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Title Extending chemical complemenation to bacteria and furthering nuclear receptor based protein engineering and drug discovery
Publication Date
Date Accessioned
Degree PhD
Discipline/Department Chemistry and Biochemistry
Degree Level doctoral
University/Publisher Georgia Tech
Abstract Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression. In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA. In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.
Subjects/Keywords Protein engineering; Nuclear receptor; Chemical complementation; Drug discovery; Nuclear receptors (Biochemistry); Developmental pharmacology; Protein engineering; Yeast Genetics
Contributors Doyle, Donald (Committee Chair); Barry, Bridgette (Committee Member); Bommarius, Andreas (Committee Member); Ledoux, Joe (Committee Member); Matsumura, Ichiro (Committee Member); Oyelere, Adegboyega (Committee Member)
Country of Publication us
Record ID handle:1853/29652
Repository gatech
Date Indexed 2020-05-13
Issued Date 2009-05-18 00:00:00
Note [degree] Ph.D.; [advisor] Committee Chair: Doyle, Donald; Committee Member: Barry, Bridgette; Committee Member: Bommarius, Andreas; Committee Member: Ledoux, Joe; Committee Member: Matsumura, Ichiro; Committee Member: Oyelere, Adegboyega;

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…Cited 1 5 10 14 CHAPTER 2 THE POWER OF GENETIC SELECTION 2.1 2.2 2.3 2.4 2.5 Genetic Selection Yeast Two-Hybrid Systems Chemical Complementation Bacterial Two-Hybrid Systems Literature Cited CHAPTER 3 23 23 25 30 35 41 BACTERIAL CHEMICAL

COMPLEMENTATION: ELIMINATING BACKGROUND 47 3.3 Bacterial Chemical Complementation: Bacteria vs. Yeast 3.2 Developing Bacterial Chemical Complementation 3.3 Results of Eliminating Bacterial Chemical Complementation Background 3.3.1 Effects of Expression Vectors…

…on Background Activation 3.3.2 Effect of Promoters on Background 3.3.3 Effects of Integrating the Reporter on Background Growth 3.4 Materials and Methods 3.5 Literature Cited 47 49 57 58 58 61 63 67 CHAPTER 4 BACTERIAL CHEMICAL COMPLEMENTATION

…LIGAND DEPENDENT ACTIVATION 70 4.1 Optimizing Bacterial Chemical Complementation for Ligand Activation 4.1.1 Enhancing the Interaction Between GBD and Gal1p 70 70 viii 4.2 4.3 4.4 4.5 4.6 4.1.2 Optimizing the Various Components of the BCC system…

Complementation Future of Bacterial Chemical Complementation Materials and Methods Literature Cited 76 82 85 87 93 97 99 100 105 LIGAND-RECEPTOR CHARGE REVERSAL VIA GENETIC ENGINEERING 113 Engineering Nuclear Receptors and Orthogonal Ligand Receptor Pairs…

…BREAST CANCER THERAPEUTICS 151 6.1 Estrogen Receptor and Breast Cancer 6.2 Motive for Dual Inhibiting Compounds as Breast Cancer Therapeutics 6.3 Using Chemical Complementation to Assess Activity of Dual Inhibiting Compounds towards Estrogen Receptor…

…Binding Pocket Residues and their interaction with 9cRA 13 Figure 2.1: General Yeast Two-Hybrid System 27 Figure 2.2: Genetic Complementation 32 Figure 2.3: Chemical Complementation 34 Figure 2.4: Negative Chemical Complementation 34 Figure…

…Figure 3.2: Bacterial Chemical Complementation (BCC) 50 Figure 3.3: Applications of Bacterial Chemical Complementation 53 Figure 3.4: Bacterial Chemical Complementation 56 Expression and Reporter Vectors Figure 3.5: Initial Results of…