secretary, Department of Health and Human Services Formulation of boronic acid compounds
US7119072B2 (en) 2002-01-30 2006-10-10 Boehringer Ingelheim (Canada) Ltd. Macrocyclic peptides active against the hepatitis C virus
US6642204B2 (en) 2002-02-01 2003-11-04 Boehringer Ingelheim International Gmbh Hepatitis C inhibitor tri-peptides
US7091184B2 (en) 2002-02-01 2006-08-15 Boehringer Ingelheim International Gmbh Hepatitis C inhibitor tri-peptides
ATE325611T1 (en) 2002-09-09 2006-06-15 Trigen Ltd BORIC ACID SALTS AND THEIR USE IN THE PROVISION OF MEDICATIONS FOR THROMBOSIS TREATMENT
US20050288253A1 (en) 2002-09-09 2005-12-29 Trigen Limited Boronic acid salts
US20050075279A1 (en) 2002-10-25 2005-04-07 Boehringer Ingelheim International Gmbh Macrocyclic peptides active against the hepatitis C virus
WO2004092162A1 (en) 2003-04-11 2004-10-28 Vertex Pharmaceuticals, Incorporated Inhibitors of serine proteases, particularly hcv ns3-ns4a protease
AU2011203054B2 (en) * 2003-04-11 2012-04-26 Vertex Pharmaceuticals, Incorporated Inhibitors of Serine Proteases, Particularly HCV NS3-NS4A Protease
US7405210B2 (en) 2003-05-21 2008-07-29 Osi Pharmaceuticals, Inc. Pyrrolopyridine-2-carboxylic acid amide inhibitors of glycogen phosphorylase
CN1791599A (en) 2003-05-21 2006-06-21 贝林格尔.英格海姆国际有限公司 Hepatitis c inhibitor compounds
US7576206B2 (en) 2003-08-14 2009-08-18 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7223745B2 (en) 2003-08-14 2007-05-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
MY148123A (en) * 2003-09-05 2013-02-28 Vertex Pharma Inhibitors of serine proteases, particularly hcv ns3-ns4a protease
CA2536182C (en) 2003-09-22 2012-07-24 Boehringer Ingelheim International Gmbh Macrocyclic peptides active against the hepatitis c virus
ES2358333T3 (en) 2004-01-21 2011-05-09 Boehringer Ingelheim International Gmbh MACROCYCLIC PEPTIDES WITH ACTION AGAINST THE VIRUS OF HEPATITIS C.
DE602005023965D1 (en) 2004-03-08 2010-11-18 Prosidion Ltd PYRROLOPYRIDINE-2-CARBOXYLIC HYDRAZIDE AS INHIBITORS OF GLYCOPE PHOSPHORYLASE
EP1765360B1 (en) * 2004-06-14 2009-11-11 Anacor Pharmaceuticals, Inc. Anti-viral uses of borinic acid complexes
UY29016A1 (en) 2004-07-20 2006-02-24 Boehringer Ingelheim Int ANALOGS OF INHIBITING DIPEPTIDES OF HEPATITIS C
CA2573346C (en) 2004-07-20 2011-09-20 Boehringer Ingelheim International Gmbh Hepatitis c inhibitor peptide analogs
ES2324173T3 (en) 2004-12-02 2009-07-31 Prosidion Limited AMIDAS OF THE ACID PIRROLOPIRIDIN-2-CARBOXILICO.
US7468383B2 (en) 2005-02-11 2008-12-23 Cephalon, Inc. Proteasome inhibitors and methods of using the same
AU2006228957A1 (en) * 2005-04-01 2006-10-05 Methylgene Inc. Inhibitors of histone deacetylase
JP2009503084A (en) * 2005-08-01 2009-01-29 フェノミックス コーポレーション Hepatitis C serine protease inhibitor and use thereof
RU2419626C2 (en) 2006-05-23 2011-05-27 Айрм Ллк Compounds and compositions as canal activating protease inhibitors
EP1886685A1 (en) 2006-08-11 2008-02-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods, uses and compositions for modulating replication of hcv through the farnesoid x receptor (fxr) activation or inhibition
US20100120716A1 (en) * 2006-12-06 2010-05-13 Phenomix Corporation Macrocyclic hepatitis c serine protease inhibitors and uses therefor
EP2117537A1 (en) 2007-02-09 2009-11-18 Irm Llc Compounds and compositions as channel activating protease inhibitors
US7442830B1 (en) 2007-08-06 2008-10-28 Millenium Pharmaceuticals, Inc. Proteasome inhibitors
US7838673B2 (en) 2007-10-16 2010-11-23 Millennium Pharmaceuticals, Inc. Proteasome inhibitors
DK2730581T3 (en) 2008-06-17 2016-08-15 Millennium Pharm Inc Boronate Ester Compounds and Pharmaceutical Compositions thereof.
AR075090A1 (en) 2008-09-29 2011-03-09 Millennium Pharm Inc ACID DERIVATIVES 1-AMINO-2-CYCLLOBUTILETILBORONICO PROTEOSOMA INHIBITORS, USEFUL AS ANTI-BANKER AGENTS, AND PHARMACEUTICAL COMPOSITIONS THAT UNDERSTAND THEM.
CA2785300A1 (en) 2009-12-22 2011-07-21 Cephalon, Inc. Proteasome inhibitors and processes for their preparation, purification and use
TW201138786A (en) * 2010-02-19 2011-11-16 Glaxo Group Ltd Therapeutic compounds
CN102892291A (en) 2010-03-31 2013-01-23 米伦纽姆医药公司 Derivatives of 1-amino-2-cyclopropylethylboronic acid
WO2012067663A1 (en) * 2010-11-18 2012-05-24 Glaxo Group Limited Compounds
WO2012107589A1 (en) 2011-02-11 2012-08-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for the treatment and prevention of hcv infections
ES2735542T3 (en) * 2011-08-17 2019-12-19 Glaxosmithkline Llc Therapeutic procedures
CA2844086A1 (en) 2011-08-19 2013-02-28 Glaxo Group Limited Benzofuran compounds for the treatment of hepatitis c virus infections
US10092574B2 (en) 2012-09-26 2018-10-09 Valorisation-Recherche, Limited Partnership Inhibitors of polynucleotide repeat-associated RNA foci and uses thereof
AU2015264272A1 (en) 2014-05-20 2016-11-24 Millennium Pharmaceuticals, Inc. Boron-containing proteasome inhibitors for use after primary cancer therapy
US10253049B2 (en) * 2014-10-01 2019-04-09 Merck Patent Gmbh Boronic acid derivatives
RU2717558C2 (en) * 2014-10-01 2020-03-24 Мерк Патент Гмбх Boronic acid derivatives
BR112017006420A2 (en) * 2014-10-01 2017-12-19 Merck Patent Gmbh boronic acid derivatives
US10526315B2 (en) 2016-06-21 2020-01-07 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives
WO2017222915A1 (en) 2016-06-21 2017-12-28 Inception 4, Inc. Heterocyclic prolinamide derivatives
WO2022198196A1 (en) 2021-03-15 2022-09-22 Maze Therapeutics, Inc. Inhibitors of glycogen synthase 1 (gys1) and methods of use thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995022985A1 (en) * 1994-02-23 1995-08-31 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Method for reproducing in vitro the proteolytic activity of the ns3 protease of hepatitis c virus (hcv)
WO1996034976A1 (en) * 1995-05-01 1996-11-07 Vertex Pharmaceuticals Incorporated Methods, nucleotide sequences and host cells for assaying exogenous and endogenous protease activity
WO1998000548A1 (en) * 1996-06-28 1998-01-08 Lg Chemicals Co., Ltd. Hepatitis c surrogate virus for testing the activity of hepatitis c virus protease, a recombinant gene and a use thereof
US5721133A (en) * 1990-04-13 1998-02-24 Schering Corporation Protease assays
WO1998037180A2 (en) * 1997-02-22 1998-08-27 Abbott Laboratories Hcv fusion protease and polynucleotide encoding same
WO2000008469A1 (en) * 1998-08-05 2000-02-17 Agouron Pharmaceuticals, Inc. Reporter gene system for use in cell-based assessment of inhibitors of the hepatitis c virus protease
WO2000066623A2 (en) * 1999-05-04 2000-11-09 Boehringer Ingelheim (Canada) Ltd. Surrogate cell-based system and method for assaying the activity of hepatitis c virus ns3 protease

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR9712544B1 (en) * 1996-10-18 2013-10-22 SERINE PROTEASE INHIBITORS, PHARMACEUTICAL COMPOSITION UNDERSTANDING THE SAME AND ITS USES
GB9623908D0 (en) * 1996-11-18 1997-01-08 Hoffmann La Roche Amino acid derivatives
AU757783B2 (en) * 1997-08-11 2003-03-06 Boehringer Ingelheim (Canada) Ltd. Hepatitis C inhibitor peptides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721133A (en) * 1990-04-13 1998-02-24 Schering Corporation Protease assays
WO1995022985A1 (en) * 1994-02-23 1995-08-31 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Method for reproducing in vitro the proteolytic activity of the ns3 protease of hepatitis c virus (hcv)
WO1996034976A1 (en) * 1995-05-01 1996-11-07 Vertex Pharmaceuticals Incorporated Methods, nucleotide sequences and host cells for assaying exogenous and endogenous protease activity
WO1998000548A1 (en) * 1996-06-28 1998-01-08 Lg Chemicals Co., Ltd. Hepatitis c surrogate virus for testing the activity of hepatitis c virus protease, a recombinant gene and a use thereof
WO1998037180A2 (en) * 1997-02-22 1998-08-27 Abbott Laboratories Hcv fusion protease and polynucleotide encoding same
WO2000008469A1 (en) * 1998-08-05 2000-02-17 Agouron Pharmaceuticals, Inc. Reporter gene system for use in cell-based assessment of inhibitors of the hepatitis c virus protease
WO2000066623A2 (en) * 1999-05-04 2000-11-09 Boehringer Ingelheim (Canada) Ltd. Surrogate cell-based system and method for assaying the activity of hepatitis c virus ns3 protease

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHO YOUNG-GYU ET AL: "In vivo assay for hepatitis C viral serine protease activity using a secreted protein." JOURNAL OF VIROLOGICAL METHODS, vol. 72, no. 1, May 1998 (1998-05), pages 109-115, XP000979032 ISSN: 0166-0934 *
HIROWATARI Y ET AL: "A novel method for analysis of viral proteinase activity encoded by hepatitus C virus in cultured cells" ANALYTICAL BIOCHEMISTRY,US,ACADEMIC PRESS, SAN DIEGO, CA, vol. 225, 1995, pages 113-120, XP002096985 ISSN: 0003-2697 *
LIN CHAO ET AL: "The hepatitis C virus NS3 serine proteinase and NS4A cofactor: Establishment of a cell-free trans-processing assay." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 92, no. 17, 1995, pages 7622-7626, XP002158507 1995 ISSN: 0027-8424 *
OVERTON HILARY ET AL: "Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays." JOURNAL OF GENERAL VIROLOGY, vol. 76, no. 12, 1995, pages 3009-3019, XP002158506 ISSN: 0022-1317 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005024051A1 (en) * 2003-06-23 2005-03-17 Vanderbilt University Cell-based assay for identifying peptidase inhibitors

Also Published As

Publication number Publication date
WO2001002424A2 (en) 2001-01-11
CA2376965A1 (en) 2001-01-11
WO2001002424A3 (en) 2001-07-19
AU5788800A (en) 2001-01-22
AU5920400A (en) 2001-01-22
WO2001002601A3 (en) 2001-07-26
EP1196436A2 (en) 2002-04-17

Similar Documents

Publication Publication Date Title
WO2001002601A2 (en) Cell-based assay systems for examining hcv ns3 protease activity
Major et al. The molecular virology of hepatitis C
De Francesco Molecular virology of the hepatitis C virus
Kolykhalov et al. Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing
Kwong et al. Hepatitis C virus NS3/4A protease
Shimoike et al. Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation
Bartenschlager The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy
Lin et al. The hepatitis C virus NS3 serine proteinase and NS4A cofactor: establishment of a cell-free trans-processing assay.
Reed et al. Molecular characterization of hepatitis C virus
Cheney et al. Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture
Lee et al. Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells
EP1694694B1 (en) Replication competent hepatitis c virus and methods of use
Shanmugam et al. Efficiency of E2-p7 processing modulates production of infectious hepatitis C virus
Butkiewicz et al. Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3
De Francesco et al. Biochemical and immunologic properties of the nonstructural proteins of the hepatitis C virus: implications for development of antiviral agents and vaccines
EP1102992A1 (en) Reporter gene system for use in cell-based assessment of inhibitors of the hepatitis c virus protease
Zhang et al. Role of protein conformation in the processing of dengue virus type 2 nonstructural polyprotein precursor
US20100068698A1 (en) Production of infectious hepatitis c virus particles in cell culture
US20090087832A1 (en) Nucleic acids and new polypeptides associated with and/or overlapping with hepatitis C virus core gene products
US20080286752A1 (en) Methods for the production of HCV, assaying HCV entry, and screening drugs and cellular receptors for HCV
Pacini et al. Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells
Filocamo et al. Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric Sindbis viruses
Wang et al. Hepatitis C virus encoded proteins: targets for antiviral therapy
Zemel et al. Mutations at vicinity of catalytic sites of hepatitis C virus NS3 serine protease gene isolated from hepatocellular carcinoma tissue
Mao et al. Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU BR CA CN CZ EE HU IL IN JP KR LT LV MX NO NZ PL RO SG SI SK TR UA VN ZA

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AU BR CA CN CZ EE HU IL IN JP KR LT LV MX NO NZ PL RO SG SI SK TR UA VN ZA

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP