Discovery and characterization of novel estrogen receptor agonist ligands and development of biochip

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Nuclear Receptors NRs) are protein transcription factors that have been shown to be involved in processes as diverse as drug development and discovery, reproduction, homeostasis, metabolism and cell death. NRs play such varied roles that, consequently, their dysfunction is associated with a large number of diseases.

The importance of NRs in disease and their ability to bind small lipophilic molecules through structurally conserved domains make them highly desirable and successful drug targets. The focus of this work was to identify novel ligands for estrogen receptors ERs), classic receptors with well defined biological actions and therapeutic utility, and orphan NRs, for which ligands and functions are frequently unknown. The first part of the thesis describes studies directed towards identifying and characterizing novel ligands for ER. Specifically, we describe the development of a robust fluorescence resonance energy transfer-based screening assay that was used to identify two novel ER agonists from a screen of approximately 10,000 small molecules.

One of these agonists has an unusual bicyclic scaffold that represents a new class of ligands for ER. Characterization of this class of ligands was performed utilizing biochemical, cellular, in vivo and crystallographic methods. A second agonist, a structural analog of conjugated equine estrogen drugs utilized in hormone replacement therapy is also characterized. Understanding the structure activity relationships of this compound through in vitro assays and structural studies provides us with insights into the activities of several components of these important drugs.

From our experiences with conventional fluorescent-based screening and our understanding of their limitations, we developed a biochip assay SAMDI) combining self-assembled monolayers on gold surfaces and MALDI-TOF mass spectrometry detection to identify new NR ligands. The drug development and discovery, testing and analysis of this biochip platform for ER and other orphan NRs are described. Lastly, this new assay is applied towards tackling the difficult problem of identifying orphan receptor ligands, and the results of a preliminary screen are presented. The work described in this thesis can hopefully contribute to advances in NR drug discovery and NR ligand characterization to ultimately identify new drugs that can be used to investigate or treat NR associated diseases.