Development of an HTS assay to identify FXR antagonists

描述(申请人提供)：人类核激素受体(NR)超家族的成员是类固醇、甲状腺激素、维甲酸、氧化类固醇、脂肪酸和胆汁酸的受体。几乎所有具有确定配体的NRs都已成功地成为治疗各种疾病的药物靶点，包括糖尿病、血脂异常、炎症和癌症。FXR是一种天然产物，可作为胆汁酸的生理受体。FXR在胆汁酸动态平衡以及糖脂代谢中起着关键作用。FXR已被认为是治疗血脂异常(动脉粥样硬化)、糖尿病和胆结石的药物靶点。虽然已发现一些合成的FXR激动剂，但尚未鉴定出选择性拮抗剂。因此，可用于表征这种受体的化学工具是有限的。建议的研究集中在开发鉴定FXR拮抗剂的方法，这些拮抗剂可以作为化学工具来充分表征FXR的生物学功能，并验证该受体是否为潜在的药物靶点。为了开发鉴定FXR拮抗剂所需的分析，将解决以下具体目标：(1)开发和验证生化分析，以高通量筛选形式检测FXR拮抗剂；(2)开发和验证二次分析，以表征在FXR高通量筛选中确定的拮抗剂“HITS”；以及(3)使用Lopac 1280化学文库对FXR分析流程方案进行验证筛选。 与公共卫生相关：这项建议侧重于开发筛查试验来识别特定的FXR拮抗剂。FXR是一种核受体，其功能是胆汁酸的生理受体。目前没有选择性的FXR拮抗剂存在，拮抗剂的鉴定将有助于表征该受体在新陈代谢调节中的作用。

DESCRIPTION (provided by applicant): Members of the human nuclear hormone receptor (NR) superfamily serve as receptors for steroids, thyroid hormones, retinoids, oxysterols, fatty acids, and bile acids. Nearly all of the NRs with identified ligands have been successful targets for drugs treating a variety of diseases including diabetes, dyslipidemia, inflammation, and cancer. FXR is a NR that functions as a physiological receptor for bile acids. FXR plays a critical role in bile acid homeostasis as well as glucose and lipid metabolism. FXR has been implicated as a drug target for the treatment of dyslipidemia (atheroscelerosis), diabetes, and gallstones. Although a few synthetic agonists have been identified for FXR, no selective antagonists have yet been characterized. Thus, the chemical tools available to characterize this receptor are limited. The proposed research is focused on development of assays to identify FXR antagonists that can be used as chemical tools to fully characterize the biological function of FXR as well as to validate this receptor as a potential drug target. In order to develop the assays required for identification of FXR antagonists the following specific aims will be addressed: (1) Develop and validate a biochemical assay to detect FXR antagonists in a high-throughput screen format.; (2) Develop and validate secondary assays for the purpose of characterizing antagonist "hits" identified in an FXR high throughput screen; and (3) Perform a validating screen of the FXR assay flow scheme using the LOPAC 1280 chemical library. PUBLIC HEALTH RELEVANCE: This proposal focuses on development of screening assays to identify specific FXR antagonists. FXR is a nuclear receptor that functions as a physiological receptor for bile acids. No selective FXR antagonists currently exist and identification of antagonists will allow for characterization of the role of this receptor in regulation of metabolism.