Abstract: Provided are methods of assessing activity of a promoter region. The methods include culturing a cell including a nucleic acid, the nucleic acid including a region that encodes an enzyme donor (ED) operably coupled to a promoter region, under conditions in which the ED is expressed when the promoter region is active. The methods further include contacting the ED, if expressed, with an enzyme acceptor (EA) to form ED-EA complexes having enzymatic activity. The methods further include detecting the level of the enzymatic activity to assess activity of the promoter region. Activity of the promoter region may be indicative, and therefore may be used to assess, the activity of a cellular signaling pathway of interest and/or of endogenous or exogenous (e.g., introduced) transcription factors of interest. Cells, compositions, and kits that find use, e.g., in practicing the methods of the present disclosure, are also provided.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP The invention also encompasses red fluorescent PCAs based on Discosoma red fluorescent protein (RFP PCA) and a kindling fluorescent protein PCA (KFP1 PCA) derived from Anemonia sulcata.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: The present invention describes rapid methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). We have demonstrated an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. Also we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. Also, we screened a large cDNA collection using automated PCA, combined with quantitative detection of protein-protein complexes. The invention enables bait-vs.-library, library-vs.-library and defined gene screening in any type of cell or cellular context, and using a wide range of reporters and detection methods.

Abstract: A method of assaying protein-protein interactions associated with proteins involved in lipid pathways using a protein fragment complementation assays, said method comprising the steps of: (a) identifying protein molecules that interact with said protein associated with lipid pathways; (b) selecting a protein reporter molecule; (c) effecting fragmentation of said protein reporter molecule such that said fragmentation results in reversible loss of reporter function; (d) fusing or attaching fragments of said protein reporter molecule separately to said interacting protein molecules as defined in step (a); (e) transfecting cells with nucleic acid constructs coding for the products of step (d); (f) reassociating said reporter fragments through interactions of the protein molecules that are fused or attached to said fragments; and (g) measuring directly or Indirectly the activity of said reporter molecule resulting from the reassociation of said reporter fragments.

Abstract: The cell-based assays described in the present invention can be used to directly assess the sensitivity and specificity of the gene annotation reagent against its target, and to determine if a non-targeted gene participates in a pathway of interest or is functionally linked to another gene or protein. The combination of annotation reagents with such cell-based assays is useful for mapping genes (proteins) into cellular pathways on a genome-wide scale. Preferred assay embodiments include fluorescence or luminescence assays in intact (live or fixed) cells. Such fluorescence or luminescence assays include high-throughput or high-content assays for protein activity, subcellular localization, post-translational modifications, or interactions of proteins. Suitable assays may include protein-protein interaction assays; protein translocation assays; and post-translational modification assays.

Abstract: The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP The invention also encompasses red fluorescent PCAs based on Discosoma red fluorescent protein (RFP PCA)and a kindling fluorescent protein PCA (KFP1 PCA) derived from Anemonia sulcata.

Abstract: This invention provides principles, methods and compositions for ascertaining the mechanism of action of pharmacologically important compounds in the context of network biology, across the entire scope of the complex pathways of living cells. Importantly, the principles, methods and compositions provided allow a rapid assessment of the on-pathway and off-pathway effects of lead compounds and drug candidates in living cells, and comparisons of lead compounds with well-characterized drugs and toxicants to identify patterns associated with efficacy and toxicity. The invention will be useful in improving the drug discovery process, in particular by identifying drug leads with desired safety and efficacy and in effecting early attrition of compounds with potential adverse effects in man.

Abstract: The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: This invention provides principles, methods and compositions for ascertaining the mechanism of action of pharmacologically important compounds in the context of network biology, across the entire scope of the complex pathways of living cells. Importantly, the principles, methods and compositions provided allow a rapid assessment of the on-pathway and off-pathway effects of lead compounds and drug candidates in living cells, and comparisons of lead compounds with well-characterized drugs and toxicants to identify patterns associated with efficacy and toxicity. The invention will be useful in improving the drug discovery process, in particular by identifying drug leads with desired safety and efficacy and in effecting early attrition of compounds with potential adverse effects in man.

Abstract: The present invention provides protein single-color and multi-color protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter such as monomeric enzymes and fluorescent proteins, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. The development of such assays provides for a broad, flexible and biologically relevant platform for drug discovery.

Abstract: The present invention discloses a method of treating an individual or animal with diabetes and/or obesity. The method comprises administering to the individual or animal a therapeutically effective amount of a protein tyrosine kinase inhibitor. Preferably, the preventative and therapeutic methods of the present invention involve administering—to a mammal in need thereof—a therapeutically effective amount of an inhibitor of a c-Src-family protein tyrosine kinase. The invention pertains to pharmaceutical compositions containing an inhibitor of a c-Src-family protein tyrosine kinase or an analog or metabolite thereof, or an inhibitor of another protein tyrosine kinase, and a pharmaceutically acceptable carrier. Purines and pyrimidines and other molecules useful in the treatment of diabetes and obesity are provided herein, in particular, pyrazolopyrimidines, cyanoquinolines, phenylaminopyrimidines, anilinoquinazolines and related compounds.

Abstract: The present invention describes rapid and efficient methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). Examples are given that demonstrate the utility of the invention and the specific advantages of PCA that are not met by other library screening methods. In a first example, we demonstrate an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. In another example we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. In yet another example we demonstrate cDNA library screening in mammalian cells using a bait-vs.-library strategy combined with fluorescence detection.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP The invention also encompasses red fluorescent PCAs based on Discosoma red fluorescent protein (RFP PCA) and a kindling fluorescent protein PCA (KFPL PCA) derived from Anemonia sulcata.