Evaluation of Vdr-coactivator Inhibitors Using Biochemical and Cell-based Assays

Abstract

ABSTRACT EVALUATION OF VDR–COACTIVATOR INHIBITORS USING BIOCHEMICAL AND CELL–BASED ASSAYS by Athena Baranowski The University of Wisconsin–Milwaukee, 2013 Under the Supervision of Dr. Alexander Arnold The vitamin D receptor (VDR) is a ligand–dependent transcription factor, which belongs to the nuclear receptor superfamily. VDR–mediated gene regulation is governed by coregulators (coactivators and corepressors). VDR coregulator binding inhibitors (CBIs), which were discovered using high throughput screening (HTS), were evaluated using cell–based assays and biochemical assays to determine their ability to inhibit the interaction between VDR and steroid receptor coactivator–2 (SRC–2). Determining their ability to inhibit the VDR–SRC–2 interaction can lead to the development of novel and safer pharmaceutical treatments for VDR–related disorders, such as cancer, autoimmune diseases, and cardiovascular disease; therefore, the study of transcription, toxicity, and protein–protein interactions are critical to the development of pharmaceutical treatments. The methods used to study CBIs include a cell–based transcription assay, which was based on the formation of β–lactamase in the Hek 293T cell line. The readout of this assay was fluorescence resonance energy transfer (FRET) of a cell–permeable substrate in the event of transcription inhibition, and a blue fluorescent signal in the event of transcription activation. Active CBIs, such as compound WL052410G (31b), exhibit a reduced blue to green fluorescent ratio. A luciferase–based viability assay, which detected ATP levels from metabolically active Hek 293T cells, was used to determine the toxicity of the CBIs, in hopes of detecting partial toxicity for pharmaceutical targets of VDR, such as compound WL052410G (31b). Different co–immunoprecipitation pull–down assays were developed to precipitate and isolate target proteins, such as VDR, SRC–2, and SRC–2–3, using affinity–based tagging, in order to determine the ability of CBIs to disrupt the protein–protein interaction between VDR and SRC–2 coactivator. The VDR–SRC–2 interaction was visualized using a fluorescently tagged antibody, which was realized using a laser imager. The most active compounds for all the assays performed belong to a series of 3–indolyl–methanamines and include a highly selective and active compound, WL052410G (31b).