Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-02-23
2003-03-04
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S039000, C435S325000, C435S334000, C435S339000
Reexamination Certificate
active
06528252
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to the identification of protease inhibitors.
BACKGROUND OF THE INVENTION
An undesirable level of protease activity occurs in a variety of conditions, including rheumatoid arthritis and other autoimmune diseases, tumor invasion, inflammation, and diseases caused by infections agents which produce proteases, such as HIV, rhinoviruses, hepatitis viruses, and herpes viruses. Protease inhibitors, particularly specific inhibitors, can be used to treat such conditions.
Thus, there is a need in the art for convenient and rapid methods of identifying protease inhibitors.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods for identifying protease inhibitors. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a method of screening a test substance for the ability to inhibit a protease having a proteolytic activity. The method encompasses the steps of contacting a target cell comprising a protease having a proteolytic activity with a viral display package in the presence of a test substance, wherein the viral display package comprises (a) a transferable label and (b) a recombinant viral envelope protein comprising in sequential order (i) a receptor-binding polypeptide which binds to a receptor on the surface of the target cell, (ii) a protease cleavage site for the protease expressed by the target cell, and (iii) a fusion-mediating polypeptide, such that proteolytic cleavage of the cleavage site does not permit substantial transfer of the transferable label from the viral display package to the target cell; and detecting the transferable label, if any, in the target cell, wherein detection of transferable label or a greater amount of transferable label in the target cell in the presence of the test substance relative to the absence of the test substance identifies the test substance as a protease inhibitor.
A “protease”, also called an endoprotease, is an enzyme which hydrolyzes a peptide bond between a pair of amino acids located in a polypeptide chain. The proteolytic activity of the protease does not permit substantial transfer of the transferable label from the viral display package to the target cell.
The “viral display package” comprises a transferable label and a recombinant viral envelope protein. The “transferable label” is a label whose presence is detectable in the target cell as a result of fusion of the viral display package and the target cell membrane. Thus, if the protease produced by the target cell cleaves the protease cleavage site in the recombinant envelope protein, the virus display package does not transfer label to the target cell. Alternatively, where the protease is inhibited, it is unable to cleave the cleavage site, and the virus display package can infect the target cell and transfer label.
Preferably, the transferable label is a gene encoding a selectable marker or is a reporter gene which encodes a detectable product.
The “sequential order” of components comprising a recombinant viral envelope protein occurs sequentially from the N- to C-terminus.
The “receptor-binding polypeptide” is capable of binding to a cognate receptor on the surface of a target cell, and thereby initiating gene delivery to the target cell. For example, the receptor binding polypeptide is capable of binding to a cognate receptor on a target cell, such as a viral envelope receptor on the surface of the target cell. The receptor-binding polypeptide should minimally include the receptor-binding domain of a protein, i.e., the portion of the protein that retains the ability to bind to the receptor on the cell. Thus, receptor-binding polypeptide (e.g., a displayed polypeptide) may be a receptor-binding domain of a viral envelope protein, such as a 4070A envelope protein or Moloney murine leukemia virus envelope protein, which (a) binds to a cell-surface receptor and (b) initiates gene delivery thereby. The underlying envelope protein (on which the receptor binding polypeptide is displayed) must retain its membrane fusion ability but does not necessarily need to retain its receptor-binding capability.
Certain alterations, such as mutations, deletions, or additions, can be made to the receptor-binding polypeptide which do not significantly affect its functions, as described above (a) and (b). One example of a receptor-binding domain useful in the context of this invention is the binding domain for the Pit-2/Ram-1 receptor of the 4070A virus. Other receptor-binding domains include the CAT-1 receptor-binding domain of the Moloney murine leukemia virus, GALV receptor-binding domain, or any of those from MLV envelope proteins.
The “fusion-mediating polypeptide” may be a fusion-mediating domain of a viral envelope protein or a substantially intact viral envelope protein and can be derived either from the same type of viral envelope protein as the receptor-binding polypeptide or from a different viral envelope protein. Viruses such as murine leukemia viruses (e.g., Moloney murine leukemia virus or 4070A murine leukemia virus) are particularly useful as sources of viral envelope proteins. In another embodiment, the recombinant viral envelope protein is a viral envelope protein of a murine leukemia virus, such as a Moloney murine leukemia virus or a 4070A, in which a protease cleavage site has been inserted between the receptor-binding and the fusion-mediating polypeptides.
The “transferable label,” if any, is detected in the target cell. Detection of the transferable label or of a greater amount of transferable label in the target cell in the presence of the test substance relative to the absence of the test substance identifies the test substance as a potential protease inhibitor. The “amount” of label transferred in the presence of an inhibitor is considered a “greater amount” if it is at least 10, 20, 25, 50, 75, 85, 90, 95, 98, 99, or 100%, or at least 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold greater than the amount of transferable label that is transferred from the viral display package to a target cell in the absence of the protease inhibitor. If no amount of label is transferred in the absence of the protease inhibitor, then any detectable label in the presence of the inhibitor is indicative of inhibition and is a “greater amount.”
A “protease cleavage site” according to the invention is a contiguous sequence of amino acids connected by peptide bonds which contains a pair of amino acids which is connected by a peptide bond that is hydrolyzed by a particular protease. Optionally, a protease cleavage site according to the invention includes one or more amino acids on either side of the peptide bond to be hydrolyzed, to which the catalytic site of the protease also binds (Schecter and Berger,
Biochem. Biophys. Res. Commun.
27, 157-62, 1967). Thus, the protease “cleavage site” also may include a protease recognition site, and both sites may be included within an amino acid linker sequence.
Alternatively, a “plurality” of target cells (i.e. more than one target cell) is contacted with a plurality of viral display packages (i.e. more than one viral display package) in the presence of a test substance. In this case, detection of transferable label in a number of target cells or in a greater number of target cells in the presence of the test substance relative to the absence of the test substance identifies the test substance as a potential protease inhibitor. A “greater number” of target cells in which the transferable label is detected is any number of target cells which is at least 1, 2, 5, 10, 20, 50, or 100 more than the number of target cells in which the transferable label is detected in the absence of a protease inhibitor.
In a specific embodiment, an otherwise intact envelope protein has a protease cleavage site inserted at a position that abrogates the function of the envelope protein when cleaved.
In another embodiment, the invention encompasses an MLV envelope protein which is capable of binding to a target recept
Chadwick Mark P.
Russell Stephen J.
BioFocus Discovery Limited
Palmer & Dodge LLP
Park Hankyel T.
Williams Kathleen M.
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