Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-02-23
2001-03-06
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C530S323000
Reexamination Certificate
active
06197536
ABSTRACT:
DESCRIPTION
The present invention relates to molecular biology and to hepatitis C virus (HCV) virology. More specifically, the invention has as its subject a process for producing, in a pure form and in high quantities, polypeptides having the proteolytic activity of HCV NS3 protease, and a method for the effective reproduction in vitro of the proteolytic activity of these polypeptides in order to define an enzymatic assay capable of selecting, for therapeutic purposes, compounds inhibiting the enzyme activity associated with NS3.
As is known, the hepatitis C virus (HCV) is the main etiological agent of non-A, non-B hepatitis (NANB). It is estimated that HCV causes at least 90% of post-transfusional NANB viral hepatitis and 50% of sporadic NANB hepatitis. Although great progress has been made in the selection of blood donors and in the immunological characterisation of blood used for transfusions, there is still a high number of HCV infections among recipients of blood transfusions (one million or more infections every year throughout the world). Approximately 50% of HCV-infected individuals develop liver cirrhosis within a period that can range from 5 to 40 years. Furthermore, recent clinical studies suggest that there is a correlation between chronic HCV infection and the development of hepatocellular carcinoma.
HCV is an enveloped virus containing an RNA positive genome of approximately 9.4 kb. This virus is a member of the Flaviviridae family, the other members of which are the flaviviruses and the pestiviruses.
The RNA genome of HCV has recently been mapped. Comparison of sequences from the HCV genomes isolated in various parts of the-world has shown that these sequences can be extremely heterogeneous. The majority of the HCV genome is occupied by an open reading frame (ORF) that can vary between 9030 and 9099 nucleotides. This ORF codes for a single viral polyprotein, the length of which can vary from 3010 to 3033 amino acids. During the viral infection cycle, the polyprotein is proteolytically processed into the individual gene products necessary for replication of the virus.
The genes coding for HCV structural proteins are located at the 5′-end of the ORF, whereas the region coding for the non-structural proteins occupies the rest of the ORF.
The structural proteins consist of C (core, 21 kDa), E1 (envelope, gp37) and E2 (NS1, gp61). C is a non-glycosylated protein of 21 kDa which probably forms the viral nucleocapsid. The protein E1 is a glycoprotein of approximately 37 kDa, which is believed to be a structural protein for the outer viral envelope. E2, another membrane glycoprotein of 61 kDa, is probably a second structural protein in the outer envelope of the virus.
The non-structural region starts with N
S
2 (p24), a hydrophobic protein of 24 kDa whose function is unknown.
NS3, a protein of 68 kDa which follows NS2 in the polyprotein, is predicted to have two functional domains: a serine protease domain within the first 200 amino-terminal amino acids, and an RNA-dependent ATPase domain at the carboxy terminus.
The NS4 gene region codes for NS4A (p6) and NS4B (p26), two hydrophobic proteins of 6 and 26 kDa, respectively, whose functions have not yet been fully clarified.
The NS5 gene region also codes for two proteins, NS5A (p56) and NSSB (p65), of 56 and 65 kDa, respectively. Amino acid sequences present in all the RNA-dependent RNA polymerases can be recognised within the NS5 region. This suggests that the NS5 region contains components of the viral replication machinery.
Various molecular biological studies indicate that the signal peptidase, a protease associated with the endoplasmic reticulum of the host cell, is responsible for proteolytic processing in the non-structural region, that is to say at sites C/E1, E1/E2 and E2/NS2.
The serine protease in NS3 is responsible for cleavage at the junctions between NS3 and NS4A, between NS4A and NS4B, between NS4B and NS5A and between NS5A and NS5B. In particular it has been found that the cleavage made by this serine protease leaves a cysteine or a treonine residue on the amino-terminal side (position P1) and an alanine or serine residue on the carboxy-terminal side (position P1′) of the cleavage site. It has been shown that the protease contained in NS3 is a heterodimeric protein in vivo, forming a complex with the protein NS4A. Formation of this complex increases proteolytic activity on sites NS4A/NS4B and NS5A/NS5B, and is a necessary requisite for proteolytic processing of site NS4B/NS5A.
A second protease activity of HCV appears to be responsible for the cleavage between NS2 and NS3. This protease activity is contained in a region comprising both part of NS2 and the portion of NS3 containing the serine protease domain, but does not use the same catalytic mechanism as the latter.
A substance capable of interfering with the proteolytic activity associated with the protein NS3 might constitute a new therapeutic agent. In effect, inhibition of this protease activity would involve stopping the proteolytic processing of the non-structural region of the HCV polyprotein and, consequently, would prevent viral replication of the infected cells.
This sequence of events has been verified for the homologous flavivirus, which, unlike HCV, infects cell line cultures. In this case, it has been shown that genetic manipulations involving generation of a protease no longer capable of carrying out its catalytic activity, abolishes the ability of the virus to replicate (1).
Furthermore, it has been widely shown, both in vitro and in clinical studies, that compounds capable of interfering with the HIV protease activity are capable of inhibiting replication of this virus (2).
The methods used to generate molecules with therapeutic potential are known to those operating in this field. Generally speaking, collections of compounds containing a large number of single chemical entities with a high molecular diversity are made to undergo an automatised assay in order to identify single active agents, which then undergo further chemical modifications in order to improve their therapeutic potential. Other approaches may include rational modification of substrates or ligands of specific target protein, with the aim of developing high binding affinity compounds capable of altering or abolishing the biological activity of the protein under examination. Determination of the three-dimensional structure of a target protein, by means of methods known in the sector as X-ray crystallography or nuclear magnetic resonance (NMR) allows rational design of molecules capable of binding specifically to the protein and which, as a result of this, have the ability to interfere with the biological properties of that protein.
Research on compounds capable of interfering with the biological activity of the protease contained in the hepatitis C virus NS3 protein is hampered by the difficulty in producing sufficient amounts of purified protein with unaltered catalytic properties, and by the need to use co-factors to enhance the activity of the enzyme in vitro.
There is therefore a need in the specific field for a process to produce NS3, or similar products, in larger amounts that has been possible in the past, and with an in vitro activity sufficient to select inhibitors.
The present invention consists of isolated and purified polypeptides, with the proteolytic activity of the HCV protein NS3, characterised by the fact that they have an amino acid sequence chosen from among the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
The invention also comprises expression vectors—to produce the polypeptides represented by sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 which have the proteolytic activity of HCV NS3—comprising:
a polynucleotide coding for one of said polypeptides;
functional regulation, transcription and translation sequences in said host cell, operatively bonded to said polynucleotide coding for one of said polypeptides; and
optionally, a selectable marker.
The invention also extends to a
Bianchi Elisabetta
De Francesco Raffaele
Narjes Frank
Pessi Antonello
Steinkühler Christian
Browdy and Neimark
Istituto di Ricerche di Biologia Moleculare S.p.A.
Nashed Nashaat T.
LandOfFree
Methodology to produce, and purify and assay polypeptides... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methodology to produce, and purify and assay polypeptides..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methodology to produce, and purify and assay polypeptides... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2476476