The vitamin D receptor (VDR) is a ligand-activated transcription factor and is partially responsible for the regulation of the endocrine system. This includes the transcriptional regulation of genes involved in the production of the parathyroid hormone (PTH) and the regulation of the blood calcium level. High levels of PHT can cause hypercalcemia in the case of primary and tertiary hyperparathyroidism. The VDR-mediated gene regulation, activated by its ligand 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is governed by the recruitment of coactivators. Our hypothesis is that small molecules, with the ability to inhibit the interaction between VDR and certain coactivators, are able to selectively modulate VDR-mediated transcription. Our long term objective is the application these VDR-coactivator inhibitors to study the role of VDR-coactivator binding for specific VDR- regulated genes. Ultimately, these molecules can be developed into new treatments for hypercalcemic hyperparathyroidism. No small molecule or non-peptide inhibitors are known for the VDR-coactivator interaction. The objective of this application is to identify a reversible, selective, and non-toxic inhibitor of the VDR-coactivator interaction with the ability to inhibit VDR-mediated transcription in cells in collaboration with the Molecular Library Probe Production Centers Network (MLPCN). The first aim is to carry out a high throughput screen (HTS) using fluorescence polarization (FP) to identify hit molecules with the ability to inhibit the interaction between VDR and a specific fluorescent coactivator peptide. The second aim describes the execution of secondary assays to validate biochemically active molecules in a cellular context and to exclude false-positive HTS hit molecules. This includes a VDR-mediated transcription assay, a cytotoxicity assay, and two FP assays using different detection wavelengths. The investigations concerning the mode of binding (allosteric vs. orthosteric), the selectivity among other nuclear receptors, and the mode of action (reversible vs. irreversible) of small molecules with the ability to inhibit VDR-mediated transcription is described as aim three. These small molecule characteristics will be addressed by employing a VDR ligand competition assay, FP assays with other nuclear receptors and fluorescent coactivator peptides, and a dialysis based reversibility assay. We are proposing aim four in case that only sub-optimal inhibitors are identified among the MLPCN small molecule collection. This aim describes the development of sub-optimal small molecule inhibitors to meet the proposal objective using structure activity relationship (SAR) studies supported by chemistry and purchase in case those structural analogs are commercially available. The discovery of VDR-coactivator inhibitors will support our long term investigations and provide the basis for the development of new treatments for hypercalcemic hyperparathyroidism. PUBLIC HEALTH RELEVANCE: High calcium blood levels (hypercalcemia) caused by high blood levels of parathyroid hormone (hyperparathyroidism) cause serious health problems including kidney failure, hypertension, dementia, and coma. The vitamin D receptor, activated by its endogenous ligand 1,25- dihydroxyvitamin D3, has been identified to play a crucial role in the regulation of these endocrine factors. The development and investigation of novel vitamin D receptor modulators (VDR-coactivator inhibitors) will increase our understanding of the VDR-dependency of hyperparathyroidism and hypercalcemia and represents a new approach for potential treatments.