Purified active HCV NS2/3 protease

Details Purified active HCV NS2/3 protease Purified active HCV NS2/3 protease

2001-12-14

2004-11-09

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S184000, C435S188000, C435S219000, C435S023000, C530S412000

Reexamination Certificate

active

06815159

ABSTRACT:

BACKGROUND OF THE INVENTION
Hepatitis C Virus (HCV) is an important cause of chronic liver disease leading to cirrhosis and end-stage liver disease in humans. Over 150 million people worldwide are persistently infected with HCV and the number of deaths attributable to chronic infection is likely to rise dramatically over the next 10-20 years. Currently available therapies are of limited efficacy and are unsatisfactory. These therapies have involved use of interferon alpha, either alone or in combination with other antiviral agents such as ribavirin. Given that a low response rate, in addition to high patient relapse and side effects, are observed, new therapies are required that may afford long-term treatment benefits.
The cloned and characterized partial and complete sequences of the HCV genome have been analyzed to provide appropriate targets for prospective antiviral therapy. HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is approximately 9600 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2/3 junction and is henceforth referred to as NS2/3 protease. The second one is a serine protease contained within the N-terminal region of NS3, henceforth referred to as NS3 protease, and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3/4 A cleavage site, and in trans, for the remaining NS4A/4B, NS4B/5A, NS5A/5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
Most of the HCV encoded enzymes have been evaluated as targets for the development of new antiviral therapies, namely the NS3 protease, helicase and ATPase activities, as well as the NS5B RNA-dependent RNA polymerase activity (Dymock, B. W. et al. (2000)
Antiviral Chemistry
&
Chemotherapy.
11 (2):79-96 and Walker, M. A. (1999)
Drug Discovery Today
4(11): 518-529). The only viral enzyme that has not been extensively characterized so far is the NS2/3 protease, probably because it acts co-translationally.
NS2/3 protease is responsible for autocleavage at the NS2 and NS3 junction between amino acids Leu1026 and Ala1027 (Hirowatari, Y., et al (1993)
Arch. Virol.
133:349-356 and Reed, K. E., et al. (1995)
J. Virol.
69 (7) 4127-4136). This cleavage appears to be essential for productive replication in vivo as shown by the absence of HCV infection in a chimpanzee following inoculation with a clone devoid of the NS2/3 protease activity (Kolykhalov, A. A., et al (2000)
J. Virol.
74 (4) 2046-2051). It also appears that generation of a functional NS2 and an authentic NS3 protease N-terminal sequence are somehow linked to NS5A phosphorylation (Liu, Q., et al. (1999)
Biochem. Biophys. Res. Commun.
254, 572-577 and Neddermann P., et al. (1999)
J. Virol.
73(12):9984-9991).
The minimal region of the HCV open reading frame required for the autocleavage activity has been reported to be located somewhere between amino acids 898 and 907 for the N-terminal boundary and amino acid 1206 for the C-terminal boundary (Hijikata, M. et al (1993)
J. Virol.
67 (8):4665-4675.; Grakoui, A., et al. (1993)
Proc. Natl. Acad. Sci. USA
90:10583-10587; Santolini, E., et al (1995)
J. Virol.
69 (12): 7461-7471; and Liu, Q., et al (1999)
Biochem. Biophys. Res. Commun.
254, 572-577; Pallaoro et al., (2001)
J. Virol.
75(20); 9939-46). Interestingly, the NS2/3 protease activity is independent of the NS3 protease activity (Grakoui, A., et al. (1993)
Proc. Natl. Acad. Sci. USA
90:10583-10587; Hijikata, M. et al (1993)
J. Virol.
67 (8):4665-4675) but the NS3 protease domain cannot be substituted by another non-structural protein (Santolini, E., et al (1995)
J. Virol.
69 (12): 7461-7471). Mutagenesis studies have shown that the residues His952 and Cys993 are essential for the cis-cleavage activity (Grakoui, A., et al. (1993)
Proc. Natl. Acad. Sci. USA
90:10583-10587; Hijikata, M. et al (1993)
J. Virol.
67 (8):4665-4675). Gorbalenya, A. E, et al. (1996)
Perspect. Drug Discovery Design.
6:64-86)) have suggested that the NS2/3 protease could be a cysteine protease. However, the observation that the activity is stimulated by metal ions and inhibited by EDTA led to the suggestion that the NS2/3 protease is a metalloprotease (Grakoui, A., et al. (1993)
Proc. Nat. Acad. Sci. USA
90:10583-10587; Hijikata, M. et al (1993)
J. Virol.
67 (8):4665-4675)). Studies with classical protease inhibitors in an in vitro transcription and translation assay (Pieroni, L. et al (1997)
J. Virol.
71 (9): 6373-6380) have not yet allowed for a definitive classification.
Processing at the NS2/3 junction has been reported (Darke, P. L. et al (1999)
J. Biol. Chem.
274 (49) 34511-34514 and WO 01/16379; Grakoui, A., et al (1993).
Proc. Natl. Acad. Sci. USA
90:10583-10587; Hijikata, M., et al. (1993)
J. Virol.
67 (8):4665-4675; Pieroni, L., et al (1997)
J. Virol.
71 (9): 6373-6380 and Santolini, E. et al (1995)
J. Virol.
69 (12): 7461-7471) following expression of the NS2/3 region in cell-free translation systems, in
E. coli
, in insect cells infected with baculovirus recombinants and/or in mammalian cells (transient transfection or vaccinia virus T7 hybrid system). However, processing has not been reported in an isolated recombinant enzyme until very recently (Pallaoro et al., (2001)
J. Virol.
75(20); 9939-46; Thibeault et al., J. Biol. Chem. 276 (49):46678-46684).
Grakoui et al. (1993)
Proc. Natl. Acad. Sci. USA,
90:10583-10587 and Komoda et al. (1994)
Gene,
145:221-226 have both disclosed the expression of HCV polypeptides, including the NS2/3 protease, in
E. coli
. Following expression, processing was assessed from SDS-PAGE and immunoblot analyses of cell lysates.
Komoda, using HCV polyproteins fused to maltose-binding protein (MBP) at their N-terminus and dihydrofolate reductase (DHFR) at their C-terminus, also reported on the partial purification of the DHFR-fused products from cell lysates by affinity chromatography for N-terminal sequencing purpose only.
Thus, the biochemical characterization of the NS2/3 protease as well as mechanistic and structural studies has been hampered due to the unavailability of a pure recombinant form of the enzyme. Before any potential inhibitors of NS2/3 protease can be identified in a high throughput-screening format, there must be a reliable source of purified, active NS2/3 protease.
WO 01/68818 published on Sep. 20, 2001 {as well as Pallaoro et al., (2001)
J. Virol.
75(20); 9939-46} have described a process for the purification of recombinant active NS2/3 protease. However, their refolding method needs to be carried out at 4° C. to avoid auto-catalysis.
The method of the present invention, also disclosed in Thibeault et al., J. Biol. Chem. 276 (49):46678-46684, discloses a purification method that proceeds in 2 steps, can be carried out at room temperature and leads in the first instance to a soluble inactive NS2/3 protease (stable at RT) that can be scaled up and stored safely without auto-cleavage.
It is therefore an advantage of this invention to provide a method for the purification of refolded inactive NS2/3 protease.
It is a further advantage of this invention that the soluble inactive protease can be further a

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