Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-02-25
2001-10-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S023000, C435S183000, C435S219000, 36
Reexamination Certificate
active
06303287
ABSTRACT:
TECHNICAL FIELD OF INVENTION
The present invention relates to compositions and crystals of a hepatitis C virus protease in complex with its viral cofactor. This invention also relates to methods of using the structure coordinates of hepatitis C virus protease in complex with a synthetic NS4A to solve the structure of similar or homologous proteins or protein complexes.
BACKGROUND OF THE INVENTION
Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A, non-B hepatitis, with an estimated human seroprevalence of 1% globally [Choo, Q.-L. et al., “Isolation of a cDNA Clone Derived From a Blood-Borne Non-A, Non-B Viral Hepatitis Genome”,
Science
, 244, pp. 359-362 (1989); Kuo, G. et al., “An Assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A, Non-B Hepatitis”,
Science
, 244, pp. 362-364 (1989); Purcell, R. H., “Hepatitis C virus: Historical perspective and current concepts”,
FEMS Microbiology Reviews
, 14, pp. 181-192 (1994); Van der Poel, C. L., “Hepatitis C Virus. Epidemiology, Transmission and Prevention in Hepatitis C virus. Current Studies in Hematology and Blood Transfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million individuals may be infected in the United States alone [Alter, M. J. and Mast, E. E., “The Epidemiology of Viral Hepatitis in the United States,
Gastroenterol. Clin. North Am
., 23, pp. 437-455 (1994)].
Upon first exposure to HCV only about 20% of infected individuals develop acute clinical hepatitis while others appear to resolve the infection spontaneously. In most instances, however, the virus establishes a chronic infection that persists for decades [Iwarson, S. “The Natural Course of Chronic Hepatitis”,
FEMS Microbiology Reviews
, 14, pp. 201-204 (1994)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [Kew, M. C., “Hepatitis C and Hepatocellular Carcinoma”,
FEMS Microbiology Reviews
, 14, pp. 211-220 (1994); Saito, I., et al. “Hepatitis C Virus Infection is Associated with the Development of Hepatocellular Carcinoma”,
Proc. Natl. Acad. Sci. USA
87, pp. 6547-6549 (1990)]. Currently, there are no broadly effective treatments for the debilitating progression of chronic HCV.
The HCV genome encodes a polyprotein of 3010-3033 amino acids (
FIG. 1
) [Choo, Q.-L., et al. “Genetic Organization and Diversity of the Hepatitis C Virus”,
Proc. Natl. Acad. Sci. USA
, 88, pp. 2451-2455 (1991); Kato, N. et al., Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis”,
Proc. Natl. Acad. Sci. USA
, 87, pp. 9524-9528 (1990); Takamizawa, A. et al., “Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers”,
J. Virol
., 65, pp. 1105-1113 (1991)]. The HCV nonstructural (NS) proteins provide catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [Bartenschlager, R. et al., “Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions”,
J. Virol
., 67, pp. 3835-3844 (1993); Grakoui, A. et al. “Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites”,
J. Virol
., 67, pp. 2832-2843 (1993); Grakoui, A. et al., Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products”,
J. Virol
., 67, pp. 1385-1395 (1993); Tomei, L. et al., “NS3 is a serine protease required for processing of hepatitis C virus polyprotein”,
J. Virol
., 67, pp. 4017-4026 (1993)].
The HCV NS protein 3 (NS3) contains a serine protease activity that helps process the majority of the viral enzymes, and is thus considered essential for viral replication and infectivity. It is known that mutations in the yellow fever virus NS3 protease decreases viral infectivity [Chambers, T. J. et. al., “Evidence that the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein”,
Proc. Natl. Acad. Sci. USA
, 87, pp. 8898-8902 (1990)]. The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein (
FIG. 1
) [C. Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”,
J. Virol
., 68, pp. 8147-8157 (1994)].
NS3 is associated with a cofactor, NS4A. NS4A seems critical to the activity of NS3, enhancing the proteolytic efficiency of NS3 at all of the cleavage sites. NS4A is a 54 residue amphipathic peptide, with a hydrophobic N-terminus and a hydrophilic C-terminus [Failla, C. et al., “Both NS3 and NS4A are Required for Proteolytic Processing of Hepatitis C Virus Nonstructural Proteins”,
J. Virol
., 68, pp. 3753-3760 (1994)]. Its function appears complex, possibly assisting in the membrane-localization of NS3 and other viral replicase components [Lin, C. et al. “A Central Region in the Hepatitis C Virus NS4A Protein Allows Formation of an Active NS3-NS4A Serine Proteinase Complex In Vivo and In Vitro”,
J. Virol
., 69, pp. 4373-4380 (1995b); Shimizu, Y. et al., “Identification of the Sequence on NS4A Required for Enhanced Cleavage of the NS5A/5B Site by Hepatitis C Virus NS3 Protease”,
J. Virol
., 70, pp. 127-132 (1996); Tanji,.Y. et al., “Hepatitis C Virus-Encoded Nonstructural Protein NS4A has Versatile Functions in Viral Protein Processing”,
J. Virol
., 69, pp. 1575-1581 (1995)] but its best characterized function is that of a cofactor for the NS3 protease.
The current understanding of HCV has not led to satisfactory treatments for HCV infection. The prospects for effective anti-HCV vaccines remain uncertain. The only established therapy for HCV disease is interferon treatment. However, interferons have significant side effects [Janssen, H. L. A., et al. “Suicide Associated with Alfa-Interferon Therapy for Chronic Viral Hepatitis”,
J. Hepatol
., 21, pp. 241-243 (1994)]; Renault, P. F. and Hoofnagle, J. H., “Side effects of alpha interferon. Seminars in Liver Disease 9, 273-277. (1989)] and induce long term remission in only a fraction (~25%) of cases [Weiland, O. “Interferon Therapy in Chronic Hepatitis C Virus Infection”,
FEMS Microbiol. Rev
., 14, pp. 279-288 (1994)]. Thus, there is a need for more effective anti-HCV therapies.
The NS3 protease is considered a potential target for antiviral agents. However, drug discovery efforts directed towards the NS3 protein have been hampered by the lack of structural information about NS3 and its complex with NS4A. Such structural information would provide valuable information in discovery of HCV NS3 protease inhibitors. However, efforts to determine the structure of HCV NS3 protease have been hampered by difficulties in obtaining sufficient quantities of pure active enzyme [Steinkuhler, C. et al., “In Vitro Activity of Hepatitis C Virus Protease NS3 Purified from Recombinant Baculovirus-Infected Sf9 Cells”,
J. Biol Chem
., pp. 637-6273 (1996)]. There have been no crystals reported of any NS3 or NS3 protease domain protein. Thus, x-ray crystallographic analysis of such proteins has not been possible.
SUMMARY OF THE INVENTION
Applicants have solved this problem by providing, for the first time, compositions comprising a hepatitis C virus (HCV) NS3 protease-like polypeptide completed with a NS4A-like peptide and methods for making such compositions.
The invention also provides crystals of a HCV NS3 protease-like polypeptide/NS4A-like peptide complex and methods for making such crystals.
The invention also provides the structure coordinates of a HCV NS3 protease-like polypeptide/NS4A-lik
Fox Ted
Kim Joseph L.
Lin Chao
Morgenstern Kurt A.
Thomson John A.
Achutamurthy Ponnathapu
Fish & Neave
Haley, Jr., Esq. James F.
Joslyn Kristin M.
Rao Manjunath N.
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