EP1322325A1 - Inhibition de la maturation et de la replication du virus de l'hepatite c - Google Patents

Inhibition de la maturation et de la replication du virus de l'hepatite c

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Publication number
EP1322325A1
EP1322325A1 EP01952780A EP01952780A EP1322325A1 EP 1322325 A1 EP1322325 A1 EP 1322325A1 EP 01952780 A EP01952780 A EP 01952780A EP 01952780 A EP01952780 A EP 01952780A EP 1322325 A1 EP1322325 A1 EP 1322325A1
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EP
European Patent Office
Prior art keywords
hsp90
cleavage
hcv
processing
activity
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
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EP01952780A
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German (de)
English (en)
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EP1322325A4 (fr
Inventor
Lloyd H. Waxman
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Merck and Co Inc
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Merck and Co Inc
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Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1322325A1 publication Critical patent/EP1322325A1/fr
Publication of EP1322325A4 publication Critical patent/EP1322325A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • HCV Hepatitis C virus
  • the HCV genome encodes a single polyprotein of approximately 3000 amino acids, containing the viral proteins in the order: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
  • the NS proteins are thought to be non-structural and are involved with the enzymatic functions of viral replication and processing of the viral polyprotein. Release of the individual proteins from the polyprotein precursor is mediated by both cellular and viral proteases.
  • proteolytic release of mature NS4A, NS4B, NS5A and NS5B is catalyzed by the chymotrypsin-like serine protease contained within the N-terminal domain of NS3, while host cell proteases release C, El, E2, and p7, and create the N- terminus of NS2 at amino acid 810.
  • chymotrypsin-like serine protease contained within the N-terminal domain of NS3
  • host cell proteases release C, El, E2, and p7, and create the N- terminus of NS2 at amino acid 810.
  • the present invention features methods for inhibiting HCV replication and processing by targeting heat shock protein 90 (HSP90).
  • HSP90 is a cellular chaperone protein that was found to be an essential factor in NS2/3 self-cleavage.
  • HSP90 can be targeted using compounds inhibiting the ability of HSP90 to facilitate NS2/3 cleavage.
  • a first aspect of the present invention describes a method of inhibiting HCV replication or processing in a cell infected with HCV comprising the step of providing to the cell an effective amount of an HSP90 inhibitor.
  • the effective amount is an amount sufficient to cause a detectable decrease in HCV replication.
  • replication is inhibited at least about 20%, at least about 50%, at least about 75 % , or at least about 90% .
  • Another aspect of the present invention describes a method of inhibiting NS2/3 cleavage in a polypeptide comprising NS2/3 activity.
  • the method involves providing to the polypeptide an effective amount of an HSP90 inhibitor.
  • the effective amount is an amount sufficient to cause a detectable decrease in NS2/3 cleavage.
  • NS2/3 cleavage is inhibited at least about 20%, at least about 50%, at least about 75%, or at least about 90%.
  • a polypeptide "comprising NS2/3 activity" is made up in whole, or in part, by an amino acid region that is derived from a naturally occurring NS2/3 region and possesses NS2/3 autocatalytic activity.
  • An amino acid region derived from a naturally occurring region contains the sequence of a naturally occurring NS2/3 region or is designed based on a naturally occurring region.
  • the NS2/3 autocatalytic activity can be present on a longer length polypeptide.
  • HCV replication in a patient infected with HCV involves the step of administering to the patent an effective amount of a HSP90 inhibitor.
  • Patients that can be infected with HCV include humans and chimpanzees.
  • the subject is a human.
  • Another aspect of the present invention describes a method of identifying a NS2/3 processing inhibitor comprising the steps of: (a) measuring the ability of a compound to inhibit HSP90 association to a polypeptide comprising NS2/3 activity; and (b) measuring the ability of the compound to inhibit NS2/3 cleavage.
  • Another aspect of the present invention describes a method of identifying an HCV replication inhibitor comprising the steps of: (a) measuring the ability of a compound to inhibit HSP90 activity; and (b) measuring the ability of the compound to inhibit HCV replication.
  • Another aspect of the present invention describes a method of identifying an HCV replication inhibitor comprising the steps of: (a) measuring the ability of a compound to inhibit HSP90 association to a polypeptide comprising NS2/3 activity; and (b) measuring the ability of the compound to inhibit HCV replication.
  • Figure 1 Physical association of NS2/3 with HSP90.
  • A Co- immunoprecipitation of NS2/3 with HSP90-specific antibody.
  • [ 35 S] methionine- labeled NS2/3 (810-1615BK), Ubi-849-1207J-BLA, or firefly luciferase synthesized in reticulocyte lysate were immunoprecipitated with anti-HSP90 mAb 3G3 or with the control IgM TEPC-183.
  • B Geldanamycin interferes with the association of NS2/3 and HSP90.
  • HSP90 inhibitors inhibit NS2/3 cleavage in a cell-based assay.
  • Cloned Jurkat cells expressing a fusion protein of NS2/3 in which ⁇ -lactamase activity is the indicator of successful NS2/3 cleavage were treated with either geldanamycin or radicicol. Inhibitor treatment was for 5 hours, followed by addition of cycloheximide to stop protein synthesis (30 minutes) and subsequent addition of the ⁇ -lactamase substrate, CCF2. After 2 hours, ⁇ -lactamase activity was quantified by fluorescence readings (460nm/530nm ratio). Lower 460nm/530nm ratios indicate inhibition of NS2/3 cleavage.
  • the geldanamycin (triangles) IC 50 is 40 nM and the radicicol (circles) IC 50 is 13 nM. DETAILED DESCRIPTION OF THE INVENTION
  • HSP90 is a chaperone protein identified herein as a target for inhibiting HCV replication or processing. Targeting of HSP90 can be achieved using compounds that inhibit the ability of HSP90 to facilitate NS2/3 cleavage.
  • Chaperone proteins can prevent incorrect interactions within and between non-native proteins and are thought to increase the yield but not rate of folding reactions of many newly synthesized proteins. (Haiti, (1996) Nature 381, 571.)
  • chaperone proteins help modulate the activities of a variety of signaling proteins, including tyrosine kinases such as p60 src (Whitesell, et al, (1994) P.N.A.S. USA 91, 8324), steroid hormone receptors (for review see Pratt, et al, (1997) Endocr. Rev.
  • HSP90 is a chaperone protein involved in regulating the activity of different proteins.
  • HSP90 functions as part of a chaperone complex involving partner proteins that assists cellular protein folding and preventing irreversible side- reactions.
  • HSP90 inhibitors physically associate with HSP90 and inhibit NS2/3 cleavage and/or HCV replication.
  • the association between HSP90 may be a direct association or may be mediated by other factors such as partner proteins.
  • Preferred HSP90 inhibitors achieve a level of inhibition of at least about 20%, at least about 50%, at least about 75%, or at least about 90%.
  • HSP90 activity can be inhibited using a variety of compounds that are well known in the art. Such compounds may be used in methods for inhibiting NS2/3 cleavage and/or HCV replication.
  • the Example section provided below illustrates the ability of compounds well known in the art such as geldanamycin and radicicol to inhibit NS2/3 cleavage.
  • Geldanamycin and radicicol are compounds that have been described as inhibitors of HSP90 activity. (Roe, et al, (1999) J. Med. Chem. 42, 260-266; and Scheibel, et al, (1998) Biochemical Pharmacology 56, 675-682.)
  • Numerous different derivatives of geldanamycin and radicicol are well known in the art. The ability of such derivatives to inhibit NS2/3 cleavage and HCV replication can be determined using standard techniques.
  • Geldanamycin is an ansamycin antibiotic having anti-tumor activity.
  • Geldanamycin has been described in different references as exerting an anti-tumor drug effect by binding in the ATP- binding site present in the N-terminal domain of HSP90.
  • Radicicol also exerts an anti-tumor drug effect by binding to HSP90 at the N-terminal domain ATP-binding site.
  • Derivatives of radicicol indicated to have anti-tumor activity are described in different references. (See, for example, U.S. Patent No. 5,597,846 and U.S. Patent No. 5,977,165, both of which are hereby incorporated by reference herein.) These derivatives provide a class of compounds containing members that are expected to have activity in inhibiting NS2/3 cleavage and/or HCV replication. The ability of a particular compound to inhibit NS2/3 cleavage or HCV replication can be determined using techniques well known in the art.
  • NS2/3 cleavage is part of HCV polyprotein processing leading to the production of an active NS3 protease.
  • cleavage between amino acidsl026 and 1027 separating NS2 from NS3 has been found to be dependent upon protein regions of both NS2 and NS3 flanking the cleaved site.
  • the cleavage is independent of the catalytic activity of the NS3 protease, as demonstrated with mutational studies.
  • NS2/3 cleavage can be measured in different systems using polypeptides comprising NS2/3 catalytic activity.
  • polypeptides comprising NS2/3 catalytic activity.
  • polypeptides include naturally occurring NS2/3 regions having a sufficient amount of NS2/3 for cleavage and derivatives thereof.
  • the NS2/3 cleavage regions can be present along with other polypeptide regions such as those present in an HCV polypeptide or those not present in an HCV polypeptide. Fusion proteins containing NS2/3 cleavage regions can be used to provide for NS2/3 activity and, for example, to provide markers assisting in assaying for NS2/3 activity.
  • the NS2/3 cleavage reaction has been studied in bacterial, mammalian and insect cells, and following in-vitro translation of the protein.
  • ⁇ assay formats can be employed to identify compounds targeting HSP90 that inhibit NS2/3 processing or HCV replication.
  • An example of a general assay format involves first identifying a compound that interacts with HSP90 and then determining whether such a compound also inhibits NS2/3 cleavage or HCV replication.
  • HSP90 Compounds interacting with HSP90 can directly bind to HSP90 or can interact with an HSP90 partner protein such as that present in a chaperone complex.
  • HSP90 partner protein such as that present in a chaperone complex.
  • the ability of a compound to interact with HSP90 can be measured in a variety of ways such as carrying out binding assays and physical association assays.
  • Binding assays measure the ability of a compound to bind to HSP90 or a HSP90 chaperone complex. Different binding assay formats can be performed to measure the ability of a compound to bind to HSP90 or an HSP90 chaperone complex. Examples of different assay formats include competitive and non- competitive. A preferred target for a binding assay is the ATP binding site.
  • HSP90 or an HSP90 chaperone complex can be labeled with a detectable moiety and used to evaluate the ability of other compounds to bind to HSP90 or an HSP90 chaperone complex.
  • Suitable detectable moieties include radioisotopes and fluorescent groups.
  • a particular detectable moiety is preferably selected and positioned on a compound such that the moiety will not substantially affect binding to HSP90 or a HSP90 chaperone complex.
  • Physical association assays measure HSP90 association with a polypeptide comprising NS2/3 fragments acted on by HSP90 or a HSP90 chaperone complex. Techniques for measuring association of HSP90 and NS2/3 include those using binding agents specific for HSP90 or NS2/3.
  • binding agents can be used, for example, to indicate the presence of HSP90 and NS2/3 in a gel or column fraction.
  • An example of specific binding agents are antibodies.
  • Antibodies specific for HSP90 and NS2/3 can be produced using standard immunological techniques. General techniques for producing and using antibodies are described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Harlow, et al, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • Compounds identified as interacting with HSP90 or an HSP90 chaperone complex can be further tested for an effect on NS2/3 processing or HCV replication. Inhibition of NS2/3 processing can be assayed for by measuring NS2/3 autocleavage.
  • Inhibition of HCV replication can be assayed for by measuring a change in HCV levels in a subject infected with HCV.
  • Subjects susceptible to HCV infection include chimpanzees. (Major, et al, (1999) Journal of Virology 73, 3317- 3325.)
  • a preferred target for inhibiting NS2/3 processing or HCV replication is the HSP90 ATP binding site.
  • Compounds binding to other sites for example, those sites affecting the tertiary structure of HSP90, or other HSP90 activities, can be targeted and tested for their ability to inhibit NS2/3 processing or HCV replication.
  • Assays can be performed using individual compounds or a preparation containing different compounds.
  • a preparation containing different compounds wherein one or more compounds achieves a desired effect such as interacting with HSP90, inhibiting NS2/3 cleavage, or inhibiting HCV replication can be divided into smaller groups to identify specific compound(s) having a desired effect.
  • a test preparation contains at least 10 different compounds in an assay that measures interaction with HSP90, inhibition of NS2/3 cleavage, or inhibition of HCV replication.
  • Compounds targeting HSP90 can be formulated and administered to a patient using the guidance provided herein along with techniques well known in the art.
  • the preferred route of administration ensures that an effective amount of compound reaches the target.
  • Guidelines for pharmaceutical administration in general are provided in, for example, Remington 's Pharmaceutical Sciences 18' * Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics 2 nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby incorporated by reference herein.
  • compositions having appropriate functional groups can be prepared as acidic or base salts.
  • Pharmaceutically acceptable salts in the form of water- or oil- soluble or dispersible products
  • Pharmaceutically acceptable salts include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases.
  • salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thio
  • Compounds can be administered using different routes including oral, nasal, by injection, transdermal, and transmucosally.
  • Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents.
  • these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.
  • compositions When administered by nasal aerosol or inhalation, compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. Such compositions may be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents.
  • the compounds may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • the injectable solutions or suspensions may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable non-toxic, parenterally-acceptable diluents or solvents such as Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • these compositions When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • Suitable dosing regimens for the therapeutic applications of the present invention are selected taking into factors well known in the art including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed.
  • Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • the daily dose for a patient is expected to be between 0.01 and 1,000 mg per adult patient per day.
  • Example 1 Production of NS2/3 HCV residues 810-1615 of the BK strain, which includes all of NS2 and most of NS3 (termed 810-1615BK), was produced from the plasmid pCTTE 810- 1615BK (a gift from Dr. Nicola La Monica) and has been described previously by Pieroni, et al., (1997) J. Virol 71, 6373. Following plasmid linearization with BLP1, RNA was transcribed with T7 RNA polymerase and purified. Protein translation was in rabbit reticulocyte lysate, 30°C for 40 minutes using [ S]-methionine as a label. Translation was then blocked by the addition of cycloheximide (250 ⁇ M final) and the samples were immediately frozen on dry ice.
  • pCTTE 810- 1615BK a gift from Dr. Nicola La Monica
  • Example 2 Expression of NS2/3 Fusion Protein
  • the plasmid pM3A derived from pCDNA (Livitrogen), encodes a fusion protein termed Ubi-849-1207J-BLA that contains ubiquitin at its N-terminus, followed by NS2/3 residues 849-1207 (J strain) linked to bacterial TEM-1 ⁇ - lactamase at the C-terminus.
  • the NS3 protease domain in this construct has the inactivating mutation SI 165 A, which does not affect NS2/3 processing activity.
  • RNA synthesis for this construct is driven by the T7 promoter, and RNA was separately prepared for translation as in Example 1.
  • ubiquitin Upon translation in rabbit reticulocyte lysate, the ubiquitin is immediately cleaved from the protein by cellular ubiquitin hydrolases. (Hochstrasser, (1996) Annual Review of Genetics 30, 405.)
  • the cleavable linkage (SEQ. ID. NO. 1) is present as ubiquitiniArgHisGlySerGluPhe-NS2/3.
  • Translation of this construct inevitably produced some processed NS2 and NS3 products, since NS2/3 processing for the J strain does not require detergent or membranes as does NS2/3 from the BK strain. Translations were limited to 30 minutes for that reason. Quantification of processing at room temperature was by comparison of samples prepared immediately after addition of cycloheximide with later time samples.
  • NS2/3 (810-1615BK) was synthesized with [ 35 S]-Met-labeling in rabbit reticulocyte lysate as described in Example 1. Lysate containing translated, [ 35 S]-methionine labeled NS2/3 810-1615BK was centrifuged through a spin column containing P-6 polyacrylamide gel (Bio-Rad; exclusion limit 6000 Da) equilibrated in 20 mM Tris-HCl buffer (pH 7.5). Rabbit reticulocyte lysate (Promega) was filtered with Amicon Microcon-10 units to generate a 10 kDa filtrate.
  • Example 4 Inhibition of NS2/3 Processing by Depleting ATP or Using ATP Analogs
  • rabbit reticulocyte lysate containing translated [ 35 S] -labeled NS2/3 (810-1615BK) was depleted of ATP by treatment with glucose plus hexokinase before processing was initiated by the addition of Triton X-100 to 1%. While neither glucose nor hexokinase alone had a significant effect, the combination of the two, which consumes ATP in the phosphorylation of glucose, inhibited processing by 60%. The results are shown in Table 2. Table 2
  • [ S]-methionine labeled NS2/3 810-1615BK was synthesized in rabbit reticulocyte lysate and 5 ⁇ l aliquots were directly combined with 1 ⁇ l 500 mM glucose, 0.5 unit yeast hexokinase, or glucose plus hexokinase, and incubated for 30 minutes at room temperature. Similarly, lysate containing NS2/3 was incubated with Mg/ATP ⁇ S or Mg/AMP-PNP, for final concentrations of the nucleoside analogs of 5 mM. A stock solution of Triton X-100 at 10% (w/v) was used to initiate autoprocessing of the 810- 1615BK NS2/3. Following addition of SDS sample buffer, samples were heated to 100°C for 5 minutes and proteins separated on SDS/14% polyacrylamide gels. Quantification of products was by phosphorimaging of the dried gels.
  • [ S]-methionine labeled NS2/3 8101615BK was synthesized in reticulocyte lysate in the presence of either 1 orlO ⁇ M geldanamycin, herbimycin A, or radicicol. After blocking further synthesis with cycloheximide, an aliquot was removed and processing was initiated with the addition of Triton X-100 to 1%. After 30 minutes the reaction was terminated with SDS sample buffer and heated to 95°C.
  • NS2/3 processing reactions were performed with 810-1615BK.
  • the inhibitory effects of geldanamycin and radicicol were titrated using techniques described previously for peptide inhibition titrations. (Darke, et al, (1999) J. Biol. Chem. 274, 34511.) Inhibitors were dissolved in DMSO and protected from light. Dilutions were in DMSO, such that the final concentration of DMSO was 2% for in vitro experiments and 1% for cell-based assays. Titration of geldanamycin and radicicol yielded ECso's in the low micromolar range (Table 4), similar to what has been observed in analogous in vitro studies of other proteins acted on by HSP90.
  • the IC 50 values were determined by first expressing the product level found as a fraction of the no-inhibitor control product level, then fitting the equation
  • Protein G-agarose (Boehringer) was used to immobilize goat anti- mouse immunoglobulin M (IgM) (5 mg/ml gel) overnight at 4°C.
  • IgM immunoglobulin M
  • lysate containing translated [ 35 S]-labeled NS2/3 was incubated with the beads essentially as described. (McGuire, et al, (1994) Molecular and Cellular Biology 14, 2438.) Following binding for 2 hours at 4°C, the beads were washed, suspended in SDS sample buffer, and heated to 95°C. Immunoprecipitates were resolved on SDS/14% polyacrylamide gels.
  • HSP90 is essential for NS2/3 processing in living cells.
  • HSP90 inhibitors Through the use of a neomycin-selectable transfection vector, stable expression of NS2/3 in Jurkat cells was obtained.
  • the complete uncleaved protein has an in vivo half -life estimated to be 5-10 minutes.
  • the plasmid pUbBla3X NS2/3-3A was transfected into Jurkat cells.
  • the CMV promoter-driven ORF of the plasmid encodes a 91 kDa protein, ubiquitin- ubiquitin-ubiquitin-NS2/3- ⁇ -lactamase, with the C-termini of the 3 ubiquitin domains rendered non-cleaveable to ubiquitin-C-terminal hydrolases (ubiquitin C-terminal sequence ArgLeuArgGlyVal, SEQ. ID. NO. 2).
  • the NS2/3 region includes HCV residues 849-1207.
  • the ⁇ -lactamase domain is TEM-1 from E. coli. Expression of the ⁇ -lactamase moiety is readily detected with the fluorogenic, cell-permeant substrate, CCF2. (Zlokarnik, et al, (1998) Science 279, 84.)
  • Transfectants were sorted by FACS using treatment with CCF2 to indicate ⁇ -lactamase expression (Zlokarnik, et al, (1998) Science 279, 84), and individual clones were grown under G418 selection.
  • the full fusion protein expressed is highly unstable to ubiquitin-directed proteosomal degradation due to its ubiquitin N-terminal tag, while the C-terminal product of NS2/3 cleavage, NS3- ⁇ - lactamase, is stable for hours.
  • NS2/3 mutation C993A which is incapable of processing, reduces ⁇ -lactamase activity of the cells approximately 8-fold.
  • the protein region essential for NS2/3 cleavage activity has been approximately mapped to amino acids 898 to 1207 of the HCV open reading frame.
  • the conserved cleavage site sequence is ArgLeuLeu AlaProIle (SEQ. ID. NO. 3). Cys993 and His952 have been identified as essential residues.
  • NS2/3 self-cleavage separates the destabilizing ubiquitin degradation signal at the N-terminus from the NS3- ⁇ -lactamase C-terminal product, thus stabilizing the ⁇ -lactamase activity within the cell.
  • inhibitory potency of HSP90 inhibitors toward NS2/3 processing in mammalian cells was measured, as shown in Figure 2.
  • Geldanamycin and radicicol are potent inhibitors of NS2/3 cleavage in this context, with IC 50 values of 40 nM and 13 nM, respectively. In addition, inhibition is nearly complete at the highest concentrations tested (Figure 2).

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Abstract

L'invention concerne des procédés permettant d'inhiber la réplication et la maturation de HCV par ciblage d'une protéine de stress 90 (HSP90). HSP90 est une protéine chaperonne cellulaire dont on a découvert qu'elle constitue un facteur essentiel de l'auto-clivage de NS2/3. HSP90 peut être ciblée au moyen de composés inhibant la capacité de HSP90 à faciliter le clivage de NS2/3.
EP01952780A 2000-07-20 2001-07-16 Inhibition de la maturation et de la replication du virus de l'hepatite c Withdrawn EP1322325A4 (fr)

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