Abstract: Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)-based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the G? subunit and a G?? interacting protein (?? IP) for the binding to the G?? dimer. These biosensors comprises (1) a ?? IP and (2) a G? or G? protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.

Abstract: Systems and methods for the monitoring of G protein activation at various cell compartments, such as the plasma membrane and the endosomes, and in a G? protein subunit family-selective manner are described. These systems and methods also allows the monitoring of G protein-coupled receptor (GPCR)-mediated as well as non-receptor guanine nucleotide exchange factor (GEF)-mediated G protein activation, and are based on the use of the G protein-binding domains of specific effectors of G proteins and cellular compartment markers, tagged with suitable energy (BRET) donors and acceptors.

Abstract: Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intracellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.

Abstract: Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intracellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.

Abstract: Described herein is a bioluminescence resonance energy transfer (BRET) based system and method to monitor ternary complex formation in real-time in live cells with high sensitivity and accuracy. This system transfers energy simultaneously between a luciferase donor and intermediate and terminal acceptors, appropriately chosen to also enable transfer from the intermediate to terminal acceptor while minimizing contaminating signals. The system may also be adapted for quaternary complex detection by including a protein complementation assay (PCA) component. The system is broadly applicable to the detection of any protein ternary/quaternary complex such as those involving nuclear receptors, GPCRs, Receptor Tyrosine Kinase (RTKs), multimeric enzymes or structural proteins.

Abstract: The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and/or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

Abstract: Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intra-cellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.

Abstract: Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)-based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the Got subunit and a G?? interacting protein (?? IP) for the binding to the G?? dimer. These biosensors comprises (1) a ?? IP and (2) a G? or G? protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.

Abstract: Described herein is a bioluminescence resonance energy transfer (BRET) based system and method to monitor ternary complex formation in real-time in live cells with high sensitivity and accuracy. This system transfers energy simultaneously between a luciferase donor and intermediate and terminal acceptors, appropriately chosen to also enable transfer from the intermediate to terminal acceptor while minimizing contaminating signals. The system may also be adapted for quaternary complex detection by including a protein complementation assay (PCA) component. The system is broadly applicable to the detection of any protein ternary/quaternary complex such as those involving nuclear receptors, GPCRs, Receptor Tyrosine Kinase (RTKs), multimeric enzymes or structural proteins.

Abstract: There are provided herein transgenic non-human animals and cells comprising a transgene encoding either a mutated human melanocortin type-4 receptor (hMC4R) protein, wherein the mutated protein is misfolded and retained intracellularly, or a wild-type human melanocortin type-4 receptor (hMC4R) protein. Transgenes and targeting constructs used to produce such transgenic animals and cells are also provided, as well as methods for using the transgenic animals in pharmaceutical screening and as commercial research animals for modeling obesity.

Abstract: Disclosed herein is a compound of Formula I: wherein X, R1, R2, and R3 are as defined herein, or a pharmaceutically acceptable salt thereof to allow the drug to penetrate the cell membrane; or a prodrug, or the compound is labeled with a detectable label or an affinity tag thereof. Also disclosed is a pharmaceutical composition, a method of treating a disorder mediated by melanocortin-4 receptors, and a method of treating obesity using the compounds described.

Abstract: Disclosed is a compound of Formula I: or a salt thereof, in which X, X1, X2, R1, R2, and R3 are described herein. Also disclosed are pharmaceutical compositions and methods of using the compounds of Formula I to treat disorders mediated by melanocortin-4 receptors.

Abstract: The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and/or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

Abstract: The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and/or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

Abstract: Disclosed herein is a compound of Formula I: wherein X, R1, R2, and R3 are as defined herein, or a pharmaceutically acceptable salt thereof to allow the drug to penetrate the cell membrane; or a prodrug, or the compound is labeled with a detectable label or an affinity tag thereof. Also disclosed is a pharmaceutical composition, a method of treating a disorder mediated by melanocortin-4 receptors, and a method of treating obesity using the compounds described.

Abstract: There are provided herein transgenic non-human animals and cells comprising a transgene encoding either a mutated human melanocortin type-4 receptor (hMC4R) protein, wherein the mutated protein is misfolded and retained intracellularly, or a wild-type human melanocortin type-4 receptor (hMC4R) protein. Transgenes and targeting constructs used to produce such transgenic animals and cells are also provided, as well as methods for using the transgenic animals in pharmaceutical screening and as commercial research animals for modeling obesity.

Abstract: The present invention relates to novel biosensors that are based on bioluminescence resonance energy transfer (BRET). These biosensors may be used to monitor rapid interaction and conformational changes within G protein-coupled receptor/G protein complexes and, in this way, reflect the activation status of the receptor. Advantageously, the biosensors may be used as a highly sensitive and quantitative assay for the identification of ligands (agonists, antagonists, inverse agonists, partial agonists, etc.) targeting G protein-coupled receptors (GPCRs) as well as for the analysis of the activation status of these receptors. Moreover, multiplexing different biosensors within receptors/G protein complexes allows for mapping ligand textures. Additionally, the biosensors permit the direct, real-time examination of interactions between receptors and G protein in their natural environment, the living cell.

Abstract: Disclosed herein is a compound of Formula I: wherein X, R1, R2, and R3 are as defend herein, or a pharmaceutically acceptable salt thereof to allow the drug to penetrate the cell membrane; or a prodrug, or the compound is labeled with a detectable label or an affinity tag thereof. Also disclosed is a pharmaceutical composition, a method of treating a disorder mediated by melanocortin-4 receptors, and a method of treating obesity using the compounds described.

Abstract: The present invention relates to a novel biosensor. A resonance energy transfer (RET) biosensor comprising a beta(?)-arrestin tagged with a first and a second chromophore, wherein said first chromophore is a fluorophore and said second chromophore is a fluorophore or a bioluminophore is described.

Abstract: Disclosed herein is a compound of Formula I: wherein X, R1, R2, and R3 are as defend herein, or a pharmaceutically acceptable salt thereof to allow the drug to penetrate the cell membrane; or a prodrug, or the compound is labeled with a detectable label or an affinity tag thereof. Also disclosed is a pharmaceutical composition, a method of treating a disorder mediated by melanocortin-4 receptors, and a method of treating obesity using the compounds described.