A cell-based method, a cell-based platform or system or a multiplexed platform, for monitoring the activity of an enzyme, a protease, a viral protease, or an HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, comprising (or made by a method 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, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease is enzymatically active (the detectable moiety is cleaved off by the enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease);
- (2) the cell-based method, cell-based platform or system or multiplexed platform 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;
- (3) the cell-based method, cell-based platform or system or multiplexed platform of (1) or (2), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold;
- (4) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (3), further comprising screening for an inhibitor of an enzyme, a protease, a viral protease or an HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease by:
- (a) providing a compound to be screened as an inhibitor of an enzyme, a protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, or providing a nucleic acid to be screened as encoding an inhibitor of an enzyme, a protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease;
- (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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, is enzymatically active (the detectable moiety is cleaved off by the enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease) and the enzymatic activity of the enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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;
- (5) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (4), further comprising 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;
- (6) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (5), further comprising 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 enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, to one of the divided cell samples;
- (7) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (6), wherein the amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease under physiologic (cell culture) conditions comprises SEQ ID NO:3 or SEQ ID NO:4;
- (8) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (7), wherein the HIV-1 protease comprises SEQ ID NO:5 or SEQ ID NO:6;
- (9) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (8), wherein the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;
- (10) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (9), wherein the cell is a mammalian cell, a monkey cell or a human cell, or a lymphocyte or a hepatocyte, or a T cell, and optionally the cells are genetically bar-coded;
- (11) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (10), wherein the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;
- (12) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (11), wherein the detectable moiety comprises an epitope for an antibody, or a FLAG tag;
- (13) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (12), wherein the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a plate-reader, a flow cytometry or microscope visualization;
- (14) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (13), wherein the compound to be screened as an inhibitor of the enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;
- (15) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (14), wherein the compound to be screened as an inhibitor of the enzyme, protease, viral protease or HIV-1 protease, or Hepatitis C Virus (HCV) or Dengue Virus (DenV) 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;
- (16) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (15), wherein the protease or enzyme is an HIV-1 protease, or a viral, a microbial or a mammalian protease or enzyme, or a NS2/NS3 or NS3/NS4A protease of HCV, or any Hepatitis C Virus (HCV) or Dengue Virus (DenV) protease, or a West Nile Virus, a Yellow Fever Virus, or any Flaviviridae virus;
- or
- (17) the cell-based method, cell-based platform or system or multiplexed platform of any of (1) to (16), wherein two or more, or a plurality of, enzymes or proteases are screened in the same cell, wherein optionally they are variants of the same enzyme or protease, or different enzymes or proteases, or a combination thereof.