Compositions and methods for identifying enzyme and transport protein inhibitors
AssigneeSan Diego State University Research Foundation
InventorRoland Wolkowicz
Life of this patent
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Abstract
The invention is directed to compositions to screen for small molecule drugs that inhibit proteases, such as viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., HIV proteases. In alternative embodiments, the invention provides cell-based assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelop protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products.
Claims15
A cell-based method for monitoring the activity and subcellular location of a protease, comprising:
- (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
- (i) an amino acid motif or subsequence susceptible to cleavage by the protase comprising SEQ ID NO:3;
- (ii) a transmembrane domain;
- (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell;
- and
- (iv) a detectable moiety,
- wherein the amino acid motif or subsequence susceptible to cleavage by the protease is positioned within the scaffold protein such that when the detectable moiety is cleaved away from or off from the scaffold protein by the protease the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
- (b) providing a nucleic acid encoding the protease operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous protease;
- (c) inserting or transfecting the nucleic acid of (a) into the cell, and also (b) into the cell if the cell does not already express a heterologous or endogenous;
- (d) co-expressing the nucleic acid of (a), and (b) in the cell, or expressing the nucleic acid of (a) in the cell if the cell already expresses a heterologous or endogenous protease;
- and
- (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,
- (f) wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the protease is enzymatically active, and the detectable moiety can be located at the site of the intracellular cleavage event, if any.
Dependencies are inferred from the claim text.