Development automated assay-regulators insulin synthesis

DESCRIPTION (provided by applicant): We propose to develop an automated screen for small molecules that modulate insulin promoter activity. The assay is based upon a mouse insulinoma, MIN6, that normally expresses insulin mRNA. This cell was engineered to stably contain two cassettes that use a fluorescent reporter protein to monitor insulin promoter activity and a housekeeping gene activity. The secondary assay to be used to confirm the hits will be to verify modulation of endogenous insulin mRNA expression relative to control mRNAs. Like all cultured insulin-producing cells, MIN6 cells produce substantially less insulin mRNA and protein than do normal a-cells in the intact pancreas. Thus, it should be feasible to identify compounds that can either stimulate or suppress insulin production. The assay will be marketed as a tool for researchers to discover small molecule modulators of insulin mRNA synthesis. Such bioactive molecules will be used subsequently to probe the regulatory pathways that control insulin secretion and should contribute to cell-based or pharmacologic treatments for type-I and -II diabetes. Preliminary data indicate that the Insulin promoter-eGFP reporter transgene mimics the activity of the endogenous insulin gene and a pilot screen of 8,000 small synthetic compounds yielded small molecule hits that increased and decreased insulin gene expression. Calculated Z? values of 0.74 for increase and 0.43 for decrease in eGFP were obtained. The proposed Aims are to 1) Optimize the assay and verify its performance in a pilot screen of 50,000 compounds, and 2) Optimize image analysis algorithms and deliver a stand-alone software package to enable investigators to derive reliable numerical data on insulin gene activity from the images. P The project proposes to develop an automated screen for small molecules that modulate insulin promoter activity. The assay will be marketed as a tool for researchers to discover small molecule modulators of insulin mRNA synthesis. Such bioactive molecules will be used subsequently to probe the regulatory pathways that control insulin secretion and should contribute to cell-based or pharmacologic treatments for type-I and -II diabetes. Preliminary data indicate that the Insulin promoter-eGFP reporter transgene mimics the activity of the endogenous insulin gene and a pilot screen of 8,000 small synthetic compounds yielded small molecule hits that increased and decreased insulin gene expression. Calculated Z' values of 0.74 for increase and 0.43 for decrease in eGFP were obtained. The proposed Aims are to 1) Optimize the assay and verify its performance in a pilot screen of 50,000 compounds, and 2) Optimize image analysis algorithms and deliver a stand-alone software package to enable investigators to derive reliable numerical data on insulin gene activity from the images. Project Narrative: The project proposes to develop an automated screen for small molecules that modulate insulin promoter activity. The assay will be marketed as a tool for researchers to discover small molecule modulators of insulin mRNA synthesis. Such bioactive molecules will be used subsequently to probe the regulatory pathways that control insulin secretion and should contribute to cell-based or pharmacologic treatments for type-I and -II diabetes