Small Molecule Screen for Circadian and Metabolic Transcription Factors

描述（由申请人提供）：觉醒、内分泌状态和代谢受到身体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又通过保守的启动子区域由核受体（NR）家族的几个孤儿成员调节。 这些受体中的两种已经在其配体结合结构域（LBD）中用天然存在的可交换脂质结晶。LBD具有其他NR的典型结构特征，例如类固醇、类维生素A或甲状腺受体，提供了令人信服的证据表明受体作为小分子配体响应性转录因子起作用。 然而，这两个密切相关的孤儿NR的药理学活性配体尚未被鉴定。 在这两种受体上成功发现配体将对哺乳动物昼夜节律生物学的研究产生重大影响，并将促进开发用于睡眠和情绪障碍、糖尿病和其他形式的代谢疾病以及癌症的新型小分子疗法。 本提案的第一个目标是在384孔格式中对这两个靶点实施均质的溶液相荧光共振能量转移（FRET）测定。 已鉴定并检测了测定的生化组分。 该测定应对激动剂和拮抗剂均产生响应。 将筛选25，000种化合物的文库，并通过二次试验确认命中，以找到阳性对照配体，用于HTS的试验验证和优化。 如有必要，将通过药物化学合成对命中进行修饰，以达到足够的效价（EC50 < 5 fM）用于确证性测定。 这些发现，以及一个经过验证的检测迁移到一个专门的HTS设施，有可能大大加快在这些目标的配体发现。 与公共卫生的相关性在健康和疾病方面，人类生理学受到调节身体功能各个方面的自主日常周期的严重影响。 这些研究的结果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.