SBIR-STTR Award

The long-term goal of this project is to develop tools for high throughput testing of drug compounds from combinatorial libraries that target G-Protein-Coupled Receptors (GPCR's) associated with appetite control and eating behavior. The development of drugs aimed at reducing obesity and controlling eating disorders represents a major target of the pharmaceutical industry. Novel targets for such drug development arise from the recent explosion of information on molecules and neural circuits involved in appetite regulation. This proposal focuses on developing testing platforms for two GPCR families associated with feeding behavior--the Melanocortin Receptors (MCRs) and the Orexin Receptors (OXR). High throughput testing of GPCR's requires an efficient and highly reliable system that allows multiplexing of receptors in order to test numerous compounds against numerous targets. This project will take advantage of a novel transfection technology that is licensed by Originus, Inc. from the University of Michigan termed Surface Transfection and Expression Protocol (STEP), which has significant advantages over transient and even stable expression methods In Phase I, we will use existing ligands to validate the platform. Future screening of new drugs will be carried out in a Phase I submission. The Specific Aims for Phase I are: (1) The use and optimization of STEP transfection for 384 well microplate transfection to test the expression and ligand activation of individually expressed feeding-related receptors including MCRs and one of the OXR's. (2) Multiplexing via the co-expression of multiple feeding-related receptors belonging to the same receptor family (MCR's) in order to enhance efficiency of testing of potential targets. Phase II will build on the information from Phase I to test other receptors with different intracellular coupling, to multiplex both within and across receptor families and to facilitate searching for drugs that are agonists and antagonists and inverse agonists. Originus has worked with several partners from pharmaceutical industry in the past and has a specific partner interested in the development of multiplexed GPCR-expressing microplates. Moreover, this project will be the prototype for the future creation of multiplexed GPCR-bearing microplates beyond the feeding-related ones. These would be used for testing a wide range of compounds targeted at modifying cell signaling in a variety of disorders.

Thesaurus Terms:
appetite, cell surface receptor, drug screening /evaluation, receptor expression, transfection combinatorial chemistry, drug discovery /isolation, orexin biotechnology, cell line, high throughput technology

The development of drugs aimed at reducing obesity and controlling eating disorders represents a major target of the pharmaceutical industry and has become one of the most exciting areas in drug discovery. The long term goal of this project is to develop tools for high throughput testing of drug compounds from combinatorial libraries that target G-Protein-Coupled Receptors (GPCR's) associated with appetite control and eating behavior. Novel targets for such drug development arise from the recent explosion of information on molecules and neural circuits involved in appetite regulation. This proposal focuses on developing testing platforms for GPCR families associated with feeding behavior. A distinguishing feature of the drug-discovery strategy we are pursuing is that is based on an efficient and highly reliable system that allows multiplexing of receptors in order to test numerous compounds against numerous targets. This strategy has been enabled by a novel transfection technology that is licensed by Originus, Inc. from the University of Michigan termed Surface Transfection and Expression Protocol (STEP), which has significant advantages over transient and even stable expression methods. Under Phase I funding, testing platforms for two GPCR families associated with feeding behavior-the Melanocortin Receptors (MCRs) and the Orexin Receptors (OXRs) were developed. STEP transfection of 384-we 11 microplates to test the expression and ligand activation of individually expressed MCRs and OXRs (Specific Aim 1) and multiplexing via co-expression of multiple MCRs (Specific Aim 2) were validated in two different cell lines. Under Phase II funding, we propose to build on the information from Phase I to validate platforms for other feeding-related GPCRs with different intracellular coupling, to extend the testing to additional cell lines, to develop new assays for HTS, to optimize allelic variants of the GPCRs, and to multiplex GPCRs of the same or different families in physiological relevant combinations. Finally we propose to initiate our own internal efforts for HTS of multiplexed GPCRs with single ligands per microplate well from a small library of known compounds and for ultraHTS (uHTS) of multiplexed GPCRs using pools of drugs from larger chemical libraries in each microplate well. The proposed experiments have been carefully designed based on interactions with many potential business partners and will allow Originus to demonstrate the efficacy of the STEP platforms for screening multiplexed feeding-related GPCRs and other GPCRs outside the immediate area of obesity drug screening. The proposed Phase II work will be the basis for the implementation of more broadly HTS and uHTS of libraries and testing of potential lead compound in collaboration with selected partners during Phase III.

Project Terms:
3'5'-cyclic ester of AMP; Adenosine Cyclic 3',5'-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine, cyclic 3',5'-(hydrogen phosphate); Adrenal Medullary Paraganglioma; Adrenal Medullary Pheochromocytoma; Adrenal Pheochromocytoma; Agonist; Alkaline Phosphatase; Anti-Obesity Agents; Anti-Obesity Drugs; Antiobesity Agents; Antiobesity Drugs; Appetite; Appetite Regulation; Area; Assay; Bioassay; Biologic Assays; Biological Assay; Biotechnology; Bone structure of ilium; Businesses; Categories; Cell Line; Cell Lines, Strains; CellLine; Cells; Chromaffin Neoplasm; Chromaffin Paraganglioma; Chromaffin Tumor; Collaborations; Complement; Complement Proteins; Coupled; Coupling; Cyclic AMP; Desire for food; Development; Drug Compounding; Drug Evaluation, Preclinical; Drug Industry; Drug Preparation; Drug Screening; Drugs; Eating Behavior; Eating Disorders; Elements; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Explosion; Family; Feeding behaviors; Funding; G Protein-Complex Receptor; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; G-Proteins; GPCR; GPR; GTP-Binding Proteins; GTP-Regulatory Proteins; Gene variant; Genetic Diversity; Genetic Variation; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Hcrt protein; Hcrt/ORX; Hcrts/ORXs; Health; High Throughput Assay; Human; Human, General; Ilium; In Vitro; Individual; Industry; Industry, Pharmaceutic; Ingestive Behavior; Intraadrenal Paraganglioma; Investigators; Lead; Libraries; Licensing; Ligands; Man (Taxonomy); Man, Modern; Medication; Methods; Michigan; Molecular Target; Nerve Cells; Nerve Unit; Neural Cell; Neuroblastoma; Neuroblastoma (Schwannian Stroma-Poor); Neurocyte; Neurons; Obesity; Orthophosphoric-monoester phosphohydrolase (alkaline optimum); Pathway interactions; Pb element; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Industry; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Phenotype; Pheochromocytoma; Physiologic; Physiological; Preclinical Drug Evaluation; Programs (PT); Programs [Publication Type]; Protocol; Protocols documentation; Receptor Protein; Research Personnel; Researchers; Scientific Advances and Accomplishments; Screening procedure; Signal Pathway; Surface; System; System, LOINC Axis 4; Technology; Testing; Transcription Activation; Transcriptional Activation; Transfection; Universities; Up-Regulation; Variation (Genetics); Wood; Wood material; Work; adenosine 3'5' monophosphate; adiposity; alkaline phosphomonoesterase; allelic variant; base; cAMP; calcium flux; calcium mobilization; chromaffinoma; combinatorial; corpulence; corpulency; corpulentia; cultured cell line; design; designing; drug candidate; drug development; drug discovery; drug/agent; experiment; experimental research; experimental study; feeding; feeding-related behaviors; glycerophosphatase; heavy metal Pb; heavy metal lead; high throughput screening; hypocretin; hypocretin/orexin; hypocretins/orexins; melanocortin receptor; neural circuit; neural circuitry; neuronal; novel; nutrient intake activity; obese; obese people; obese person; obese population; orexin; pathway; programs; prototype; receptor; receptor coupling; release of sequestered calcium ion into cytoplasm; research study; response; scientific accomplishments; scientific advances; screening; screenings; small molecule libraries; tool