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US 2017/0159102 A1Published application
Compositions and method for identifying enzyme and transport protein inhibitors
OwnerRoland Wolkowiczinventor
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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.
Claims16
A cell-based method for monitoring the activity of a protease, a viral protease, or an HIV-1 protease (PR), comprising:
- (1) (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 protease, viral protease or HIV-1 protease (PR), under physiologic or cell culture conditions;
- (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, viral protease or HIV-1 protease (PR) is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the protease, viral protease or HIV-1 protease (PR) 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, viral protease or HIV-1 protease (PR) operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous protease, viral protease or HIV-1 protease (PR);
- (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous protease, viral protease or HIV-1 protease (PR);
- (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, viral protease or HIV-1 protease (PR);
- and
- (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,
- wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease, viral protease or HIV-1 protease (PR) 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, viral protease or HIV-1 protease (PR) is enzymatically active (the detectable moiety is cleaved off by the protease, viral protease or HIV-1 protease (PR));
- (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif, wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface;
- or
- (3) the method of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold.
13 more elements
(canceled)
6-13. (canceled)
A cell-based method for monitoring the activity of a protease comprising:
- (1) (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 protease under physiologic or cell culture conditions;
- (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 cleavage away from (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 (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous protease;
- (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,
- 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 (the detectable moiety is cleaved off by the protease);
- or
- (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,
- wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.
13 more elements
18-28. (canceled)
A cell-based method for monitoring the activity of a cell's ER and/or trans-Golgi network comprising:
- (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
- (i) a transmembrane domain;
- (ii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell;
- and
- (iii) a detectable moiety;
- (b) inserting or transfecting the scaffold protein-encoding nucleic acid of (a) into the cell;
- (d) expressing the nucleic acid of (a);
- and
- (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,
- wherein the scaffold protein is expressed on the extracellular surface of the cell when the activity of the cell's ER and trans-Golgi network is functioning;
- or
- (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,
- wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.
10 more elements
33-90. (canceled)
Dependencies are inferred from the claim text.