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

项目摘要 需要阐明在不同神经元群体内调节细胞过程的方法 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的，非遗传的，小分子方法- 转录因子-化学诱导邻近（TF-CiP）-利用细胞类型和电路特异性 内源性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”转录因子异源二聚化，所述转录因子天然结合靶 基因，与“劫持”转录因子，增强或抑制靶基因的转录。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， 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5-羟色胺合成的限速酶TPH 2的表达， 作为调节肾上腺素能神经元亚群中血清素水平的一种手段。虽然中部的人口 多巴胺能神经元相对较小，这些神经元将投射发送到整个大脑， 在调节情绪、焦虑、睡眠和社会行为方面发挥重要作用。实现电路特异性， 调节降钙素的小分子将是对现有疗法的改进， 由于不加选择地靶向中枢和外周的血清素信号传导而产生的不良副作用 神经系统TPH 2的转录受应激、性激素和几种转录因子的调节。 我们利用这些知识沿着单细胞RNA测序和投影映射数据， 作为候选TF-CiP转录因子的来源的多巴胺能神经元。在目标1中，我们将筛选 在化学诱导的异源二聚化作用下协同调节TPH 2转录的转录因子 FRB和FKBP的标签该筛选将使用Tph 2-Venus报告基因和邻苯二甲醛- 介导的5-羟色胺可视化作为在肾上腺素能细胞中的读数。在目标2中，我们将使用结构 生物学家和化学家合成用于TPH 2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，使用表面等离子体的结合验证 共振和等温滴定量热法，小分子功能化，以及两个 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TPH 2转录和5-羟色胺合成的功效。在目标4中，我们将测试TF-CiP 使用鼠社会行为测定和脑免疫染色的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个神经药物，是能够 靶向神经元或回路的特定子集，而无需外源遗传编码蛋白的帮助。

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.