US 6,333,167 B1 · Methods and reagents for identifying inhibitors of proteolysis of membrane-associated proteins

Abstract

The present disclosure provides DNA constructs, genetically modified host cells and methods for identifying inhibitors of proteolysis of membrane-associated protease substrates including, but not limited to, amyloid precursor protein, a Notch protein and sterol response element binding proteins. The methods rely on chimeric proteolytic substrates containing a portion of the proteolytic substrate protein (including the proteolytic cleavage site) and a transcription repressor protein portion. Release of the repressor results in blockage of the transcription of a chimeric reporter gene which includes repressor-responsive transcription regulatory sequences and a reporter protein coding region. A specific protease inhibitor is identified by increased reporter protein activity.

Claims48

Dependent claims

A method of evaluating a compound for the ability to inhibit proteolysis of a proteolytic substrate, comprising:

(a) culturing a recombinant eukaryotic host cell which expresses (i) a chimeric protein comprising a transcription repressor portion and a protease recognition portion, said protease recognition portion comprising a proteolytic cleavage site of a proteolytic substrate of interest, wherein the chimeric polypeptide is attached to a non-nuclear membrane, and (ii) a reporter gene operably linked to a transcription regulatory sequence which is responsive to said transcription repressor;
(b) adding a test compound to the recombinant host cell of step (a);
and
(c) monitoring expression of a reporter gene product, wherein an increase in expression in the presence of the test compound added in step (b) over expression in the absence of the test compound is an indication that the test compound inhibits proteolysis of the proteolytic substrate.

1 more element

Dependencies are inferred from the claim text.

Methods and reagents for identifying inhibitors of proteolysis of membrane-associated proteins

Life of this patent

Methods and reagents for identifying inhibitors of proteolysis of membrane-associated proteins

Life of this patent

Abstract

Claims48