A High Throughput Ultrafiltration LC-MS Platform for the Discovery of Vitamin D Receptor Ligands

Abstract

Vitamin D, a pleiotropic acting member of the nuclear receptor superfamily has demonstrated the ability to affect many auxiliary biological sites beyond the classically described homeostasis of calcium and phosphorous. Apart for the genomic actions of vitamin D, a myriad of rapid response mechanisms have been elucidated including signal transduction pathways acting in an autocrine/paracrine manner. Drug discovery efforts have been increasingly promising for many diseases states through the structural modification of the secosteroid structure. However, toxicity of high doses of vitamin D and analogs has spurred an investigation into potent nonsecosteroid binding partners for the vitamin D receptor (VDR) with the promise of alleviating these side effects. This dissertation describes the development of a VDR ultrafiltration liquid chromatography mass spectrometry-screening (LC-MS) assay. With the VDR platform in place a high throughput approach was developed, which significantly expands our laboratories, screening capabilities while simultaneously identifying new ligands for the VDR. An evaluation of a metabolomics software platform was also presented, which greatly reduces the complexity of the LC-MS data sets. This is accomplished through the noise filtering, peak detection, peak alignment, and cross sample peak matching algorithms within the vendor software package LC-Solutions. Currently, a 100-fold improvement in sampling capacity has been established however in the foreseeable future further advancement of this assay can be realized with continual advancement of materials and equipment. A variety of compounds were discovered as ligands for the VDR during this process. In particular a class of compounds described as indenoisoquinolines has demonstrated binding to the VDR and have also displayed affinity towards another nuclear receptor RXR. This expands the therapeutic value of this class as dual acting chemoprevention or chemotherapeutic agents. In addition the prenylated flavoniods found in hops have shown the ability to out compete an established ligand for VDR (curcumin).