Small molecule regulation of endogenous transcription factors for circuit-specific neuromodulation

内源转录因子的小分子调节用于电路特异性神经调节

• 批准号: 10260250
• 负责人: Gerald R. Crabtree
• 金额: $ 126.18万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10260250
• 关键词: Address; Affect; Animals; Anxiety; Area; BRAIN initiative; Behavior; Binding; Biological Assay; Brain; Brain region; Calorimetry; Cell physiology; Cells; Chemicals; Chromatin; Data; Dimerization; Enzymes; Fibroblasts; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Gonadal Steroid Hormones; Heterodimerization; Human; Immunofluorescence Immunologic; Intervention; Knock-in; Knowledge; Laboratories; Ligands; Light; Link; Mediating; Medicine; Methods; Molecular; Moods; Mus; Neurologic; Neurons; Neuropharmacology; Neurosciences; Organism; Peripheral Nervous System; Pharmaceutical Preparations; Pharmacologic Substance; Population; Process; Proteins; Publishing; Regulation; Reporter; Resources; Role; Selective Serotonin Reuptake Inhibitor; Serotonin; Serotonin Production; Signal Transduction; Social Behavior; Specificity; Stress; Structural Biologist; Surface Plasmon Resonance; TPH2; Tacrolimus Binding Proteins; Testing; Therapeutic Uses; Titrations; Transcriptional Regulation; Validation; Venus; Virus Diseases; Visualization; Work; base; cell type; cytotoxicity; disability; drug action; effective therapy; efficacy testing; in vivo; innovation; insight; interdisciplinary approach; nervous system disorder; neuroregulation; neurotransmission; non-genetic; novel; promoter; protein degradation; protein transport; side effect; single-cell RNA sequencing; sleep behavior; small molecule; therapeutic development; tool; transcription factor; transcriptome sequencing; transgene expression; virtual; virus genetics

PROJECT SUMMARY Methods for regulating cellular processes within distinct populations of neurons are needed to elucidate relationships between molecular mechanisms, circuits, and behavior; and to develop cell type- or circuit- selective treatments for neurological disorders. We propose a novel, non-genetic, small molecule method— transcription factor-chemically induced proximity (TF-CiP)—that harnesses the cell type- and circuit-specificity of endogenous transcription factors to regulate gene expression in subsets of neurons. TF-CiP utilizes a bifunctional small molecule to heterodimerize an “anchor” transcription factor, which naturally binds to a target gene, with a “hijacked” transcription factor, which enhances or represses transcription of the target gene. Cell specificity is determined from the intersection of expression of each transcription factor. TF-CiP can theoretically be adapted for any organism and because transcription factors are well conserved, it is possible that the same TF-CiP small molecule can be used to modulate neuronal processes across animal species. We will develop TF-CiP to regulate the expression of the rate-limiting enzyme for brain serotonin synthesis, TPH2, as a means to tune serotonin levels in subsets of serotonergic neurons. Although the population of central serotonergic neurons is relatively small, these neurons send projections throughout the brain and serve important roles in regulating mood, anxiety, sleep, and social behavior. Achieving circuit-specificity for a serotonin-modulatory small molecule would be an improvement over current therapies, which can have undesirable side-effects due to indiscriminate targeting of serotonin signaling in the central and peripheral nervous systems. TPH2 transcription is regulated by stress, sex hormones, and several transcription factors. We leverage this knowledge along with single-cell RNA-sequencing and projection mapping data in serotonergic neurons as a resource for candidate TF-CiP transcription factors. In Aim 1, we will screen for transcription factors that regulate TPH2 transcription cooperatively upon chemically-induced heterodimerization of their FRB and FKBP tags. This screen will employ a Tph2-Venus reporter and orthophthalaldehyde- mediated serotonin visualization as readouts in serotonergic cells. In Aim 2, we will work with structural biologists and chemists to synthesize bifunctional TF-CiP molecules for TPH2 transcriptional regulation. This will involve virtual binding screens of over 8 million small molecules, binding validation using surface plasmon resonance and isothermal titration calorimetry, small molecule functionalization, and chemical linkage of two transcription factor-binding molecules. In Aim 3, TF-CiP molecules will be screened for cytotoxicity, selectivity, and efficacy at regulating TPH2 transcription and serotonin synthesis in cells. In Aim 4, we will test TF-CiP molecules for efficacy and selectivity in vivo using murine social behavior assays and brain immunostaining, respectively. Successful completion of this study will provide the first neuropharmaceutic that is capable of targeting specific subsets of neurons or circuits without the aid of exogenous genetically-encoded proteins.

项目概要 需要调节不同神经元群体内细胞过程的方法来阐明 分子机制、电路和行为之间的关系；并开发细胞类型或电路 神经系统疾病的选择性治疗。我们提出了一种新颖的非遗传小分子方法—— 转录因子化学诱导接近 (TF-CiP)——利用细胞类型和电路特异性 内源转录因子调节神经元亚群中的基因表达。 TF-CiP 利用 双功能小分子使“锚定”转录因子异二聚化，该转录因子自然地与靶标结合 基因，带有“劫持”的转录因子，可以增强或抑制目标基因的转录。细胞 特异性由每个转录因子表达的交集确定。 TF-CiP 可以 理论上适用于任何生物体，并且由于转录因子非常保守，因此有可能 相同的 TF-CiP 小分子可用于调节动物物种的神经元过程。我们 将开发 TF-CiP 来调节大脑血清素合成限速酶 TPH2 的表达， 作为调节血清素能神经元亚群中血清素水平的一种手段。虽然中部地区人口 血清素能神经元相对较小，这些神经元向整个大脑发送投射并服务 在调节情绪、焦虑、睡眠和社交行为方面发挥着重要作用。实现电路特异性 血清素调节小分子将是对当前疗法的改进，它可以 由于不加区别地针对中枢和外周血清素信号传导而产生不良副作用 神经系统。 TPH2 转录受到应激、性激素和多种转录因子的调节。 我们利用这些知识以及单细胞 RNA 测序和投影映射数据 血清素能神经元作为候选 TF-CiP 转录因子的资源。在目标 1 中，我们将筛选 在化学诱导的异二聚化过程中协同调节 TPH2 转录的转录因子 他们的 FRB 和 FKBP 标签。该屏幕将采用 Tph2-Venus 报告基因和邻苯二甲醛- 介导的血清素可视化为血清素能细胞中的读数。在目标 2 中，我们将与结构性合作 生物学家和化学家合成用于 TPH2 转录调控的双功能 TF-CiP 分子。这 将涉及超过 800 万个小分子的虚拟结合屏幕，使用表面等离子体激元进行结合验证 共振和等温滴定量热法、小分子功能化以及两者的化学连接 转录因子结合分子。在目标 3 中，将筛选 TF-CiP 分子的细胞毒性、选择性、 以及调节细胞中 TPH2 转录和血清素合成的功效。在目标 4 中，我们将测试 TF-CiP 使用小鼠社会行为测定和脑免疫染色来确定体内功效和选择性的分子， 分别。这项研究的成功完成将提供第一种能够 无需外源基因编码蛋白的帮助即可针对神经元或电路的特定子集。