Methods and compositions for the positive selection of protein destabilizers

Abstract

The present application relates, in some aspects, to the development of an assay that uses cell survival and/or cell viability as a phenotypic identifier to positively select for agents that destabilize a protein of interest.

What is claimed is: 
     
         1 . A method of identifying a test agent that destabilizes a protein of interest, the method comprising,
 providing cells expressing a recombinant fusion protein comprising a protein of interest and an enzyme, wherein the enzyme converts an exogenous substrate that is not toxic to the cells into a product that is toxic to the cells;   contacting a first portion of the cells with a test agent;   culturing the first portion of the cells in the presence of the exogenous substrate;   culturing, separately from the first portion of cells, a second portion of the cells in the presence of the exogenous substrate, wherein the second portion of the cells is not contacted with the test agent; and   determining a level of cell survival in each of the cultured portions, wherein a greater level of cell survival in the first portion compared to the second portion indicates that the test agent destabilizes the protein of interest.   
     
     
         2 . The method of  claim 1 , wherein the protein of interest is an oncoprotein. 
     
     
         3 . The method of  claim 2 , wherein the oncoprotein is selected from the group consisting of MYC, Ikaros family zinc finger protein 1 (IKZF1), Ikaros family zinc finger protein 3 (IKZF3), Interferon regulatory factor 4, mutant p53, N-Ras, K-Ras, c-Fos, c-Jun, and estrogen receptor (ER). 
     
     
         4 . The method of any one of the preceding claims, wherein the protein of interest is a protein associated with neurodegeneration. 
     
     
         5 . The method of any one of the preceding claims, wherein the protein of interest is a prion. 
     
     
         6 . The method of any one of the preceding claims, wherein the protein of interest is an amyloid protein. 
     
     
         7 . The method of any one of the preceding claims, wherein the enzyme is selected from the group consisting of deoxycytidine kinase, thymidylate kinase, thymidine kinase-guanylate kinase fusion, and FKBP-Caspase9 fusion. 
     
     
         8 . The method of any one of the preceding claims, wherein the exogenous substrate is a synthetic compound. 
     
     
         9 . The method of  claim 8 , wherein the synthetic compound is a nucleoside analog. 
     
     
         10 . The method of  claim 9 , wherein the nucleoside analog is selected from the group consisting of bromovinyl-deoxyuridine, azidothymidine (AZT), and Ganciclovir. 
     
     
         11 . The method of  claim 8 , wherein the synthetic compound is AP1903. 
     
     
         12 . The method of any one of the preceding claims, wherein the test agent is selected from the group consisting of a small organic molecule, an amino acid, a protein, a nucleoside, a nucleotide, a nucleic acid, or an analog or derivative thereof. 
     
     
         13 . The method of any one of the preceding claims, wherein the test agent is an immunomodulatory drug (IMiD). 
     
     
         14 . The method of  claim 13 , wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. 
     
     
         15 . The method of any one of  claims 1 - 12 , wherein the test agent is an estrogen receptor antagonist. 
     
     
         16 . The method of  claim 15 , wherein the estrogen receptor antagonist is Fulvestrant. 
     
     
         17 . The method of any one of the preceding claims, wherein the cells further express a reporter protein. 
     
     
         18 . The method of  claim 17 , wherein the reporter protein is a fluorescent protein or a bioluminescent protein. 
     
     
         19 . The method of  claim 18 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein, tdTomato, and red fluorescent protein. 
     
     
         20 . The method of  claim 18 , wherein the bioluminescent protein is selected from the group consisting of renilla luciferase (RLuc) and firefly luciferase (FLuc). 
     
     
         21 . A method of identifying a test agent that destabilizes a protein of interest, the method comprising,
 providing cells expressing a recombinant fusion protein comprising a protein of interest and an enzyme, wherein the enzyme converts an exogenous substrate that is not toxic to the cells into a product that is toxic to the cells;   contacting the cells with a library of test agents;   culturing the cells in the presence of the exogenous substrate;   determining cell survival in the cultured cells, wherein survival of a cell indicates that a test agent from the library of test agents destabilizes the protein of interest; and   isolating the surviving cell.   
     
     
         22 . The method of  claim 21 , further comprising identifying the test agent from the surviving cell. 
     
     
         23 . The method of  claim 21  or  claim 22 , wherein the library of test agents is selected from the group consisting of a library of nucleic acids, a library of peptides, a library of antibodies or antigen-binding fragments thereof, and a library of small organic molecules. 
     
     
         24 . The method of  claim 23 , wherein the library of nucleic acids comprises DNA, cDNA, RNA, siRNA, shRNA, miRNA, sgRNA, mRNA, or some combination thereof. 
     
     
         25 . The method of any one of  claims 21 - 24 , wherein the library of test agents is a viral library. 
     
     
         26 . The method of  claim 25 , wherein contacting the cells with the library of agents comprises transducing the cells with the viral library. 
     
     
         27 . The method of  claim 25  or  claim 26 , wherein the viral library is selected from the group consisting of a lentiviral library, a retroviral library, an adenoviral library, an adeno-associated viral library, a herpes simplex viral library, and a baculoviral library. 
     
     
         28 . The method of any one of  claims 22 - 27 , wherein the test agent is identified by nucleic acid sequencing. 
     
     
         29 . The method of any one of  claims 22 - 27 , wherein the test agent is identified by peptide sequencing. 
     
     
         30 . The method of any one of  claims 21 - 29 , wherein the cells further express a reporter protein. 
     
     
         31 . The method of any one of  claims 21 - 30 , wherein the test agent destabilizes the protein of interest by degrading the protein of interest. 
     
     
         32 . The method of any one of  claims 21 - 30 , wherein the test agent destabilizes the protein of interest by degrading an mRNA molecule encoding the protein of interest or encoding a protein that regulates the stability of the protein of interest. 
     
     
         33 . A method of identifying a test agent that destabilizes a protein of interest, the method comprising,
 providing a mixture of cells comprising a first portion of cells expressing a recombinant fusion protein comprising a protein of interest and an enzyme and a second portion of cells expressing a recombinant protein comprising the enzyme, wherein the enzyme converts an exogenous substrate that is not toxic to the cells into a product that is toxic to the cells;   contacting the mixture of cells with a test agent;   culturing the mixture of cells in the presence of the exogenous substrate; and   determining a level of cell survival for each of the portions in the cultured mixture, wherein a ratio of the number of cells in the first portion to the number of cells in the second portion in the cultured mixture greater than 1 indicates that the test agent destabilizes the protein of interest.   
     
     
         34 . The method of  claim 33 , further comprising:
 providing an aliquot of the mixture of cells that has not been contacted with the test agent;   culturing the aliquot in the presence of the exogenous substrate; and   determining the level of cell survival for each of the portions in the cultured aliquot, wherein a ratio of the number of cells in the first portion to the number of cells in the second portion in the cultured aliquot that is less than the ratio in the cultured mixture indicates that the test agent destabilizes the protein of interest.   
     
     
         35 . The method of  claim 33 , wherein the recombinant fusion protein of the first portion of cells comprises a first reporter protein and the recombinant protein of the second portion of cells comprises a second reporter protein, wherein the second reporter protein is different from the first reporter protein. 
     
     
         36 . The method of  claim 35 , wherein the first reporter protein and the second reporter protein are each independently a fluorescent protein or a bioluminescent protein. 
     
     
         37 . The method of  claim 36 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein, tdTomato, and red fluorescent protein. 
     
     
         38 . The method of  claim 36 , wherein the level of cell survival is determined by flow cytometry. 
     
     
         39 . The method of  claim 36 , wherein the bioluminescent protein is selected from the group consisting of renilla luciferase (RLuc) and firefly luciferase (FLuc). 
     
     
         40 . The method of any one of  claims 33 - 39 , wherein the ratio of the first portion to the second portion in the mixture is less than 1 prior to culturing. 
     
     
         41 . The method of  claim 40 , wherein the ratio of the first portion to the second portion is approximately 0.1, 0.01, or 0.001. 
     
     
         42 . The method of any one of  claims 33 - 41 , wherein the protein of interest is an oncoprotein. 
     
     
         43 . The method of  claim 42 , wherein the oncoprotein is selected from the group consisting of MYC, Ikaros family zinc finger protein 1 (IKZF1), Ikaros family zinc finger protein 3 (IKZF3), Interferon regulatory factor 4, mutant p53, N-Ras, K-ras, c-Fos, c-Jun, and estrogen receptor (ER). 
     
     
         44 . The method of any one of  claims 33 - 41 , wherein the protein of interest is a protein associated with neurodegeneration. 
     
     
         45 . The method of any one of  claims 33 - 41 , wherein the protein of interest is a prion. 
     
     
         46 . The method of any one of  claims 33 - 41 , wherein the protein of interest is an amyloid protein. 
     
     
         47 . The method of any one of  claims 33 - 46 , wherein the enzyme is selected from the group consisting of deoxycytidine kinase, thymidylate kinase, thymidine kinase-guanylate kinase fusion, and FKBP-Caspase9 fusion. 
     
     
         48 . The method of any one of  claims 33 - 47 , wherein the exogenous substrate is a synthetic compound. 
     
     
         49 . The method of  claim 48 , wherein the synthetic compound is a nucleoside analog. 
     
     
         50 . The method of  claim 49 , wherein the nucleoside analog is selected from the group consisting of bromovinyl-deoxyuridine, azidothymidine (AZT), and Ganciclovir. 
     
     
         51 . The method of  claim 48 , wherein the synthetic compound is AP1903. 
     
     
         52 . The method of any one of  claims 33 - 51 , wherein the test agent is an IMiD. 
     
     
         53 . The method of  claim 52 , wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. 
     
     
         54 . The method of any one of  claims 33 - 51 , wherein the test agent is an estrogen receptor antagonist. 
     
     
         55 . The method of  claim 54 , wherein the estrogen receptor antagonist is Fulvestrant. 
     
     
         56 . A DNA vector comprising in operable linkage:
 (a) a promoter;   (b) a nucleotide sequence encoding a fusion protein comprising a protein of interest fused to a cytotoxic element selected from the group consisting of deoxycytidine kinase, thymidylate kinase, thymidine kinase-guanylate kinase fusion, and FKBP-Caspase9 fusion;   (c) an internal ribosomal entry site (IRES); and   (d) a nucleotide sequence encoding a reporter protein.   
     
     
         57 . The DNA vector of  claim 56 , wherein the fusion protein comprises a linker fusing the protein of interest to the cytotoxic element. 
     
     
         58 . The DNA vector of  claim 57 , wherein the linker comprises a peptide linker. 
     
     
         59 . The DNA vector of  claim 58 , wherein the peptide linker comprises one or more iterations of the amino acid sequence Glycine-Glycine-Serine. 
     
     
         60 . The DNA vector of claim any one of  claims 56 - 59 , wherein the reporter protein is a fluorescent protein or a bioluminescent protein. 
     
     
         61 . The DNA vector of  claim 60 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein, tdTomato, and red fluorescent protein. 
     
     
         62 . The DNA vector of  claim 60 , wherein the bioluminescent protein is selected from the group consisting of renilla luciferase (RLuc) and firefly luciferase (FLuc). 
     
     
         63 . The DNA vector of any one of  claims 56 - 62 , wherein the promoter is an inducible promoter. 
     
     
         64 . The DNA vector of any one of  claims 56 - 63 , wherein the promoter is a eukaryotic promoter or a synthetic promoter. 
     
     
         65 . The DNA vector of  claim 64 , wherein the promoter comprises cytomegalovirus (CMV) promoter. 
     
     
         66 . The DNA vector of any one of  claims 56 - 65 , wherein the protein of interest is derived from an ORFeome of an organism. 
     
     
         67 . The DNA vector of any one of  claims 56 - 66 , wherein the protein of interest comprises an oncoprotein. 
     
     
         68 . The DNA vector of  claim 67 , wherein the oncoprotein is selected from the group consisting of MYC, Ikaros family zinc finger protein 1 (IKZF1), Ikaros family zinc finger protein 3 (IKZF3), Interferon regulatory factor 4, mutant p53, N-Ras, K-Ras, c-Fos, c-Jun, and estrogen receptor (ER). 
     
     
         69 . The method of any one of  claims 56 - 66 , wherein the protein of interest is a protein associated with neurodegeneration. 
     
     
         70 . The method of any one of  claims 56 - 66 , wherein the protein of interest is a prion. 
     
     
         71 . The method of any one of  claims 56 - 66 , wherein the protein of interest is an amyloid protein. 
     
     
         72 . An isolated host cell comprising the DNA vector of any one of  claims 56 - 71 . 
     
     
         73 . The isolated host cell of  claim 72 , wherein the host cell is a bacterial cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell. 
     
     
         74 . A method of identifying a test agent that destabilizes a protein of interest, the method comprising,
 providing cells comprising a DNA vector comprising, in operable linkage, an inducible promoter and a nucleotide sequence encoding a fusion protein comprising a protein of interest fused to a cytotoxic element;   contacting a first portion of the cells with a test agent;   culturing the first portion of the cells in the presence of an inducer of the inducible promoter;   culturing, separately from the first portion of cells, a second portion of the cells in the presence of the inducer, wherein the second portion of the cells is not contacted with the test agent; and   determining a level of cell survival in each of the cultured portions, wherein a greater level of survival in the first portion compared to the second portion indicates that the test agent destabilizes the protein of interest.   
     
     
         75 . The method of  claim 74 , wherein the inducible promoter is a chemically inducible promoter. 
     
     
         76 . The method of  claim 75 , wherein the chemically inducible promoter is selected from the group consisting of an ethanol-inducible promoter, an IPTG-inducible promoter, a tetracycline-inducible promoter, a steroid-inducible promoter, and a metal-inducible promoter. 
     
     
         77 . The method of any one of  claims 74 - 76 , wherein the inducer is any compound that directly or indirectly interacts with the inducible promoter to initiate or enhance transcription of the nucleotide sequence. 
     
     
         78 . The method of  claim 74 , wherein the inducible promoter is a heat-shock inducible promoter. 
     
     
         79 . The method of  claim 78 , wherein the inducer is heat.