Modeling, Synthesis, and Biological Evaluation of Potential RXT Selective Agonist

Arizona State University-Tempe Campus, Tempe AZ

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Abstract

We propose to employ rational drug design to synthesize novel retinoid X receptor (RXR) analogs (rexinoids) that preferentially bind to RXR, and are superior in biological potency and specificity to current rexinoids. These novel compounds will possess decreased toxicity as well as attenuated antagonism of other nuclear receptor pathways that employ RXR as a heterodimeric partner in transcriptional signaling. We will also utilize the newly synthesized rexinoid compounds to probe cancer cells to elucidate unique anti- proliferative/prodifferentiation genes that are regulated by RXR-mediated pathways. Our model synthetic skeleton is bexarotene, a potent FDA-approved RXR agonist used for treatment of cutaneous T cell lymphoma (CTCL);however, patients treated with this potent RXR-agonist often suffer from side effects like hypothyroidism, raised triglycerides, and cutaneous toxicity, presumably arising through alternate RXR-involved biological pathways. Thus it is constructive to develop novel rexinoids that would not provoke these untoward side effects. Through preliminary modeling studies, we will determine whether a given compound will associate effectively with the ligand-binding domain of RXR. For compounds passing these preliminary modeling studies, we will synthesize these analogs of bexarotene that incorporate novel atoms and functional groups at specific locations on the molecule in an attempt to create an RXR agonist that is not only selective for RXR, but does not induce negative side-effects in the proposed cell culture and animal studies. We will assay these unique agonist molecules in RXR mammalian-2-hybrid assays. Those analogs that induce robust RXR homodimerization will be further evaluated in an RXRE-mediated transcriptional assay system, to determine agonist activity. Analogs with the greatest potential as RXR agonists will also be tested for their proapoptotic ability and their lack of teratogenicity. Once we have identified lead RXR-agonists, we will assay in cell culture their ability to influence transcriptional signaling by other receptors, such as TR, that utilize RXR as a heterodimeric partner. The most specific RXR-homodimerization agonists will be examined for diminished clearance rates in vivo and novel metabolites in both in vitro and in vivo pharmacokinetic profiles. In vivo toxicity will be determined in the most promising of compounds by dosing and assaying lipid and TSH profiles in a rat model, the two pathways most dysregulated in patients undergoing bexarotene therapy. Finally, microarray analysis will be used to determine gene expression profiles of CTCL cells treated with bexarotene and select analogs. The genes that display the most responsive regulation (activation or repression) will be compared to bexarotene treated cells in an effort to identify those analogs with the potential to most effectively regulate the greatest number of biomarkers associated with chemoprotection. In addition to the above aims, this project will help train undergraduate research students who will design, model, synthesize, and test novel and innovative RXR analogs with potential clinical application and significance in the treatment of CTCL.