Screening for LXR agonists-inhibitors of brain amyloidosis and inflammation

描述(申请人提供)：尽管学术界和制药行业做出了巨大努力，但抗体疫苗接种、γ-和b-分泌酶抑制剂仍远未用于临床。也有人提出了通过干扰胆固醇代谢来改善AD表型的药物，但使用这些药物的理由尚不明确，在所有情况下，解释其有益效果的分子机制也知之甚少。因此，仍然非常需要其他降低Ab值的策略。肝X核受体(LXRa和LXRb)是控制胆固醇代谢相关基因表达的转录因子。我们发现，激活的LXR调节脑胆固醇细胞内和细胞外运输的代谢途径，从而影响APP的处理、b-淀粉样蛋白的沉积及其从大脑中的清除。此外，我们和其他人已经证明，用天然或合成的LXR配体处理神经细胞系和原代神经元可以减少抗体的分泌，并且用合成的LXR配体在体内治疗年轻的AD模型小鼠，增加了中枢神经系统中LXR反应基因的表达，并降低了他们大脑中的可溶性b-淀粉样蛋白水平。我们还发现，LXR配体的应用抑制了炎性细胞因子的分泌，并增加了单抗或脂多糖治疗后神经元的存活率。在体内，LXR配体治疗对AD表型的最终有益结果是由神经元和星形胶质细胞表达的影响抗体生成、β-淀粉样蛋白形成和清除的基因以及与抗体清除和炎症密切相关的小胶质细胞表达的基因介导的联合作用。因此，这些数据证实了用于识别LXR激动剂/激动剂的药物发现研究的设计以及它们在良好的体外和体内模型系统中的系统评估。在特定目标1中，LXR的激活状态将通过其显著增加主要LXR反应基因ABCA1转录的能力来评估。因此，在体外应用给定化合物(总共50,000用于筛选)后，ABCA1信使核糖核酸的增加将作为LXR激活的报告。符合LXR激活标准的化合物将在体外进一步筛选其减少抗体产生的能力。HITS对ABCA1表达和apoE分泌具有双重刺激作用，降低抗体和促进胆固醇外流的能力将在基于LXR基因敲除细胞的检测中得到进一步表征。此外，有效的化合物将被描述为它们对细胞因子表达的抑制作用以及对对脂肪酸和甘油三酯合成有不良影响的基因的转录活性的调节作用。在特定目标2中，将选择两种最有效的化合物在PS1APP转基因小鼠身上进行初步体内试验。公共卫生相关性：尽管学术界和制药业做出了巨大努力，但基于我们对阿尔茨海默病的理解最近取得的进展，药物仍然无法用于临床。脑胆固醇代谢及其与疾病发病机制不同方面的关系证实了基于转录因子及其反应基因控制的复杂调控网络的新治疗方法的发展。我们正在提出一种药物筛选方法，用于鉴定天然或合成的化合物，这些化合物可以激活称为核肝X受体(LXRa和LXRb)的转录因子，从而导致胆固醇代谢相关基因的上调和表达增加。这项药物发现研究基于我们和其他实验室产生的数据，这些数据证实大脑中LXR控制的胆固醇转运体和脂蛋白参与了b-淀粉样蛋白的聚集、沉积和清除。因此，LXR激动剂可用于降低抗体、抑制炎症和改善行为障碍。

DESCRIPTION (provided by applicant): Despite the enormous efforts in academia and in pharmaceutical industry, Ab vaccination, y- and b-secretase inhibitors are still far from clinical use. Drugs that ameliorate AD phenotypes by interfering cholesterol metabolism have been also suggested, but the rationale for using those is poorly defined and in all of the cases the molecular mechanisms which account for their beneficial effects are poorly understood. Therefore there remains a great need for other strategies of lowering Ab. Nuclear Liver X receptors (LXRa and LXRb) are transcription factors that control the expression of genes involved in cholesterol metabolism. We have found that activated LXR regulate metabolic pathways of brain cholesterol intra- and extracellular transport that influence APP processing, b-amyloid deposition and its clearance from brain. Moreover, we and others have shown, that treatments of neuronal cell lines and primary neurons with natural or synthetic LXR ligands decrease Ab secretion, and that in vivo treatment of young AD model mice with a synthetic LXR ligand, increased the expression of LXR responsive genes in CNS and decreased soluble b-amyloid levels in their brains. We have also found that the application of LXR ligands inhibited secretion of inflammatory cytokines and increased neuronal survival following Ab or LPS treatment. The ultimate beneficial outcome of LXR ligand treatment on AD phenotype in vivo is a combination of effects mediated by genes expressed in neurons and astrocytes influencing Ab generation, b-amyloid formation and clearance, and by genes expressed in microglia which are tightly related to both - Ab clearance and inflammation. The data therefore substantiate the design of a drug discovery study for identification of LXR activators/agonists and their systematic evaluation in well established in vitro and in vivo model systems. In Specific aim 1, the activated status of LXR will be evaluated by their ability to increase dramatically the transcription of ABCA1 - a major LXR response gene. Thus, the increased amount of ABCA1 mRNA following in vitro application of a given compound (altogether >50,000 for screening) will serve as a reporter for LXR activation. Compounds that meet the criteria for LXR activation will be further screened for their ability to reduce Ab production in vitro. "Hits" with dual stimulatory effect on ABCA1 expression and apoE secretion, ability to reduce Ab and to facilitate cholesterol efflux will be further characterized in LXR knockout cell based assays. In addition, potent compounds will be characterized for their inhibitory effects on cytokine expression and regulatory effects on transcriptional activity of genes with undesired effects on fatty acids and triglyceride synthesis. In Specific Aim 2, the two most potent compounds will be chosen for initial in vivo tests in PS1APP transgenic mice. PUBLIC HEALTH RELEVANCE: Despite the enormous efforts in academia and in pharmaceutical industry, drugs based on the recent progress in our understanding for Alzheimer disease are still not available for clinical use. Brain cholesterol metabolism and its relation to different aspects of the disease pathogenesis substantiates the development of new therapeutic approaches based on complex regulatory networks controlled by transcription factors and their responsive genes. We are proposing a drug screening approach for identification of natural or synthetic compounds that activate transcription factors called Nuclear Liver X receptors (LXRa and LXRb) and therefore lead to upregulation and increased expression of genes involved in cholesterol metabolism. This drug discovery study is based on data generated in our and other laboratories which confirm that LXR controlled cholesterol transporters and lipoproteins in brain are involved in b-amyloid aggregation, deposition and clearance. Therefore, LXR activators can be used for lowering Ab inhibition of inflammation and improvement of behavioral deficits.