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.

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.

Creating receptors for control of transcription with arbitrary small molecules has widespread applications including gene therapy, biosensors, and enzyme engineering. Using the combination of high throughput docking, codon randomization, and chemical complementation, we have created new receptors to control transcription with small molecules. Chemical complementation, a new method of protein engineering, was used to discover retinoid X receptors (RXR) variants that are activated by compounds that do not activate wild-type RXR. A first library of 32,768 RXR variants was designed for the synthetic retinoid-like compound LG335. The library produced ligand-receptor pairs with LG335 that have a variety of EC50s and efficacies. One engineered variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at 10 and #64979;fold lower LG335 concentration than wild-type RXR with 9cRA in yeast. The activity of this variant in mammalian cells correlates with its activity in yeast. A second library of 262,144 RXR variants was designed for two purposes: (i) to develop a high-throughput chemical complementation method to select variants that have high efficacies and low EC50s; and (ii) to find variants which are activated by small molecules not known to bind RXR variants. Selection conditions were manipulated to find only variants with high efficacies and low EC50s. This library was also selected for variants that activate transcription specifically in response to gamma-oxo-1-pyrenebutyric acid (OPBA), which is different from any known RXR ligand. OPBA was chosen as a potential ligand using high-throughput docking with the software program FlexX. Two variants are activated by OPBA with an EC50 of 5 mM. This is only ten-fold greater than the EC50 of wild type RXR with its ligand 9cRA (500 nM) in yeast. An improved method synthesizing LG335 and a method for quantifying intracellular ligand concentrations were developed. Although the LG335 synthetic method has an additional step, the overall yield was improved to 8% from 4% in the original publication. Liquid chromatography and mass spectrometry was used to quantify the intracellular concentration of LG335, which was found to be within four fold of the LG335 concentration in the media.

Aspergillus westerdijkiaem qui est récemment démembré d'A. ochraceus est un producteur principal de plusieurs composés de type polycétone d'importance économique. Ces composés incluent l’ochratoxin A, mellein, l'acide penicillique, asperlactone et l’isoasperlactone et quelques intermédiaires comme l'acide 6- methylsalicylique et l’acide orsellinique. La biosynthèse de ces métabolites est catalysée par un groupe d'enzymes connues comme la polycétone synthases (PKSs). Ce travail a été visé pour cloner et a caractérisé fonctionnellement les différentes genes des PKS i.e. aoks1, aolc35-12 et aomsas, et de genes de polyketide synthases-non ribosomal peptide synthase (PKS-NRPS) i.e. aolc35-6, chez A. westerdijkiae. Ces gènes ont été inactivés par l'insertion du gène d’hygromycine B phosphotransferase d’Escherichia coli dans le génome d'A. westerdijkiae, pour obtenir les mutantes ao?ks1, ao?lc35-12, ao?msas et ao?lc35-6. Les mutants ao?ks1 et ao?lc35-12 ont été trouvés déficients dans la biosynthèse d’ochratoxin A, mais produisaient encore mellein. À notre connaissance, c’est la première fois que nous avons caractérisé les gènes impliquées dans la biosynthèse d’OTA, sachant que mellein, qui était proposé dans la littérature comme un intermédiaire, joue a cune role dans la biosynthesis de l'OTA. Ensuite le mutant ao?msas n'a pas seulement perdu la capacité de produire isoasperlactone et asperlactone, mais aussi il ne produit pas l’intermédiaire acide 6-methylsalicylique. Basé sur les expériences de la caractérisation génétique et de complémentation chimiques, nous avons proposé un shéma hypothétique de la biosynthèse d’asperlactone et isoasperlactone dans lequel l'acide 6-methylsalicylique, diepoxide et aspyrone jouent le rôle d’intermédiaires. La techniques de gène knock-out et de la reverse transcription PCR (RT-PCR) ont montré que seulle gène de type PKS-NRPS « aolc35-6 » identifié chez A. westerdijkiae codant pour un intermédiaire inconnu(s) qui pourrait inciter l'expression de gène aomsas et un gène impliqué dans la biosynthèse d'acide orsellinique et d'acide penicillique. / Aspergillus westerdijkiaem which is recently dismembered from A. ochraceusm is the principal producer of several economically important polyketide metabolites. These metabolites include ochratoxin A, mellein, penicillic acid, asperlactone and isoasperlactone and some intermediates like orsellinic acid and 6-methylsalicylic acid. The biosynthesis of these metabolites is catalyzed by a group of enzymes known as polyketide synthases (PKSs). This work was aimed to clone and functionally characterized various PKS i.e. aoks1, aolc35-12 and aomsas, and polyketide synthasesnon ribosomal peptide synthase (PKS-NRPS) genes i.e. aolc35-6, in A. westerdijkiae. These genes were inactivated by the insertion of Escherichia coli hygromycin B phosphotransferase gene in the genome of A. westerdijkiae to obtain ao?ks1, ao?lc35-12, ao?msas and ao?lc35-6 mutants. ao?ks1, ao?lc35-12 mutants were found deficient in ochratoxin A biosynthesis but are still producing mellein. To our knowledge, we for the first time characterized a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. Further ao?msas mutant not only lost the capacity to produce isoasperlactone and asperlactone but also the intermediate nature product 6-methylsalicylic acid. Based on the genetic characterization and chemical complementation experiments, we have proposed a hypothetical pathway mentioning that 6-methylsalicylic acid, diepoxid and aspyrone are intermediates of isoasperlactone and asperlactone. Gene knockout technique and reverse transcription PCR (RT-PCR) shown that the only PKS-NRPS gene aolc35-6 so far identified in A. westerdijkiae encoding certain unknown intermediate(s) which induces the expression of aomsas gene and a gene involved in the biosynthesis of orsellinic acid and penicillic acid.