Compositions and method for identifying enzyme and transport protein inhibitors

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.

1 . 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.   
     
     
         2 . The method of  claim 1 , further comprising screening for an inhibitor of a protease, a viral protease or an HIV-1 protease (PR) by:
 (a) providing a compound to be screened as an inhibitor of a protease, viral protease or HIV-1 protease (PR), or providing a nucleic acid to be screened as encoding an inhibitor of a protease, viral protease or HIV-1 protease (PR);   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  1 (a) and claim  1 (b) in the cell; and   (c) 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 inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, 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)) and the enzymatic activity of the protease, viral protease or HIV-1 protease (PR) is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         3 . The method of  claim 2  further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or 
 (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the protease, viral protease or HIV-1 protease (PR) to one of the divided cell samples. 
 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 2 , wherein:
 (a) the amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease (PR) under physiologic (cell culture) conditions comprises SEQ ID NO:3 or SEQ ID NO:4;   (b) the HIV-1 protease (PR) comprises SEQ ID NO:5 or SEQ ID NO:6;   (c) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (d) the cell is a mammalian cell, a monkey cell or a human cell;   (e) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (f) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (g) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (h) the compound to be screened as an inhibitor of the protease, viral protease or HIV-1 protease (PR) comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid; or   (i) the compound to be screened as an inhibitor of the protease, viral protease or HIV-1 protease (PR) is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library.   
     
     
         6 - 13 . (canceled) 
     
     
         14 . 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.   
     
     
         15 . The method of  claim 14 , further comprising screening for an inhibitor of a protease by:
 (a) providing a compound to be screened as an inhibitor of a protease, or providing a nucleic acid to be screened as encoding an inhibitor of a protease;   (b) contacting a plurality of the cells with the compound or nucleic acid of claim  14 (a) either before, during and/or after the co-expressing the nucleic acid of claim  14 (a) and claim  14 (b) in the cell; and   (c) 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 inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, 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) and the enzymatic activity of the protease is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         16 . The method of  claim 15  further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or 
 (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the protease to one of the divided cell samples. 
 
     
     
         17 . The method of  claim 15 , wherein:
 (a) the transcriptional regulatory unit comprises a promoter;   (b) the transcriptional regulatory unit comprises an inducible promoter;   (c) the transcriptional regulatory unit comprises a constitutive promoter;   (d) the cell is a mammalian cell, a monkey cell or a human cell;   (e) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (f) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (g) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (h) the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;   (i) the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library; or   (i) the protease is an HIV-1 protease (PR), or the protease is a viral, a microbial or a mammalian protease.   
     
     
         18 - 28 . (canceled) 
     
     
         29 . 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.   
     
     
         30 . The method of  claim 29 , further comprising screening for an inhibitor of the cell's ER and trans-Golgi network by:
 (a) providing a compound or nucleic acid to be screened as an inhibitor of the cell's ER and trans-Golgi network;   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  29 (a) in the cell; and   (c) 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 (or is substantially expressed) on the extracellular surface of the cell when the cell's ER and trans-Golgi network is not inhibited.   
     
     
         31 . The method of  claim 30  further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or 
 (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and adding a known inhibitor of the cell's ER and/or trans-Golgi network to one of the divided cell samples. 
 
     
     
         32 . The method of  claim 30 , wherein:
 (a) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (b) the cell is a mammalian cell, a monkey cell or a human cell;   (c) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (d) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (e) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (f) the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;   (g) the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library; or   (h) the protease is an HIV-1 protease (PR), or the protease is a viral, a microbial or a mammalian protease.   
     
     
         33 - 90 . (canceled)