Extending chemical complementation to bacteria and furthering nuclear receptor based protein engineering and drug discovery

The bacterial chemical complementation system (BCC) was designed based on a bacterial two hybrid system in which the alpha subunit of RNA polymerase is fused to a nuclear receptor coactivator and the GBD is fused to a nuclear receptor and expressed in E. coli. The Gal4 DNA binding domain binds its response element upstream of an essential gene. Ideally, upon binding of the appropriate ligand to the NR ligand binding domain (LBD), the LBD undergoes a conformational change, recruits the coactivator-alpha RNA polymerase fusion protein, and activates transcription of an essential gene. A new bacterial strain was engineered in which a Gal4 response element controls the expression of the HIS3 gene in the bacterial strain. Once this strain was created and the background growth for this strain was reduced using various concentrations of 3-amintriazole (a competitive inhibitor of the HIS3 gene), we were able to produce activation from our ligand independent control system. However, bacterial chemical complementation did not produce ligand dependent activation.;In a second project designed to further NR based protein engineering and drug discovery, chemical complementation in S. cerevisiae was used to evaluate a library of charge reversal variants. These variants were rationally designed in an attempt to gain a better understanding of nuclear receptor function and structure and to produce an orthogonal ligand receptor pair. A library of retinoic acid receptor (RARalpha) variants were developed to alter the binding selectivity of the receptor from the natural negatively charged ligand, all-trans retinoic acid (atRA), to positively charged retinoid ligands. Single, double, and triple variants were constructed based on five residues in the binding pocket of RARgamma known to stabilize the carboxylate of atRA. Multiple variants were evaluated via chemical complementation with diverse activation profiles with the various amine based retinoids and atRA. We were able to engineer two triple variants capable of activating with the ethyl amine retinoid but not the natural atRA ligand. However these variants do not activate with the ethyl amine retinoid as well as RARalpha does. With the data obtained from evaluating the tolerability of mutations by RARalpha, further developments towards engineering an enhanced ligand-receptor pair capable of activating with higher affinities of the amine based retinoids should be possible.;In a third project CC was utilized to characterize tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. The compounds were assessed for their ability to inhibit estrogen receptor (ER) activation and thus decrease cell proliferation associated with breast cancer. The dual inhibiting compounds are composed of a covalently linked tamoxifen based moiety and a histone deacetylase inhibitor (HDACi) based moiety. Both tamoxifen alone and HDACi alone have been found to decrease proliferation in breast cancer cell lines via ER and histone deacetylases, respectively. However, covalently linking the two moieties can potentially create dual inhibiting compounds that act on various stages of the cell cycle to produce a more potent and effective drug to treat breast cancer. Several dual inhibiting compounds were found to decrease the activation of ER, by its natural ligand estradiol, better than tamoxifen alone. Additionally, when tested in both ER positive and negative breast cancer cells, the compounds were found to decrease proliferation better than tamoxifen alone or the HDACi alone. We have also established that covalent linkage of SAHA and tamoxifen enhances their anti-proliferative effects in MCF-7 cells. Overall, these compounds can have a profound impact as a potential therapeutics as an alternative method for breast cancer treatment. (Abstract shortened by UMI.)...