Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-10-17
2002-08-20
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C530S350000, C530S351000, C530S399000
Reexamination Certificate
active
06436666
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to polypeptides and methods useful for screening protease regulators such as inhibitors of hepatitis C virus (HCV) proteases.
BACKGROUND OF THE INVENTION
Protease activities have been implicated in many pathological processes of disease conditions, especially in viral infections. Protease regulators, especially protease inhibitors, are strongly desired for their potential therapeutic applications. For example, because HCV NS3 protease cleaves the non-structural HCV proteins which are necessary for HCV replication, the NS3 protease can be a target for the development of therapeutic agents against the HCV virus. Several methods are available in the field for screening protease inhibitors. However, there is a continuing need in the art to develop more effective methods and reagents for protease regulator screening.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for screening a compound capable of regulating protease activity.
It is another object of the invention to provide a polypeptide useful for screening test compounds for protease regulating activity.
It is yet another object of the invention to provide a method of forming a substrate for use in a screening assay for protease regulators.
These and other objects of the invention are provided by one or more of the embodiments provided below.
In one embodiment of the invention a method is provided for screening a compound capable of regulating protease activity. According to the method, a protease and a polypeptide are incubated in the presence and absence of a test compound. The polypeptide comprises an anchor region, a protease recognition site, and a detectable signal region. The anchor region is bound to a solid support. The protease recognition site comprises a) a protease cleavable peptide bond, and b) at least 3 amino acids on each side of the cleavable peptide bond. The signal region bound to the solid support is detected. An amount of the signal region bound to the solid support in the presence of the test compound which is greater or less than an amount detected in the absence of the test compound indicates that the test compound is capable of regulating protease activity.
In another embodiment of the invention a polypeptide consisting of a polyamino acid sequence is provided useful for screening test compounds for protease regulating activity. The polypeptide comprises an anchor region, a protease recognition site, and a detectable signal region. The anchor region is capable of binding to a solid support. The protease recognition site comprises a) a cleavable peptide bond, and b) at least 3 naturally adjacent amino acids on each side of the cleavable peptide bond.
In yet another embodiment a method is provided of forming a substrate for use in a screening assay for protease regulators. The method comprises the step of binding a polypeptide to a solid support via a protein-protein interaction. The K
d
of the protein-protein interaction is less than 10
−8
. The polypeptide comprises an anchor region, a protease recognition site, and a detectable signal region. The protease recognition site comprises a) a cleavable peptide bond, and b) at least 3 naturally adjacent amino acids on each side of the cleavable peptide bond. The polypeptide binds to the solid support via the anchor region.
The present invention provides the art with an effective and rapid method for screening protease regulators. The invention also provides polypeptides used in the method which can be easily produced recombinantly without any chemical modification. The method can be readily carried out in an automated format and is suitable for large scale drug screens. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A
is a schematic of the assay in the absence of protease cleavage.
The polypeptide is anchored on a solid support. Y represents the anchor region, ▪ represents the protease recognition site, and &Circlesolid;&Circlesolid;&Circlesolid;-represents the signal region.
FIG. 1B
is a schematic of the assay after protease cleavage. The signal region is released from the polypeptide and the solid support.
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Chien David Y.
Selby Mark J.
Shoemaker Kevin
Warne Robert L.
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Hemmendinger Lisa M.
Swartz Rodney P
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