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Title Engineering the pregnane X receptor and estrogen receptor alpha to bind novel small molecules using negative chemical complementation
Publication Date
Date Accessioned
Degree PhD
Discipline/Department Chemistry and Biochemistry
Degree Level doctoral
University/Publisher Georgia Tech
Abstract Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.
Subjects/Keywords Nuclear receptors; Chemical complementation; Negative chemical complementation; Yeast-two hybrid selection; Pregnane X receptor; Estrogen receptor; Pregnane; Protein engineering; Nuclear receptors (Biochemistry); Transcription factors; Yeast Genetics
Contributors Bahareh Azizi (Committee Chair); Donald Doyle (Committee Chair); Andreas Bommarius (Committee Co-Chair); Loren Williams (Committee Co-Chair); Adegboyega Oyelere (Committee Member); Nick Hud (Committee Member); Sheldon May (Committee Member)
Country of Publication us
Record ID handle:1853/39620
Repository gatech
Date Indexed 2020-05-13
Issued Date 2011-04-05 00:00:00
Note [degree] Ph.D.; [advisor] Committee Chair: Bahareh Azizi; Committee Chair: Donald Doyle; Committee Co-Chair: Andreas Bommarius; Committee Co-Chair: Loren Williams; Committee Member: Adegboyega Oyelere; Committee Member: Nick Hud; Committee Member: Sheldon May;

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…8 1.3 Chemical Complementation 12 1.4 Applications of Chemical Complemenation 15 1.4.1 Drug Discovery 15 1.4.2 Engineering Nuclear Receptors 15 1.5 Using Nuclear Receptors to Develop a Novel Biocatalyst 17 1.6 Summary 19 1.7 References…

…19 CHAPTER 2 ENGINEERING NUCLEAR RECEPTORS TOWARD ANTIBIOTICS: PREGNANE X RECEPTOR 27 2.1 Engineering Nuclear Receptors to Bind Antibiotics 27 2.2 Promiscuous Activity of PXR 27 2.3 PXR in Chemical Complementation 32 2.4 PXR Library for…

…Antibiotics 36 viii 2.4.1 Library Design 36 2.4.2 PXR Library Construction and Selection in Chemical Complementation 41 2.5 PXR in HEK293T Mammalian Cells 50 2.6 Summary and Future Work 52 2.7 Materials and Methods 53 2.8 References 56 CHAPTER…

Complementation 99 ix 4.3.2 hERα Variants in Chemical Complementation 103 4.3.3 Random Mutagenesis on hERα 107 4.4 Summary and Future Work 113 4.5 Materials and Methods 115 4.6 References 118 CHAPTER 5 CHARACTERIZATION OF L346 IN HUMAN ESTROGEN…

…RECEPTOR ALPHA: DETERMINING TRANSCRIPTIONAL ACTIVATION WITH STEROID LIGANDS 126 5.1 Previous Mutational Analysis on Human Estrogen Receptor Alpha 126 5.2 Analysis of L346 Variants 131 5.2.1 L346 Variants in Chemical Complementation 131 5.2.2 L346…

…Activity in Mammalian Cells Adrosterone and 17-α Estradiol 148 5.4 Summary and Future Work 152 5.5 Materials and Methods 153 5.6 References 155 CHAPTER 6 NEGATIVE CHEMICAL COMPLEMENTATION WITH ANTAGONISTS 159 6.1 Genetic Selection and Negative…

Chemical Complementation 159 6.2 Negative Chemical Complementation with Agonists 165 6.2.1 Negative Chemical Complementation with RXR 165 6.2.2 Negative Chemical Complementation with hERα 166 x 6.3 Negative Chemical Complementation with…

…Fulvestrant 174 6.4 Using Negative Chemical Complementation for Protein Engineering 177 6.5 Nuclear Receptors and Corepressors 181 6.5.1 Developing Negative Chemical Complementation with Corepressors 184 hERα with ACTR:TUPI in Negative Chemical