Compositions and method for identifying enzyme and transport protein inhibitors

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 (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.

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 (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.

An isolated, recombinant or synthetic nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:

• (a) (i) an amino acid motif or subsequence susceptible to cleavage by a protease under physiologic (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;
• or
• (b) the nucleic acid of (a), 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.

A chimeric polypeptide comprising:

• (1) (i) an amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease (PR) under physiologic (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 HIV-1 protease (PR) is positioned within the scaffold protein such that when the detectable moiety is cleavage away from (off from) the scaffold protein by the HIV-1 protease (PR) the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
• (2) the chimeric polypeptide 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 chimeric polypeptide of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold,
• where optionally the p2/p7 recognition sequence comprises or consists of ATIMMQRGN (SEQ ID NO:2), or optionally an exemplary amino-acid sequence of p2/p7 comprises AEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQAN ATIMMQRGN (SEQ ID NO:3).

A chimeric polypeptide comprising:

• (1) (i) an amino acid motif or subsequence susceptible to cleavage by the protease under physiologic (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;
• or
• (2) the chimeric polypeptide 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.

A cell-based method for monitoring the activity of any enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),
• wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
• (b) providing a nucleic acid encoding the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;
• (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;
• (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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;
• and
• (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell (e.g., by flow cytometry or any high-throughput assay),
• wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is enzymatically active (the detectable moiety is cleaved off by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV);
• (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,
• wherein the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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.

A cell-based method for monitoring the activity of an enzyme, e.g., a protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),
• wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
• (b) providing a nucleic acid encoding the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;
• and
• (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell (e.g., by flow cytometry or any high-throughput assay),
• wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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 motif,
• wherein the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, 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.

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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected);
• (b) inserting (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 (e.g., by flow cytometry or any high-throughput assay),
• 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 motif,
• wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme 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.

An isolated, recombinant or synthetic nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:

• (a) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected);
• or
• (b) the nucleic acid of (a), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,
• wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, 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.

A chimeric polypeptide comprising:

• (1) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),
• wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
• (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,
• wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, 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 chimeric polypeptide of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold,
• where optionally the p2/p7 recognition sequence comprises or consists of ATIMMQRGN (SEQ ID NO:5), or optionally an exemplary amino-acid sequence of p2/p7 comprises AEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQAN ATIMMQRGN (SEQ ID NO:6).

A chimeric polypeptide comprising:

• (1) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (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 (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),
• wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;
• or
• (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,
• wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, 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.

A cell-based method for monitoring the activity of any enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprising:

• (1) (a) providing:
• a nucleic acid encoding a chimeric (hybrid) protein operatively linked to a transcriptional regulatory unit (e.g., a promoter and/or enhancer, e.g., a doxycycline inducible promoter);
• and, a cell comprising an environment capable of supporting the expression of the chimeric (hybrid) protein by the nucleic acid,
• wherein the chimeric (hybrid) protein comprises a chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD:
• aa 1-147);
• (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof;
• and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD:
• aa 768-881),
• and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor;
• (b) inserting (transfecting) the nucleic acid of (a) into the cell,
• wherein optionally the cell does not already express a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;
• and
• (c) contacting the cell with a putative (test) enzyme inhibitor,
• wherein optionally the enzyme inhibitor comprises a small molecule, a protein, a nucleic acid, a polysaccharide and/or a lipid,
• and optionally the enzyme inhibitor is added to the cell before, during and/or after inserting (transfecting) the nucleic acid of (a) into the cell and/or expressing the chimeric protein encoded by the nucleic acid of (a) in the cell,
• and optionally cell-based method further comprises a negative control set of cells into which the nucleic acid of (a) also has been inserted and transfected and expresses the chimeric protein encoded by the nucleic acid of (a), but the negative control set of cells is not exposed to the putative (test) enzyme inhibitor or is exposed to a different putative (test) enzyme inhibitor;
• (d) determining whether the putative (test) enzyme inhibitor is an effective or sufficient inhibitor of the enzyme or enzymatically active fragment thereof by measuring the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor,
• wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor;
• (2) the method of (1), wherein the enzyme is a protease or an HIV-1 protease (PR), or a NS2/NS3 or NS3/NS4A protease of HCV;
• or
• (3) the method of (1), wherein the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a mammalian cell or a human cell.

An isolated, recombinant or synthetic nucleic acid encoding a chimeric (hybrid) protein operatively linked to a transcriptional regulatory unit (e.g., a promoter and/or enhancer, e.g., a doxycycline inducible promoter),

• wherein the chimeric (hybrid) protein comprises a chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD:
• aa 1-147);
• (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof;
• and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD:
• aa 768-881),
• and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor,
• wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor.

A chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD:

• aa 1-147);
• (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof;
• and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD:
• aa 768-881), and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor,
• wherein optionally transcription/expression of the chimeric Gal4 expression system is operably linked to a promoter and/or an enhancer, e.g., a doxycycline inducible promoter,
• wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor.

An isolated, recombinant or synthetic nucleic acid encoding a chimeric (hybrid) protein, wherein the chimeric (hybrid) protein comprises (or consists of) from N- to C-terminus:

• (a) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER) targeting,
• (ii) a tag or detection moiety, or “scaffold”, capable of being recognized on a cell surface,
• (iii) at least two transmembrane domains that span the ER membrane, with an extra loop at the ER luminal face,
• (iv) an enzyme recognition/cleavage site spanning a segment of a gp120/41 boundary, facing the ER lumen, and
• (v) an ER retention sequence or motif;
• (b) the chimeric (hybrid) protein of (a), wherein the tag or detection moiety, or “scaffold”, comprises a tag for an antibody or an antigen binding fragment thereof (the antibody binding specifically to the tag or detection moiety, or “scaffold”), or the tag or detection moiety, or “scaffold”, comprises a ligand, or the tag or detection moiety, or “scaffold”, comprises a FLAG molecule or equivalent thereof;
• (c) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises a furin enzyme recognition/cleavage site, a calcium-dependent protein convertase enzyme recognition/cleavage site, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, or an enzyme recognition/cleavage site derived from a member of the subtilisin/kexin family of proprotein convertases;
• (d) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises an enzyme recognition/cleavage site within the V3 loop of gp120;
• (e) the chimeric (hybrid) protein of (a), wherein the ER retention sequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalent thereof;
• (f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is an HIV-1 gp120/41;
• (g) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane domains that span the ER membrane consist of two transmembrane domains;
• (h) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise at least one TM of a CRR5;
• (i) the chimeric (hybrid) protein of (h), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise TM1 and TM2 from the CRR5;
• (j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:2);
• (k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary further comprises a restriction enzyme recognition site acids at both sides, or comprises additional amino-acids at both sides comprising AKRRVVQREKR (SEQ ID NO:15) and AVGIGALF (SEQ ID NO:16);
• or
• (l) the isolated, recombinant or synthetic nucleic acid encoding the chimeric (hybrid) protein is operatively linked to a transcriptional regulatory unit, or a promoter such as an inducible or constitutive promoter.

A chimeric or hybrid protein comprising (or consisting of) from N- to C-terminus:

• (a)) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER) targeting,
• (ii) a tag or detection moiety, or “scaffold”, capable of being recognized on a cell surface,
• (iii) at least two transmembrane domains that span the ER membrane, with an extra loop at the ER luminal face,
• (iv) an enzyme recognition/cleavage site spanning a segment of a gp120/41 boundary, facing the ER lumen, and
• (v) an ER retention sequence or motif;
• (b) the chimeric (hybrid) protein of (a), wherein the tag or detection moiety, or “scaffold”, comprises a tag for an antibody or an antigen binding fragment thereof (the antibody binding specifically to the tag or detection moiety, or “scaffold”), or the tag or detection moiety, or “scaffold”, comprises a ligand, or the tag or detection moiety comprises a FLAG molecule or equivalent thereof;
• (c) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises a furin enzyme recognition/cleavage site, a calcium-dependent protein convertase enzyme recognition/cleavage site, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, or an enzyme recognition/cleavage site derived from a member of the subtilisin/kexin family of proprotein convertases;
• (d) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises an enzyme recognition/cleavage site within the V3 loop of gp120;
• (e) the chimeric (hybrid) protein of (a), wherein the ER retention sequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalent thereof;
• (f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is an HIV-1 gp120/41;
• (g) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane domains that span the ER membrane consist of two transmembrane domains;
• (h) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise at least one TM of a CRR5;
• (i) the chimeric (hybrid) protein of (h), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise TM1 and TM2 from the CRR5;
• (j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:2);
• (k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary further comprises a restriction enzyme recognition site acids at both sides, or comprises additional amino-acids at both sides comprising AKRRVVQREKR (SEQ ID NO:3) and AVGIGALF (SEQ ID NO:4);
• or
• (l) the chimeric (hybrid) protein of (a), wherein the protein comprises (or consists of) a synthetic protein or peptide, recombinant protein or peptide, a peptidomimetic or a combination thereof.