WO1997031641A1 - Method of treating hepatitis delta virus infection - Google Patents

Method of treating hepatitis delta virus infection Download PDF

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Publication number
WO1997031641A1
WO1997031641A1 PCT/US1996/012501 US9612501W WO9731641A1 WO 1997031641 A1 WO1997031641 A1 WO 1997031641A1 US 9612501 W US9612501 W US 9612501W WO 9731641 A1 WO9731641 A1 WO 9731641A1
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ihdag
hdv
inhibitor
lhdag
ftase
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PCT/US1996/012501
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French (fr)
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Patrick J. Casey
James C. Otto
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Duke University
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Priority to AU66051/96A priority Critical patent/AU6605196A/en
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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses

Definitions

  • the present invention relates, in general, to hepatitis delta virus, and in particular, to a method of treating hepatitis delta virus infeccion.
  • the method involves inhibiting famesylation of the delta virus large antigen using an inhibitor of famesyltransferase.
  • Hepatitis delta virus is a satellite virus of hepatitis B virus that can cause an increase in the incidence and severity of liver disease in individuals infected with both viruses (Rizzetto, (1983) Heptology 3, 729-737; Lai, (1995) Annu. Rev. Biochem . 64, 259-286) .
  • HDV consists of the HDV RNA genome and two HDV encoded proteins, designated as the small (sHDAg) and large (IHDAg) HDV antigens, encapsulated in an envelope composed of hepatitis B surface antigens (Lai, (1995) Annu . Rev. Biochem . 64, 259-286) .
  • sHDAg and IHDAg contain identical deduced amino acid sequences for their first 195 amino acids, with IHDAg containing an additional 19 amino acid C-terminal tail (Lai, (1995) Annu. Rev. Biochem . 64, 259-286.) . Despite their sequence identity, sHDAg and IHDAg have very different functions. Whereas sHDAg is essential for HDV replication (Kuo et al, (1989) J. Virol . 63, 1945-1950) , IHDAg is a dominant inhibitor of HDV replication (Chao et al, (1990) J. Virol . 64,
  • the current model of HDV assembly indicates that IHDAg functions by directly interacting with hepatitis B surface antigens that form the envelope of the viral particle (Lai, (1995) Annu. Rev. Biochem. 64, 259-286) .
  • IHDAg is modified by an isoprenoid lipid on a cysteine located near its C-terminus (Glenn et al, (1992) Science 256, 1331-1333) , and this modification is necessary for IHDAg to facilitate HDV assembly (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175) .
  • the prenylation motif contained in IHDAg is the C-terminal tetrapeptide Cys-Arg-Pro-Gln (CRPQ) (Glenn et al, (1992) Science 256, 1331-1333) .
  • This sequence is similar to the conventional prenylation motif which consists of the C-terminal tetrapeptide CaaX, where the cysteine residue is the prenylation site, "a" are generally aliphatic residues, and X can be one of several amino acids.
  • Two distinct protein prenyltransferases modify proteins which contain a CaaX motif: famesyltransferase (FTase) , which modifies proteins with the 15-carbon farnesyl isoprenoid, and geranylgeranyltransferase-I (GGTase-I) , which modifies proteins with the 20-carbon geranylgeranyl group (Glomset et al, (1994) Annu. Rev.
  • the C-terminal amino acid in general determines which of the two isoprenoid lipids modify a CaaX motif. If X is Met, Ser or Gin, the sequence is a substrate for FTase (Moores et al, (1991) J. Biol . Chem . 266, 14603-14610) , while Leu at this position directs modification by GGTase-I (Moores et al, (1991) J. Biol . Chem .
  • GGTase- II A third protein prenyltransferase, GGTase- II, recognizes a different class of C-terminal motifs present in GTP-binding proteins of the Rab family
  • IHDAg As prenylation of IHDAg is required for HDV assembly, inhibition of the enzyme responsible for its prenylation provides a treatment of HDV infection.
  • Two reports have appeared indicating that IHDAg is modified with the geranylgeranyl isoprenoid (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175) .
  • the present invention results, at least in part, from the realizations that the predominant modification of IHDAg is famesylation rather than geranylgeranylation, and that the protein is exclusively a substrate for FTase.
  • the present invention relates to a method of inhibiting hepatitis delta virus (HDV) assembly.
  • the method comprises contacting an HDV-infected cell wich an amount of a famesyltransferase inhibitor sufficient to inhibit famesylation of IHDAg and thereby inhibit HDV assembly.
  • the invention further relates to a method of treating a patient infected with HDV. The method comprises administering to the patient an amount of a famesyltransferase inhibitor sufficient to inhibit famesylation of IHDAg and thereby effect the treatment.
  • FIG. 1 Prenylation of GST-lHDAg by bovine brain cytosol .
  • GST-lHDAg, H-Ras, and H-Ras-CVLL were incubated with bovine brain cytosol in the presence of either 2 ⁇ M [ 3 H] FPP or 2 ⁇ M [ 3 H] GGPP for 1 h at 37°C.
  • Prenylated proteins were resolved by SDS-PAGE and visualized by fluorography. The gel was exposed for either 2 days (Fig. IA) or 14 days (Fig. IB) .
  • Samples processed in the various lanes are as follows: 1 and 4, no added substrate protein; 2 and 6, H-Ras; 3 and 7, H- Ras-CVLL; 4 and 8, GST-lHDAg. Data shown are from a single experiment, which is representative of several such experiments.
  • Ras or H-Ras-CVLL were incubated with FTase (Fig. 2A) or GGTase-I (Fig. 2B) in the presence of either 2 ⁇ M [ 3 H]FPP or 2 ⁇ M [ 3 H] GGPP for 1 h at 37°C.
  • Prenylated proteins were resolved by SDS-PAGE and visualized by fluorography. Exposure time was 4 days.
  • Samples processed in the various lanes are as follows: 1 and 5, no added substrate protein; 2 and 6, H-Ras; 3 and 7, H- Ras-CVLL; 4 and 8, GST-lHDAg. Data shown are from a single experiment, which is representative of two such experiments.
  • FIG. 3 Kinetics of protein prenyltransferase modification of GST-lHDAg. Saturation curves for modification of GST-lHDAg, H-Ras, and H-Ras-CVLL by both FTase and GGTase-I were determined. Assays were conducted with either FTase and FPP (Fig. 3A) or GGTase- I and GGPP (Fig. 3B) . Reactions were stopped by addition of 2% SDS and prenylated proteins produced determined by filter binding assays. Data shown represent the mean of duplicate determinations from a single experiment, which is representative of several such experiments .
  • Fig. 4A COS cells were transfected with either an expression vector encoding MEV alone (lanes 1 and 3) or with expression vectors for both MEV and IHDAg (lanes 2 and 4) .
  • Detergent-solubilized extracts were prepared and either analyzed directly (lanes 1 and 2) or after immunoprecipitation using anti-IHDAg antibody (lanes 3 and 4) . Samples were resolved by SDS-PAGE and prenylated proteins visualized by fluorography. Exposure time was 3 days.
  • Figs. 4B-D Isoprenoid analysis of metabolically labelled IHDAg. Samples identical to those in Fig. 4A were subjected to isoprenoid analysis. Solubilized cell extract (Fig.
  • the prenylation motif at the C-terminus of the HDV large antigen IHDAg is necessary for the protein to interact with hepatitis B surface antigen in vi tro (Hwang et al, (1993) J. Virol . 67, 7659-7662) .
  • Prenylation of IHDAg is also required for HDV particle formation (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175; (Chang et al, (1994) J. Virol . 68, 646-653) . It has now been demonstrated that IHDAg is exclusively a substrate for FTase and that the protein is farnesylated.
  • the present invention thus provides a method of preventing viral particle formation and thereby inhibiting HDV infection using inhibitors of FTase.
  • a number of laboratories have developed potent
  • FTase inhibitors for use in the treatment of cancers that contain oncogenic Ras proceins (James et al, (1993) Science 260, 1937-1942; Gibbs et al, (1993) J. Biol . Chem. 268, 7617-7620; Tamanoi . (1993) Trends Biochem . Sci . 18, 349-353) . Many of these inhibitors are effective against certain tumors containing activated Ras proteins, and have been demonstrated to be relatively non-toxic in animals (Kohl et al, (1994) Proc . Na tl . Acad . Sci . USA 91, 9141-9145;
  • FTase inhibitors as well as others, to block the pathological consequences of HDV infections.
  • Compounds that can be used to inhibit famesylation of IHDAg by FTase include substrate analogs, farnesyl diphosphate analogs, bisubstrate analogs, non- competitive and other types of inhibitors (see Graham, Review, Oncologic, Endocrine and Metabolic, Exp. Opin. Ther. Patents 5 (12) : 1269-1285 (1995) , hereinafter "the Graham review” the contents of which is incorporated herein by reference) .
  • Bisubstrate inhibitors suitable for use in the invention are described in Manne et al, Oncogene 10:1763 (1995) , Patel et al, J. Med. Chem. 38:435 (1995) and Bhide et al, Biomed. Chem. Lett. 4:2107 (1994) , the contents of which are incorporated herein by reference.
  • Additional compounds suitable for use in the invention include derivatives of pyridobenzocycloheptene described in WO 9510516, WO 9510515, WO 9510514 and in the Graham review (those disclosures being incorporated herein by reference) .
  • the famesyltransferase inhibitors referenced above can be formulated as pharmaceutical compositions in accordance with the details provided in the respective disclosures .
  • the agent will be combined with a pharmaceutically acceptable carrier or diluent, according to standard pharmaceutical practice.
  • These compounds can be administered as indicated in the respective disclosures, for example, orally, or parenterally, including via intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes, providing that the route of administration results in distribution of the agent to the liver, the site of HDV infection. Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow.
  • Bovine brain cytosol was prepared by homogenizing bovine brain in 50 mM HEPES, pH 7.4, containing a protease inhibitor mix (Moomaw et al, (1995) Methods Enzymol . 250, 12-21) , and subjecting the homogenate to centrifugation at 100000 x g for 1 h.
  • Recombinant human H-Ras and H-Ras-CVLL were expressed in E. coli and purified as described (Casey et al, (1991) Proc . Natl . Acad. Sci . USA 88, 8631-8635) .
  • Recombinant rat FTase and rat GGTase-I were expressed in Sf9 cells and purified as described (Moomaw et al, (1995) Methods Enzymol . 250, 12-21; Chen et al, (1993) J. Biol . Chem . 268, 9675-9680; Zhang et al, (1994) J. Biol . Chem . 269, 23465-23470) .
  • GST-lHDAg The GST- lHDAg fusion protein was expressed in E. coli and purified essentially as described by Lee et al , ((1994) Virology 199, 169-175) .
  • a 500 ml flask of LB media was inoculated with 1 ml of an overnight culture of JM109 cells that had been transformed with the vector pGEX-2T- IHDAg (a bacterial expression vector containing the cDNA for a fusion protein between glutathione-S-transferase and the C-terminal 117 amino acids of IHDAg (Lee et al, (1994) Virology 199, 169-175) .
  • IPTG was added to 1 mM, and the cells were grown for an additional 4 h.
  • Cells were harvested by centrifugation at 3000 x g for 15 min, resuspended in lysis buffer (PBS containing 10 mM EDTA, 10 mM DTT, 1% Triton X-100, 10% streptomycin sulfate and protease inhibitor mix) , and lysed by three passes through a
  • the reactions were initiated by the addition of either bovine brain cytosol (500 ⁇ g protein) or purified recombinant FTase (75 ng) or GGTase-I (75 ng) , incubated for 1 h at 37°C, and stopped by addition of SDS-PAGE sample buffer and heating at 65°C for 5 min. Samples were resolved on 12% SDS-PAGE gels. Proteins were visualized by Coomassie Blue staining, and the gels were prepared for fluorography by rinsing in water and soaking for 45 min in 1 M salicylic acid. Prenylated proteins were visualized by exposing the gels to Fuji RX film.
  • the concentrations of prenylation-competent GST-lHDAg, H-Ras, and H-Ras-CVLL were determined by driving the prenylation reactions to completion in the presence of excess FTase or GGTase-I and the respective prenyldiphosphate, and determining the amount of protein prenylated by the filter binding assay described below.
  • Final concentrations of substrates ranging from 0.1 to 20 ⁇ M were utilized in kinetic assays. Assay conditions were identical to those described above, with the exception that the specific activities of [ 3 H] FPP and [ 3 H] GGPP were 3 Ci/mmol.
  • Reactions were initiated by the addition of 75 ng of FTase or GGTase-I, and incubated for 15 min at 37°C. The reactions were stopped by addition of 4% SDS, proteins precipitated by addition of trichloroacetic acid (TCA) , and prenylated proteins separated from free isoprenoid by filtration through nicrocellulose filcers as described (Reiss et al, (1990) Cell 62, 81-88) . The amount of prenylated protein retained on the filters was quantitated by liquid scintillation spectroscopy. Metabolic labelling of IHDAg wi th [ 3 H] mevalona ce in transfected COS cells .
  • COS-7 cells were grown in DMEM supplemented with 8% calf serum and 2% fetal calf serum. The cells were transfected using a DEAE/Dextran- chloroquine method (Lopatta et al, (1984) Nucleic Acids Res . 12, 5707-5717) .
  • DMEM containing 0.25 mg/ml DEAE/Dextran, 5 ⁇ g/ml pMEV a mammalian expression vector containing the cDNA for a mevalonate transporter (Kim et al, (1992) J. Biol . Chem .
  • pSV-lHDAg a mammalian cell expression vector containing the cDNA for IHDAg (Ryu et al, (1992) J. Virol . 66, 2310-2315) was added to COS-7 cells (3 X 10 6 cells/100 mm dish) . Following an incubation at 37°C for 1 h, 7 ml of supplemented DMEM containing 52 ⁇ g/ml chloroquine was added to the cells. After 6 h, the media was aspirated from the cells, the cells were washed with DMEM, and fresh supplemented DMEM was added.
  • lovastatin was added to the media to a final concentration of 15 ⁇ M. Following a 1 h incubation at 37°C, the media was aspirated, and supplemented DMEM containing 20 ⁇ M lovastatin and 50 ⁇ Ci/ml [5- 3 H] mevalonate was added. After an 11 h incubation at 37°C, the media was removed from the cells and the cells were washed with PBS and harvested by scraping.
  • Cells were pelleted by centrifugation, resuspended in 50 mM Hepes, pH 7.4, containing 1 mM EDTA, 1 mM DTT, and protease inhibitor mix, flash frozen in liquid nitrogen, thawed and lysed by passing several times through a 27 gauge needle.
  • Immunoprecipi ta tion of IHDAg from COS cell extracts Cellular proteins were solubilized from the lysed cell extracts by addition of Triton X-100 and NaCl to final concentrations of 1% and 250 mM, respectively.
  • Samples were incubated on ice for 30 min, passed through a 27 g syringe several times, and insoluble material precipitated by centrifugation at 100000 x g for 1 h at 4°C.
  • SDS was added to the solubilized protein co a final concentration of 0.5% and the sample was heated at 65°C for 5 min to denature proteins.
  • the sample was then adjusted to 0.25% SDS, 125 mM NaCl, 1% Triton X- 100, and 1% sodium cholate .
  • IHDAg was immunoprecipitated by incubation of the final extract obtained with rabbit anti-lHDAg (Wang et al, (1992) J. Gen . Virol .
  • Isoprenoid analysis Isoprenoid analysis was conducted essentially as described by Farnsworth et al . (Farnsworth et al , (1990) Me thods : A Companion to Meth . Enz . 1, 231-240) . Solubilized COS cell proteins or immunoprecipitated IHDAg were precipitated in 15% TCA and the resulting protein pellets washed extensively in acetone at -20°C. Precipitated proteins were subjected to trypsin digestion, and isoprenoids cleaved from the tryptic peptides by treatment with methyl iodide in 2% formic acid.
  • H-Ras containing the C-terminus -CVLS, and thus a substrate for FTase
  • H-Ras-CVLL containing the C-terminus -CVLL, and thus a substrate for GGTase-I
  • Isoprenoid analysis was performed on IHDAg expressed in COS cells.
  • COS cells transfected with a vector expressing IHDAg were incubated with [ 3 H] mevalonate, a precursor for both FPP and GGPP, resulting in the metabolic labelling of prenylated proteins (Schmidt et al, (1984) J. Biol . Chem . 259, 10175-10180) .
  • the cells were co- transfected with a second plasmid coding for a mevalonate transport protein (MEV) and also treated with lovastatin.
  • MMV mevalonate transport protein
  • the isoprenoid modifying IHDAg was cleaved from the protein by methyliodide cleavage, and the liberated lipid identified by HPLC. Isoprenoid analysis was performed both on the total pool of prenylated proteins present in solubilized extracts from COS cells, and on immunoprecipitated IHDAg. For the total pool of prenylated proteins, approximately 20% of the protein- associated isoprenoid was farnesyl and 80% was geranylgeranyl (Fig. 4B) , a ratio consistent with previous studies (Rilling et al, (1993) Arch . Biochem . Biophys . 301, 210-215) .
  • FTase famesyltransferase specific inhibitor SCH56582
  • SCH56582 which is structurally closely related to SCH44342 described in Buss et al, Chemistry & Biology (1995) , 2:787-791
  • IHDAg glutathione-S-transferase-lHDAg fusion protein
  • the standard assay included 75 ng FTase, 2 ⁇ M farnesyl diphosphate, and either 1.5 ⁇ M IHDAg or 2.5 ⁇ M Ha-Ras, a well characterized FTase substrate.
  • SCH56582 was included in these assays at concentrations ranging from 0-500 nM. Assays were initiated by the addition of FTase, and were conducted at 37°C for 15 min.
  • the effect of the FTase specific inhibitor B581 (see Garcia et al, (1993) J. Biol. Chem. 268:18415) on the famesylation of the glutathione-S-transferase-lHDAg fusion protein (IHDAg) was also examined in vi tro .
  • the standard assay included 75ng FTase, 2 ⁇ M farnesyl diphosphate, and either 1 ⁇ M IHDAg or 2 ⁇ M Ha-Ras.
  • B581 was included in the assays at concentrations ranging from 0-500nM. Assays were initiated by the addition of FTase, and were conducted at 37°C for 15 min. The results shown in Fig. 5B demonstrate that B581 inhibits famesylation of IHDAg in vi tro with an IC S0 of ⁇ 50nM

Abstract

The present invention relates, in general, to hepatitis delta virus, and in particular, to a method of treating hepatitis delta virus infection. The method involves inhibiting farnesylation of the delta virus large antigen using an inhibitor of farnesyltransferase.

Description

METHOD OF TREATING HEPATITIS DELTA VIRUS INFECTION
This invention was made with Government support (NIH Grant No. GM46372) . Accordingly, the Government has rights in the invention.
TECHNICAL FIELD
The present invention relates, in general, to hepatitis delta virus, and in particular, to a method of treating hepatitis delta virus infeccion. The method involves inhibiting famesylation of the delta virus large antigen using an inhibitor of famesyltransferase.
BACKGROUND
Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus that can cause an increase in the incidence and severity of liver disease in individuals infected with both viruses (Rizzetto, (1983) Heptology 3, 729-737; Lai, (1995) Annu. Rev. Biochem . 64, 259-286) . HDV consists of the HDV RNA genome and two HDV encoded proteins, designated as the small (sHDAg) and large (IHDAg) HDV antigens, encapsulated in an envelope composed of hepatitis B surface antigens (Lai, (1995) Annu . Rev. Biochem . 64, 259-286) . sHDAg and IHDAg contain identical deduced amino acid sequences for their first 195 amino acids, with IHDAg containing an additional 19 amino acid C-terminal tail (Lai, (1995) Annu. Rev. Biochem . 64, 259-286.) . Despite their sequence identity, sHDAg and IHDAg have very different functions. Whereas sHDAg is essential for HDV replication (Kuo et al, (1989) J. Virol . 63, 1945-1950) , IHDAg is a dominant inhibitor of HDV replication (Chao et al, (1990) J. Virol . 64,
5066-5069) , and is required for assembly of the HDV particle (Chang et al, (1991) Proc. Na tl . Acad . Sci . USA 88, 8490-8494; Ryu et al, (1992) J. Virol . 66, 2310-2315) . As IHDAg can package into pseudo-viral particles with hepatitis B surface antigens in the absence of both HDV RNA and sHDAg (Ryu et al, (1992) J. Virol . 66, 2310-2315; Chen et al, (1992) J". Virol . 66, 2853-2859) , the current model of HDV assembly indicates that IHDAg functions by directly interacting with hepatitis B surface antigens that form the envelope of the viral particle (Lai, (1995) Annu. Rev. Biochem. 64, 259-286) .
IHDAg is modified by an isoprenoid lipid on a cysteine located near its C-terminus (Glenn et al, (1992) Science 256, 1331-1333) , and this modification is necessary for IHDAg to facilitate HDV assembly (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175) . The prenylation motif contained in IHDAg is the C-terminal tetrapeptide Cys-Arg-Pro-Gln (CRPQ) (Glenn et al, (1992) Science 256, 1331-1333) .
This sequence is similar to the conventional prenylation motif which consists of the C-terminal tetrapeptide CaaX, where the cysteine residue is the prenylation site, "a" are generally aliphatic residues, and X can be one of several amino acids. Two distinct protein prenyltransferases modify proteins which contain a CaaX motif: famesyltransferase (FTase) , which modifies proteins with the 15-carbon farnesyl isoprenoid, and geranylgeranyltransferase-I (GGTase-I) , which modifies proteins with the 20-carbon geranylgeranyl group (Glomset et al, (1994) Annu. Rev. Cell Biol . 10, 181-205; Zhang et al, (1996) Annu . Rev. Biochem . 65:241- 269) . The C-terminal amino acid (i.e. the "X") in general determines which of the two isoprenoid lipids modify a CaaX motif. If X is Met, Ser or Gin, the sequence is a substrate for FTase (Moores et al, (1991) J. Biol . Chem . 266, 14603-14610) , while Leu at this position directs modification by GGTase-I (Moores et al, (1991) J. Biol . Chem . 266, 14603-14610; Casey et al , (1991) Proc . Na tl . Acad . Sci . USA 88, 8631-8635; Yokoyama et al, (1991) Proc . Na tl . Acad . Sci . USA 88, 5302-5306) . A third protein prenyltransferase, GGTase- II, recognizes a different class of C-terminal motifs present in GTP-binding proteins of the Rab family
(Seabra et al, (1992) Cell 70, 1049-1057) . Following the prenylation of the CaaX motif, two additional processing events occur - proteolytic cleavage of the -aaX tripeptide and carboxymethylation of the new C- terminal prenylcysteine (Glomset et al , (1994) Annu.
Rev. Cell Biol . 10, 181-205; Zhang et al , (1996) Annu . Rev. Biochem . 65:241-269) .
As prenylation of IHDAg is required for HDV assembly, inhibition of the enzyme responsible for its prenylation provides a treatment of HDV infection. Two reports have appeared indicating that IHDAg is modified with the geranylgeranyl isoprenoid (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175) . The present invention results, at least in part, from the realizations that the predominant modification of IHDAg is famesylation rather than geranylgeranylation, and that the protein is exclusively a substrate for FTase.
SUMMARY OF THE INVENTION
The present invention relates to a method of inhibiting hepatitis delta virus (HDV) assembly. The method comprises contacting an HDV-infected cell wich an amount of a famesyltransferase inhibitor sufficient to inhibit famesylation of IHDAg and thereby inhibit HDV assembly. The invention further relates to a method of treating a patient infected with HDV. The method comprises administering to the patient an amount of a famesyltransferase inhibitor sufficient to inhibit famesylation of IHDAg and thereby effect the treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Prenylation of GST-lHDAg by bovine brain cytosol . GST-lHDAg, H-Ras, and H-Ras-CVLL were incubated with bovine brain cytosol in the presence of either 2 μM [3H] FPP or 2 μM [3H] GGPP for 1 h at 37°C. Prenylated proteins were resolved by SDS-PAGE and visualized by fluorography. The gel was exposed for either 2 days (Fig. IA) or 14 days (Fig. IB) . Samples processed in the various lanes are as follows: 1 and 4, no added substrate protein; 2 and 6, H-Ras; 3 and 7, H- Ras-CVLL; 4 and 8, GST-lHDAg. Data shown are from a single experiment, which is representative of several such experiments.
Figure 2. Prenylation of GST-lHDAg by purified recombinant protein prenyltransferases . GST-lHDAg, H-
Ras or H-Ras-CVLL were incubated with FTase (Fig. 2A) or GGTase-I (Fig. 2B) in the presence of either 2 μM [3H]FPP or 2 μM [3H] GGPP for 1 h at 37°C. Prenylated proteins were resolved by SDS-PAGE and visualized by fluorography. Exposure time was 4 days. Samples processed in the various lanes are as follows: 1 and 5, no added substrate protein; 2 and 6, H-Ras; 3 and 7, H- Ras-CVLL; 4 and 8, GST-lHDAg. Data shown are from a single experiment, which is representative of two such experiments.
Figure 3. Kinetics of protein prenyltransferase modification of GST-lHDAg. Saturation curves for modification of GST-lHDAg, H-Ras, and H-Ras-CVLL by both FTase and GGTase-I were determined. Assays were conducted with either FTase and FPP (Fig. 3A) or GGTase- I and GGPP (Fig. 3B) . Reactions were stopped by addition of 2% SDS and prenylated proteins produced determined by filter binding assays. Data shown represent the mean of duplicate determinations from a single experiment, which is representative of several such experiments .
Figure 4. Prenylation of IHDAg in animal cells.
Fig. 4A. COS cells were transfected with either an expression vector encoding MEV alone (lanes 1 and 3) or with expression vectors for both MEV and IHDAg (lanes 2 and 4) . Detergent-solubilized extracts were prepared and either analyzed directly (lanes 1 and 2) or after immunoprecipitation using anti-IHDAg antibody (lanes 3 and 4) . Samples were resolved by SDS-PAGE and prenylated proteins visualized by fluorography. Exposure time was 3 days. Figs. 4B-D. Isoprenoid analysis of metabolically labelled IHDAg. Samples identical to those in Fig. 4A were subjected to isoprenoid analysis. Solubilized cell extract (Fig. 4B) or samples subjected to immunoprecipitation with anti- lHDAg (Figs. 4C and D) were processed by TCA precipitation and isoprenoid lipids cleaved from prenylated proteins using methyl iodide. Farnesol (C15) and geranylgeraniol (C20) were added to the samples as internal standards, and released isoprenoids were resolved by reverse-phase HPLC. Fractions were analyzed by liquid scintillation spectroscopy. Isoprenoid analysis was performed on the following samples: Fig. 4B, detergent-solubilized extract of cells expressing both MEV and IHDAg (corresponding to Fig. 4A, lane 2) ; Fig. 4C, immunoprecipitate from cells expressing both MEV and IHDAg (corresponding to Fig. 4A, lane 4) ; Fig. 4D, immunoprecipitate from cells expressing only MEV (corresponding to Fig. 4A, lane 3) . For all panels, data shown are from a single experiment, which is representative of data obtained from two different transfections .
Figure 5. Inhibition of the famesylation of IHDAg in vi tro . Fig. 5A. SCH56582. Fig. 5B . B581.
DETAILED DESCRIPTION OF THE INVENTION
The prenylation motif at the C-terminus of the HDV large antigen IHDAg is necessary for the protein to interact with hepatitis B surface antigen in vi tro (Hwang et al, (1993) J. Virol . 67, 7659-7662) . Prenylation of IHDAg is also required for HDV particle formation (Glenn et al, (1992) Science 256, 1331-1333; Lee et al, (1994) Virology 199, 169-175; (Chang et al, (1994) J. Virol . 68, 646-653) . It has now been demonstrated that IHDAg is exclusively a substrate for FTase and that the protein is farnesylated. The present invention thus provides a method of preventing viral particle formation and thereby inhibiting HDV infection using inhibitors of FTase. A number of laboratories have developed potent
FTase inhibitors for use in the treatment of cancers that contain oncogenic Ras proceins (James et al, (1993) Science 260, 1937-1942; Gibbs et al, (1993) J. Biol . Chem. 268, 7617-7620; Tamanoi . (1993) Trends Biochem . Sci . 18, 349-353) . Many of these inhibitors are effective against certain tumors containing activated Ras proteins, and have been demonstrated to be relatively non-toxic in animals (Kohl et al, (1994) Proc . Na tl . Acad . Sci . USA 91, 9141-9145;
Kohl et al, (1995) Na ture Med . 1 , 792-797) . The present invention relates to the use of such FTase inhibitors, as well as others, to block the pathological consequences of HDV infections. Compounds that can be used to inhibit famesylation of IHDAg by FTase include substrate analogs, farnesyl diphosphate analogs, bisubstrate analogs, non- competitive and other types of inhibitors (see Graham, Review, Oncologic, Endocrine and Metabolic, Exp. Opin. Ther. Patents 5 (12) : 1269-1285 (1995) , hereinafter "the Graham review" the contents of which is incorporated herein by reference) . (See also Buss et al, (1995) Chemistry & Biology 2:787-791, the contents of which is incorporated by reference) Substrate analogs (ie CaaX analogs) suitable for use in the invention are described, for example, in USP 5420245, EP-461869-A, EP 520823-A, EP-523873-A, EP 528486-A, WO 9404561, EP 535730-A, USP 5340828, EP 535731-A, USP 5238922, USP 5326773, WO 9410137, EP 618221-A, WO 9410138, WO 9409766, WO 9426723, USP 5352705, WO 9500497, WO 9509001, WO 9509000, WO 9511917 and WO 9512612. The Graham review makes reference to the above-indicated disclosures as well as others from the technical literature (the above disclosures and the literature references cited in the Graham review being incorporated herein by reference) .
Farnesyl diphosphate analogs and antagonists suitable for use in the invention are described, for example, in USP 5362906, EP 540782-A, EP 534546-A, EP 537007-A, EP 537008-A, WO 9419357, EP 547669-A, USP 5245061, USP 5350867, EP 547670-A, USP 5260465, EP 547671-A, USP 5420157, EP 547672-A, USP 5260479, WO 9512572, WO 9316980, WO 9408940, USP 5286895, USP 5254727, WO 9418157, USP 5430055, and WO 9508546. The Graham review makes reference to these disclosures, which are also incorporated herein by reference, and others from the technical literature, the contents of which are incorporated herein by reference .
Bisubstrate inhibitors suitable for use in the invention are described in Manne et al, Oncogene 10:1763 (1995) , Patel et al, J. Med. Chem. 38:435 (1995) and Bhide et al, Biomed. Chem. Lett. 4:2107 (1994) , the contents of which are incorporated herein by reference. Additional compounds suitable for use in the invention include derivatives of pyridobenzocycloheptene described in WO 9510516, WO 9510515, WO 9510514 and in the Graham review (those disclosures being incorporated herein by reference) . Further disclosures of suitable compounds can be found in WO 9324643, USP 5436263, USP 5420334, WO 9508542, WO 9318651, USP 5276217, and USP 5349112. These disclosures and others from the technical literature are cited in the Graham review and are incorporated herein by reference.
Compounds suitable for use in the present invention can be prepared in accordance with the disclosures referenced above.
It will be clear from a review of the above- referenced documents that the amount of compound to be administered to effect inhibition of farnesyl trans erase varies with the agent. These disclosures provide ample guidance from which an optimum dose can be selected for a particular patient, given the patient's, age, weight, status and responsiveness. These disclosures also provide guidelines as to administration regimens suitable for use in inhibiting IHDAg famesylation, which will also vary with the agent and the patient.
The famesyltransferase inhibitors referenced above can be formulated as pharmaceutical compositions in accordance with the details provided in the respective disclosures . Typically, the agent will be combined with a pharmaceutically acceptable carrier or diluent, according to standard pharmaceutical practice. These compounds can be administered as indicated in the respective disclosures, for example, orally, or parenterally, including via intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes, providing that the route of administration results in distribution of the agent to the liver, the site of HDV infection. Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLES
The following experimental protocols are referenced in the Examples that follow.
Protein prenyl transf erases and protein subs tra tes . Bovine brain cytosol was prepared by homogenizing bovine brain in 50 mM HEPES, pH 7.4, containing a protease inhibitor mix (Moomaw et al, (1995) Methods Enzymol . 250, 12-21) , and subjecting the homogenate to centrifugation at 100000 x g for 1 h. Recombinant human H-Ras and H-Ras-CVLL were expressed in E. coli and purified as described (Casey et al, (1991) Proc . Natl . Acad. Sci . USA 88, 8631-8635) . Recombinant rat FTase and rat GGTase-I were expressed in Sf9 cells and purified as described (Moomaw et al, (1995) Methods Enzymol . 250, 12-21; Chen et al, (1993) J. Biol . Chem . 268, 9675-9680; Zhang et al, (1994) J. Biol . Chem . 269, 23465-23470) .
Expression and purifica tion of GST-lHDAg. The GST- lHDAg fusion protein was expressed in E. coli and purified essentially as described by Lee et al , ((1994) Virology 199, 169-175) . A 500 ml flask of LB media was inoculated with 1 ml of an overnight culture of JM109 cells that had been transformed with the vector pGEX-2T- IHDAg (a bacterial expression vector containing the cDNA for a fusion protein between glutathione-S-transferase and the C-terminal 117 amino acids of IHDAg (Lee et al, (1994) Virology 199, 169-175) . After incubation for 4 h at 37°C, IPTG was added to 1 mM, and the cells were grown for an additional 4 h. Cells were harvested by centrifugation at 3000 x g for 15 min, resuspended in lysis buffer (PBS containing 10 mM EDTA, 10 mM DTT, 1% Triton X-100, 10% streptomycin sulfate and protease inhibitor mix) , and lysed by three passes through a
French press. Cell debris was removed by centrifugation at 45000 x g for 1 h, and the supernatant was incubated with glutathione-Sepharose 4B for 1 h at room temperature. The mixture was transferred to a column, and the column was washed with 20 volumes of PBS containing 850 mM NaCl, 10 mM EDTA, 10 mM DTT, and 1% Triton X-100. GST-lHDAg was then eluted from the column with 50 mM Tris-HCl, pH 7.7, containing 5 mM glutathione, and concentrated to 40 mg/ml using an Amicon Centricon-30.
In vi tro prenyla tion reactions . GST-lHDAg (20 μg) , H-Ras (2 μg) or H-Ras-CVLL (2 μg) were utilized in prenylation reactions that contained 50 mM Tris-HCl, pH 7.7, 5 mM MgCl2, 5 μM ZnCl, 2 mM DTT and either 2 μM [3H]FPP (8 Ci/mmol) or 2 μM [3H] GGPP (8 Ci/mmol) (Chen et al, (1993) J. Biol . Chem . 268, 9675-9680; Zhang et al , (1994) J. Biol . Chem . 269, 23465-23470) . The reactions were initiated by the addition of either bovine brain cytosol (500 μg protein) or purified recombinant FTase (75 ng) or GGTase-I (75 ng) , incubated for 1 h at 37°C, and stopped by addition of SDS-PAGE sample buffer and heating at 65°C for 5 min. Samples were resolved on 12% SDS-PAGE gels. Proteins were visualized by Coomassie Blue staining, and the gels were prepared for fluorography by rinsing in water and soaking for 45 min in 1 M salicylic acid. Prenylated proteins were visualized by exposing the gels to Fuji RX film.
For kinetic determinations, the concentrations of prenylation-competent GST-lHDAg, H-Ras, and H-Ras-CVLL were determined by driving the prenylation reactions to completion in the presence of excess FTase or GGTase-I and the respective prenyldiphosphate, and determining the amount of protein prenylated by the filter binding assay described below. Final concentrations of substrates ranging from 0.1 to 20 μM were utilized in kinetic assays. Assay conditions were identical to those described above, with the exception that the specific activities of [3H] FPP and [3H] GGPP were 3 Ci/mmol. Reactions were initiated by the addition of 75 ng of FTase or GGTase-I, and incubated for 15 min at 37°C. The reactions were stopped by addition of 4% SDS, proteins precipitated by addition of trichloroacetic acid (TCA) , and prenylated proteins separated from free isoprenoid by filtration through nicrocellulose filcers as described (Reiss et al, (1990) Cell 62, 81-88) . The amount of prenylated protein retained on the filters was quantitated by liquid scintillation spectroscopy. Metabolic labelling of IHDAg wi th [3H] mevalona ce in transfected COS cells . COS-7 cells were grown in DMEM supplemented with 8% calf serum and 2% fetal calf serum. The cells were transfected using a DEAE/Dextran- chloroquine method (Lopatta et al, (1984) Nucleic Acids Res . 12, 5707-5717) . DMEM containing 0.25 mg/ml DEAE/Dextran, 5 μg/ml pMEV (a mammalian expression vector containing the cDNA for a mevalonate transporter (Kim et al, (1992) J. Biol . Chem . 267, 23113-23121) , and 5 μg/ml pSV-lHDAg (a mammalian cell expression vector containing the cDNA for IHDAg (Ryu et al, (1992) J. Virol . 66, 2310-2315) was added to COS-7 cells (3 X 106 cells/100 mm dish) . Following an incubation at 37°C for 1 h, 7 ml of supplemented DMEM containing 52 μg/ml chloroquine was added to the cells. After 6 h, the media was aspirated from the cells, the cells were washed with DMEM, and fresh supplemented DMEM was added. At 41 h post-transfection, lovastatin was added to the media to a final concentration of 15 μM. Following a 1 h incubation at 37°C, the media was aspirated, and supplemented DMEM containing 20 μM lovastatin and 50 μCi/ml [5-3H] mevalonate was added. After an 11 h incubation at 37°C, the media was removed from the cells and the cells were washed with PBS and harvested by scraping. Cells were pelleted by centrifugation, resuspended in 50 mM Hepes, pH 7.4, containing 1 mM EDTA, 1 mM DTT, and protease inhibitor mix, flash frozen in liquid nitrogen, thawed and lysed by passing several times through a 27 gauge needle. Immunoprecipi ta tion of IHDAg from COS cell extracts . Cellular proteins were solubilized from the lysed cell extracts by addition of Triton X-100 and NaCl to final concentrations of 1% and 250 mM, respectively. Samples were incubated on ice for 30 min, passed through a 27 g syringe several times, and insoluble material precipitated by centrifugation at 100000 x g for 1 h at 4°C. SDS was added to the solubilized protein co a final concentration of 0.5% and the sample was heated at 65°C for 5 min to denature proteins. The sample was then adjusted to 0.25% SDS, 125 mM NaCl, 1% Triton X- 100, and 1% sodium cholate . IHDAg was immunoprecipitated by incubation of the final extract obtained with rabbit anti-lHDAg (Wang et al, (1992) J. Gen . Virol . 73, 183-188) for 15 min, followed by incubation with protein A-sepharose 4B for 15 min. The protein A-sepharose 4B beads were pelleted by centrifugation, and were washed extensively with 0.1 M Tris, pH 7.7, containing 0.1% Triton X-100. Immunoprecipitated proteins were eluted from the beads either by addition of SDS-PAGE sample buffer (for SDS- PAGE) or by two washes with 0.2 M acetic acid (for isoprenoid analysis) . Immunoprecipitation of IHDAg was monitored by fluorography of SDS-PAGE gels.
Isoprenoid analysis . Isoprenoid analysis was conducted essentially as described by Farnsworth et al . (Farnsworth et al , (1990) Me thods : A Companion to Meth . Enz . 1, 231-240) . Solubilized COS cell proteins or immunoprecipitated IHDAg were precipitated in 15% TCA and the resulting protein pellets washed extensively in acetone at -20°C. Precipitated proteins were subjected to trypsin digestion, and isoprenoids cleaved from the tryptic peptides by treatment with methyl iodide in 2% formic acid. Following cleavage, methyl iodide was removed under mild vacuum and the solution neutralized by addition of sodium carbonate. Isoprenoids were extracted into a solution of chloroform:methanol (9:1) , and the extracted isoprenoids dried under nitrogen and resuspended in 50% acetonitrile containing 0.1% phosphoric acid. Farnesol and geranylgeraniol (15 nmol of each) were added to the samples as standards, and isoprenoids resolved by HPLC using a 50%-100% gradient of acetonitrile in 0.1% phosphoric acid. Fraccions containing [3H] -labelled isoprenoids were identified by liquid scintillation spectroscopy.
Example 1
In Vitro Prenylation of GST-lHDAg in Bovine Brain Cytosol
In order to identify the protein prenyltransferase responsible for the modification of IHDAg, it was first determined whether a fusion protein between glutathione- S transferase and the C-terminal 117 amino acids of IHDAg (GST-lHDAg) (Lee et al, (1994) Virology 199,
169-175) was a substrate for these enzymes present in bovine brain cytosol. Initial experiments indicated that GST-lHDAg was exclusively farnesylated, with no evident modification with the geranylgeranyl isoprenoid (Fig. IA, lanes 4 and 8) . Prolonged exposure of the gel revealed that a low level of geranylgeranylation of GST- lHDAg occurred (Fig. IB, lane 8) ; the level of geranylgeranylation of GST-lHDAg was approximately 1% of the level of famesylation. Two recombinant Ras proteins, H-Ras (containing the C-terminus -CVLS, and thus a substrate for FTase) and H-Ras-CVLL (containing the C-terminus -CVLL, and thus a substrate for GGTase-I) were used as control substrates for the two enzymes, and clearly demonstrated the presence of each enzyme in bovine brain cytosol (Fig IA) . These results indicated that IHDAg is a substrate for FTase.
Example 2
In Vitro Prenylation of GST-lHDAg Using
Purified Recombinant Protein Prenyltransferases
To further assess the ability of GST-lHDAg to serve as a substrate for FTase, the ability of recombinant FTase and GGTase-I to prenylate GST-lHDAg was examined. FTase efficiently transferred the farnesyl group from FPP to GST-lHDAg, and the enzyme also supported a low level of geranylgeranylation, again approximately 1% of the level of famesylation (Fig. 2A, lanes 4 and 8) . GGTase-I was unable to modify GST-lHDAg using either FPP or GGPP as substrates (Fig. 2B, lanes 4 and 8) . These results were confirmed by a more detailed kinetic analysis of the interaction of GST-lHDAg with the two enzymes (Fig. 3) . The fusion protein was found to be an excellent substrate for FTase with an apparent K„ of 0.8 μM, which compares favorably to that measured for H-Ras (Fig.3A) . GGTase-I, however, did not utilize GST-lHDAg as a substrate over the range concentrations tested (Fig. 3B) .
Example 3
Isoprenoid Analysis of IHDAg Expressed in Animal Cells
Isoprenoid analysis was performed on IHDAg expressed in COS cells. COS cells transfected with a vector expressing IHDAg were incubated with [3H] mevalonate, a precursor for both FPP and GGPP, resulting in the metabolic labelling of prenylated proteins (Schmidt et al, (1984) J. Biol . Chem . 259, 10175-10180) . In order to increase the labelling of prenylated proteins in the COS cells, the cells were co- transfected with a second plasmid coding for a mevalonate transport protein (MEV) and also treated with lovastatin. Expression of MEV serves to increase the uptake of exogenous mevalonate (Kim et al, (1992) J Biol Chem 267, 23113-23121), while lovastatin treatment inhibits the de novo synthesis of mevalonate by the cell (Endo, (1992) J. Lipid Res . 33, 1569-1582) .
[3H] Isoprenoid-labelled proteins were observed in solubilized COS cell extracts prepared from cells expressing either MEV alone or both MEV and IHDAg (Fig. 4A, lanes 1 and 2) ; in the latter condition an additional labelled protein of 27 kDa, the predicted size of IHDAg, could be faintly observed. This 27 kDa protein was readily immunoprecipitated from solubilized COS cell extracts using an anti-IHDAg antibody, confirming its identity as the viral antigen (Fig. 4A, lane 4) .
The isoprenoid modifying IHDAg was cleaved from the protein by methyliodide cleavage, and the liberated lipid identified by HPLC. Isoprenoid analysis was performed both on the total pool of prenylated proteins present in solubilized extracts from COS cells, and on immunoprecipitated IHDAg. For the total pool of prenylated proteins, approximately 20% of the protein- associated isoprenoid was farnesyl and 80% was geranylgeranyl (Fig. 4B) , a ratio consistent with previous studies (Rilling et al, (1993) Arch . Biochem . Biophys . 301, 210-215) . Isoprenoid analysis of the immunoprecipitated IHDAg, however, revealed that the protein was exclusively modified by the farnesyl isoprenoid (Fig. 4C) ; the amount of geranylgeranyl lipid in immunoprecipitated IHDAg was essentially identical to that observed when COS cells not expressing IHDAg were subjected to the same procedure (compare Fig. 4C and D) . These results are completely consistent with the in vi tro data that indicated that IHDAg was a FTase substrate. It is believed that the geranylgeranylation of IHDAg observed by others in vi tro (Lee et al, (1994) Virol ogy 199, 169-175) was mediated by FTase. Example 4
Inhibition of Famesylation of IHDAg In Vitro
The effect of the famesyltransferase (FTase) specific inhibitor SCH56582 (which is structurally closely related to SCH44342 described in Buss et al, Chemistry & Biology (1995) , 2:787-791) on the famesylation of a glutathione-S-transferase-lHDAg fusion protein (IHDAg) was examined in vi tro . The standard assay included 75 ng FTase, 2 μM farnesyl diphosphate, and either 1.5 μM IHDAg or 2.5 μM Ha-Ras, a well characterized FTase substrate. SCH56582 was included in these assays at concentrations ranging from 0-500 nM. Assays were initiated by the addition of FTase, and were conducted at 37°C for 15 min.
The results shown in Fig. 5A demonstrate that this inhibitor of famesyltransferase inhibits famesylation of IHDAg in vi tro with an IC50 of -8nm.
The effect of the FTase specific inhibitor B581 (see Garcia et al, (1993) J. Biol. Chem. 268:18415) on the famesylation of the glutathione-S-transferase-lHDAg fusion protein (IHDAg) was also examined in vi tro . The standard assay included 75ng FTase, 2μM farnesyl diphosphate, and either 1 μM IHDAg or 2 μM Ha-Ras. B581 was included in the assays at concentrations ranging from 0-500nM. Assays were initiated by the addition of FTase, and were conducted at 37°C for 15 min. The results shown in Fig. 5B demonstrate that B581 inhibits famesylation of IHDAg in vi tro with an ICS0 of ~50nM
(Ha-Ras-ICs0 of -90nM) .
All documents cited above are hereby incorporated in their entirety by reference.
One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

Claims

WHAT IS CLAIMED TS :
1. A method of inhibiting hepatitis delta virus (HDV) assembly in a cell comprising contacting an HDV- infected cell with an amount of a famesyltransferase inhibitor sufficient to effect said inhibition.
2. The method according to claim 1 wherein said inhibitor inhibits famesylation of the IHDAg antigen of HDV.
3. The method acording to claim 1 wherein said inhibitor is B581.
4. The method according to claim 1 wherein said inhibitor is a pyridobenzocycloheptene derivative.
5. A method of treating a patient infected with HDV comprising administering to the patient an amount of a famesyltransferase inhibitor sufficient to effect said treatment .
6. The method according to claim 5 wherein said inhibitor inhibits famesylation of the IHDAg antigen of HDV.
7. The method acording to claim 5 wherein said inhibitor is B581.
8. The method according to claim 5 wherein said inhibitor is a pyridobenzocycloheptene derivative.
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