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

我们建议采用合理的药物设计来合成新的维甲酸X受体(RXR)类似物(Rexinoids)，这些类似物优先与RXR结合，在生物学效力和特异性上优于现有的rexinoid。这些新化合物将具有降低的毒性以及减弱使用RXR作为转录信号中异二聚体伙伴的其他核受体途径的拮抗作用。我们还将利用新合成的rexin类化合物来探测癌细胞，以阐明由RXR介导的途径调节的独特的抗增殖/前分化基因。 我们的模型合成骨架是贝沙罗汀，这是FDA批准的用于治疗皮肤T细胞淋巴瘤(CTCL)的一种有效的RXR激动剂；然而，使用这种有效的RXR激动剂治疗的患者通常会出现副作用，如甲状腺功能减退、甘油三酯升高和皮肤毒性，可能是通过替代RXR相关的生物途径引起的。因此，开发不会引起这些不良副作用的新的rexinoid是有建设性的。通过初步的建模研究，我们将确定给定的化合物是否会有效地与RXR的配体结合结构域结合。对于通过这些初步模拟研究的化合物，我们将合成这些在分子上特定位置结合新原子和官能团的贝沙罗汀类似物，试图创造一种不仅对RXR具有选择性，而且在拟议的细胞培养和动物研究中不会引起负面副作用的RXR激动剂。我们将在RXR哺乳动物-2杂交试验中分析这些独特的激动剂分子。那些诱导RXR同源二聚的类似物将在RXRE介导的转录检测系统中进一步评估，以确定激动剂的活性。具有最大潜力作为RXR激动剂的类似物也将接受促凋亡能力和无致畸作用的测试。一旦我们确定了主要的RXR激动剂，我们将在细胞培养中测试它们影响其他受体转录信号的能力，例如利用RXR作为异二聚体伙伴的受体。将检查最特异的RXR同质二聚激动剂在体内的清除率降低以及新的代谢物在体外和体内的药代动力学曲线。体内毒性将通过在大鼠模型中剂量和分析血脂和TSH谱来确定最有希望的化合物，这两条途径在接受贝沙罗汀治疗的患者中调节最不正常。最后，微阵列分析将被用来确定贝沙罗汀和选择类似物处理的CTCL细胞的基因表达谱。显示最敏感调节(激活或抑制)的基因将与贝沙罗汀处理的细胞进行比较，以努力确定那些具有最有效地调节与化学保护相关的最大数量生物标记物的潜力的类似物。除了上述目标外，该项目还将帮助培养本科生，他们将设计、建模、合成和测试在CTCL治疗中具有潜在临床应用和意义的新颖和创新的RXR类似物。

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.