Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-04-05
2003-02-25
Allen, Marianne P. (Department: 1631)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C702S019000, C424S184100, C424S225100, C514S002600, C514S012200, C514S013800
Reexamination Certificate
active
06524589
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to crystalline HCV NS3/NS4A complex, the structure of HCV NS3/NS4A complex as determined by X-ray crystallography, the use of that structure to solve the structure of HCV NS3, NS3/NS4A complex homologues and other crystal forms of the HCV NS3/NS4A complex, mutants and co-complexes thereof and the use of HCV NS3/NS4A complex, mutants and co-complexes thereof to design inhibitors of HCV NS3/NS4A complex.
BACKGROUND OF THE INVENTION
The hepatitis C virus causes one of the world's most pandemic and insidious diseases. According to the World Health Organization, there are approximately 170 million HCV carriers worldwide with prevalence up to 0.5-10% (Release:, 1998), and in the United States, four million individuals are hepatitis C virus carriers (Alter and Mast, 1994). The hepatitis C virus (HCV) was identified in 1989 and accounted for 50-60% of the non-A, non-B transfusion associated hepatitis (Alter et al
N Engl J Med
321:1494-1500 (1989); Choo, et al.,
Science
244:359-362 (1989); Kuo, et al.,
Science
244:362-364 (1989)). To date, interferon-alpha monotherapy and interferon-alpha-2b and ribavirin combination therapy (Rebetron, Schering-Plough, Kenilworth, N.J.) are the only approved treatments. Twenty percent of the patients responded to interferon-&agr; monotherapy and 42% of the patients responded to Rebetron combination therapy (Reichard, et al.,
Lancet
351:83-87 (1998)). It is important to develop more effective antiviral agents against the various viral targets in order to effectively combat the disease.
HCV, a member of the Flaviviridae family, is a positive-sense, single-stranded RNA virus with genome size of approximately 9.4 kb (Heinz,
Arch Virol Suppl
4:163-171 (1992); Mizokami & Ohba,
Gastroenterol Jpn
28
Suppl
5:42-44 (1993); Ohba, et al.,
FEBS Lett
378:232-234 (1996); Takamizawa, et al.,
J Virol
65:1105-1113 (1991)). The genomic RNA encodes a polyprotein of approximately 3000 amino acid residues in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Lohmann, et al.,
J Hepatol
24:11-19 (1996); Simmonds,
Clin Ther
18
Suppl
B:9-36 (1996)). This polyprotein is processed by host and viral proteases (Grakoui, et al.,
J Virol
67:1385-1395 (1993); Shimotohno, et al.,
J Hepatol
22:87-92 (1995)). NS3 has been the target of interest for antiviral discovery because of its important roles in HCV maturation and replication. NS3 has two major functional domains: the amino terminal one third of the protein is a serine protease responsible for some key aspects of polyprotein processing (Shimotohno, et al.,
J Hepatol
22:87-92 (1995)), and the carboxyl terminal two thirds of NS3 share sequence similarity with those of the DEAD box family of RNA helicases (Gorbalenya, et al.,
FEBS Lett
235:16-24 (1988); Koonin & Dolja,
Crit Rev Biochem Mol Biol
28:375-430 (1993); Korolev, et al.,
Protein Sci
7:605-610 (1998)). Virus encoded helicases such as vaccinia NPH-II and plum pox potyvirus CI have been demonstrated to be important for viral replication and production of infectious virions (Fernandez, et al.,
Nucleic Acids Res
25:4474-4480 (1997); Gross & Shuman
J Virol
72:4729-4736 (1998); Gross & Shuman,
J Virol
70:8549-8557 (1996)). There are two enzymatic activities associated with the HCV helicase-NTPase and nucleic acid unwinding; the former is believed to provide an energy source for the unwinding reaction through NTP hydrolysis (Kim, et al.,
Virus Res
49:17-25 (1997); Suzich, et al.,
J Virol
67:6152-6158 (1993)). Studies of the crystal structure of HCV helicase reveal that this molecule consists of three domains—domain I contains the NTP and Mg
++
binding sites, domain II is speculated to be an nucleic acid binding site and domain III is characterized as having extensive helical structure. Between domain I and II lies a coupling region speculated to be involved in the RNA unwinding function of the holoenzyme (Kim et al.,
Cell
87:343-355 (1996); Yao, et al.,
Nat Struct Biol
4:463-467 (1997)).
In spite of these different functions during the HCV life cycle, there is no evidence that the NS3 protease and helicase are ever separated from one another in HCV infected cells. Likewise, only the 70 kDa NS3 protein containing both the protease and helicase domains has ever been detected in cells transfected with recombinant vaccinia expressing HCV nonstructural proteins (Grakoui, et al.,
J Virol
67:1385-1395 (1993)). In addition, NS3 has been found to spontaneously associate with NS4A to form a stable noncovalent NS3/NS4A complex in vivo (Failla, et al.,
J Virol
69:1769-1777 (1995); Grakoui, et al.,
J Virol
67:1385-1395 (1993)). The covalent fusion of the protease and helicase domains in NS3 as well as the spontaneous formation of a complex with NS4A suggests that there may be functional dependence between these polypeptides to achieve additional activities other than the fundamental enzymatic properties.
In order to study the enzymatic properties of HCV helicase with as much biological relevance as possible, it is desirable to generate a full length NS3/NS4A complex. Previous attempts to express a full length NS3/NS4A protein complex in recombinant baculovirus resulted in a material with a propensity to aggregate which limited its application (Sali, et al.,
Biochemistry
37:3392-3401 (1998)).
The NS3 protein and the NS3/NS4A complex are considered a valuable target for antiviral agents. However, drug discovery efforts directed towards the NS3 protein have been hampered by a lack of structural information about the holoenzyme NS3. Such structural information would provide valuable information in discovery of HCV NS3 protein inhibitors. There have been no crystals reported of a NS3 holoenzyme or NS3 holoenzyme complex. Thus, x-ray crystallographic analysis of such proteins has not previously been available.
SUMMARY OF THE INVENTION
The present invention addresses this need by providing, for the first time, compositions comprising a crystallized hepatitis C virus (HCV) NS3/NS4A polypeptide complex. The present invention relates to crystalline HCV NS3/NS4A complex, the structure of HCV NS3/NS4A complex as determined by x-ray crystallography, the use of the structure to solve the structure of HCV NS3/NS4A complex homologues and other crystal forms of a HCV NS3/NS4A complex and mutants and co-complexes thereof, and the use of the HCV NS3/NS4A complex structure and that of its homologues, mutants, and co-complexes thereof to design inhibitors of HCV NS3/NS4A complex.
One aspect of the present invention is directed to the three-dimensional structure of an isolated and purified protein designated HCV NS3 and its structure coordinates. Another aspect of the invention is to use the structure coordinates of the HCV NS3/NS4A complex crystal to reveal the atomic details of the active sites and one or more of the accessory binding sites of HCV NS3/NS4A complex.
A further aspect of the invention is to provide HCV NS3/NS4A complex mutants characterized by one or more different properties compared to wild-type HCV NS3/NS4A complex.
The invention also provides a machine-readable data storage medium encoded with the structural coordinates of a NS3/NS4A polypeptide complex or a homologue thereof. Such a homologue contains alpha carbon (C&agr;) atoms having a root mean square deviation of equivalent C&agr; atoms of less than 3.0 Å when compared to the NS3/NS4A polypeptide complex.
The invention also provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to a HCV NS3/NS4A polypeptide complex.
Another aspect of this invention is to use the structure coordinates and atomic details of HCV NS3/NS4A complex or mutants or homologues or co-complexes thereof to design, evaluate computationally, synthesize and use inhibitors of HCV NS3/NS4A complex that prevent or treat the undesirable physical and pharmacological properties of HCV. These inhibitors may be used
Prosise Winifred W.
Reichert Paul
Taremi Shahriar Shane
Weber Patricia C.
Yao Nanhua
Allen Marianne P.
Fontenot J. Darrell
Schering Corporation
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