Small Molecule Screen for Circadian and Metabolic Transcription Factors

描述(申请人提供)：觉醒、内分泌状态和新陈代谢受到体内大多数有核细胞中存在的昼夜节律转录时钟的强烈影响。核心时钟转录因子BMAL1由核受体家族的几个孤儿成员通过保守的启动子区域进行调节。其中两个受体在其配体结合域(LBD)中与自然产生的可交换脂质结晶。LBD具有其他NRs的典型结构特征，如类固醇、视黄醇或甲状腺受体，为受体作为小分子配体反应转录因子的功能提供了令人信服的证据。然而，这两个密切相关的孤儿NRs的药理活性配体尚未确定。在这两个受体上成功发现配体将对哺乳动物昼夜生物学的研究产生重大影响，并将促进针对睡眠和情绪障碍、糖尿病和其他形式的代谢性疾病以及癌症的新型小分子疗法的开发。这项提议的第一个目标是以384孔格式对这两个目标实施均相、溶液相荧光共振能量转移(FRET)分析。对该检测方法的生化成分进行了鉴定和检测。该检测方法应该对激动剂和拮抗剂都有反应。将对包含25,000种化合物的文库进行筛选，并通过二次化验确认命中结果，以便为HTS的化验验证和优化找到阳性对照配体。如有必要，HITS将通过药物化学合成进行修改，以获得足够的效力(EC50；5 FM)以进行确证分析。这些发现，以及一种经过验证的测试迁移到专门的HTS设施，有可能极大地加快在这些目标上发现配体的速度。与公共健康的相关性人类生理学，在健康和疾病方面，受到调节身体功能的各个方面的自主每日周期的严重影响。从这些研究中得出的发现有可能解决这些过程，以造福人类健康。

DESCRIPTION (provided by applicant): Wakefulness, endocrine status, and metabolism are strongly influenced by circadian transcriptional clocks present in most nucleated cells of the body. A core clock transcriptional factor, BMAL1, is in turn regulated by several orphan members of the nuclear receptor (NR) family through a conserved promoter region. Two of these receptors have been crystallized with naturally-occurring, exchangeable lipids in their ligand-binding domains (LBDs) The LBDs have structural features typical of other NRs, such as the steroid, retinoid, or thyroid receptors, providing compelling evidence that the receptors function as small molecule ligand responsive transcription factors. Pharmacologically active ligands to these two closely- related orphan NRs have yet to be identified, however. Successful ligand discovery at the two receptors will have a major impact on research in mammalian circadian biology and will facilitate development of novel small molecule therapeutics for sleep and mood disorders, for diabetes and other forms of metabolic disease, and for cancer. The first objective of this proposal is to implement a homogenous, solution phase fluorescence resonance energy transfer (FRET) assay for these two targets in a 384-well format. The biochemical components of the assay have been identified and tested. The assay should respond to both agonists and antagonists. A library of 25,000 compounds will be screened and hits confirmed by a secondary assay in order to find positive control ligands for assay validation and optimization for HTS. If necessary, hits will be modified by medicinal chemistry synthesis to achieve adequate potency (EC50 < 5 fM) for confirmatory assays. These findings, and migration of a proven assay to a dedicated HTS facility, have the potential to considerably accelerate ligand discovery at these targets. Relevance to Public Health Human physiology, in health and disease, is heavily influenced by the autonomous daily cycle that regulates all aspects of body function. The findings that emerge from these studies have to potential to address these processes for the benefit of human health.