A novel small molecule probe to study TOX-family transcriptional regulators

一种用于研究 TOX 家族转录调节因子的新型小分子探针

• 批准号: 9324512
• 负责人: JONATHAN G KAYE
• 金额: $ 26.25万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-06 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324512
• 关键词: Address; Affinity; Alpha Cell; Amino Acid Sequence; Area; B-Lymphocytes; Binding; Biological; Biological Assay; Bone Marrow; CD4 Positive T Lymphocytes; Cell Cycle Kinetics; Cell Differentiation process; Cell Line; Cell Lineage; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chromatin; Chromatin Structure; Complex; Crystallization; Cutaneous; DNA; DNA Binding; DNA Binding Domain; Data; Development; Disease; Docking; Experimental Models; Failure; Family; Family member; Fluorescence Recovery After Photobleaching; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; HMG Domain; HMG-Box Domains; HMGB Proteins; Helper-Inducer T-Lymphocyte; Human; Imidazole; Immune; Immune system; In Vitro; Innate Immune System; Investigation; Kinetics; Knock-out; Laboratories; Lead; Lymphoid Cell; Measures; Mediating; Minor Groove; Modeling; Molecular; Mus; Natural Killer Cells; Neurons; Nuclear; Nuclear Protein; Nuclear Proteins; Oligodendroglia; Permeability; Pharmacology; Play; Process; Protein Analysis; Protein Family; Protein Region; Proteins; Regulation; Reporting; Role; Specificity; Stem cells; Structural Protein; Structure; Structure-Activity Relationship; System; T-Cell Lymphoma; T-Lymphocyte; Testing; Therapeutic; Thymocyte Selection; Thymus Gland; Time; Work; base; cell growth; cell type; immune system function; insight; knock-down; malignant breast neoplasm; member; novel; overexpression; progenitor; protein function; small molecule; tool; transcriptome sequencing; tumor; tumor growth

Project Summary/Abstract The goal of this work is to characterize a unique small molecule probe of nuclear protein TOX (thymocyte selection-associated HMG box protein) to aid in understanding how this protein regulates development and function of the immune system. These studies would also provide a significant proof-of- principle that a small molecule can target the interface of a protein and the DNA minor groove. The TOX amino acid sequence is 94% identical between mice and humans, suggesting a highly conserved function. Our laboratory has previously demonstrated that many aspects of the immune system fail to develop in TOX-deficient mice, including CD4 T lymphocytes and the entire innate lymphoid cell lineage, due to a failure of specific progenitor cells to continue differentiation in thymus and bone marrow, respectively. We focus here on the DNA binding HMG-box domain of TOX and its role in regulating gene expression. This is a highly evolutionarily conserved region of the protein, and one that is shared with the other three members of the TOX subfamily of proteins. Thus, the small molecule probe we have identified has potential to have broad utility in a number of biological contexts. We have determined the crystal structure of the HMG-box domain of TOX and used these data and molecular docking to identify a small molecule, neurodazine, that we predicted would bind TOX and alter the interaction of the protein with DNA. Neurodazine is an imidazole-based cell-permeable small molecule that was identified by its ability to alter gene expression associated with neuronal cells, but has not been well studied. Here we will address whether this small molecule is active as a modulator of TOX activity. That neurodazine can influence TOX activity was suggested by novel assays we developed to detect TOX binding to DNA, and to allow an easy read out of TOX-mediated regulation of gene expression. We propose here to characterize the binding of neurodazine to TOX, including crystal structure determination of the complex, and to determine whether neurodazine specifically inhibits TOX-induced gene regulation. Most interestingly, neurodazine can enhance binding of TOX to DNA, but appears to disrupt the function of the protein. This has led to the hypothesis that neurodazine may stabilize TOX on chromatin, altering its nuclear dynamics. This will be tested in living cells using fluorescence recovery after photobleaching. Finally, two distinct cell systems will be used to determine the potential of neurodazine to inhibit TOX activity in the context of immune cells; bone marrow progenitor cell differentiation to innate lymphoid cells and TOX- dependent cutaneous T cell lymphoma cell growth. Together, these studies will provide key insights into structure-function relationships of this protein and form the basis for future development of additional small molecule modulators of the TOX-family of proteins that could be useful as probes and leads for pretherapeutics.

项目概要/摘要 这项工作的目标是表征核蛋白 TOX 的独特小分子探针 （胸腺细胞选择相关的 HMG 盒蛋白）有助于了解该蛋白如何调节 免疫系统的发育和功能。这些研究还将提供重要的证据—— 原理是小分子可以靶向蛋白质和DNA小沟的界面。毒素 小鼠和人类之间的氨基酸序列有 94% 相同，表明其功能高度保守。 我们的实验室之前已经证明，免疫系统的许多方面都无法在 TOX缺陷小鼠，包括CD4 T淋巴细胞和整个先天淋巴细胞谱系，由于 特定祖细胞分别无法在胸腺和骨髓中继续分化。我们 这里重点关注 TOX 的 DNA 结合 HMG-box 结构域及其在调节基因表达中的作用。这是 蛋白质的一个高度进化保守的区域，并且与其他三个成员共享一个区域 TOX 蛋白亚家族的成员。因此，我们发现的小分子探针有潜力 在许多生物学背景下具有广泛的用途。 我们确定了 TOX 的 HMG-box 结构域的晶体结构，并使用这些数据和 分子对接来识别小分子神经达嗪，我们预测它会结合 TOX 并改变 蛋白质与DNA的相互作用。 Neurodazine 是一种基于咪唑的细胞渗透性小分子 其通过改变与神经元细胞相关的基因表达的能力而被识别，但尚未被证实 好好研究了。在这里，我们将讨论这种小分子是否具有作为 TOX 活性调节剂的活性。 我们开发的 TOX 检测新方法表明神经达嗪可以影响 TOX 活性 与 DNA 结合，并可以轻松读取 TOX 介导的基因表达调控。我们建议 此处表征神经达嗪与 TOX 的结合，包括晶体结构测定 复合物，并确定神经达嗪是否特异性抑制 TOX 诱导的基因调控。最多 有趣的是，神经达嗪可以增强 TOX 与 DNA 的结合，但似乎会破坏 TOX 的功能。 蛋白质。这导致了这样的假设：神经达嗪可能稳定染色质上的 TOX，改变其 核动力学。这将在活细胞中使用光漂白后的荧光恢复进行测试。 最后，将使用两个不同的细胞系统来确定神经达嗪抑制 TOX 活性的潜力 在免疫细胞的背景下；骨髓祖细胞分化为先天淋巴细胞和 TOX- 依赖性皮肤T细胞淋巴瘤细胞生长。这些研究共同将为以下问题提供重要见解： 该蛋白质的结构-功能关系，并为未来开发其他小分子奠定了基础 TOX 蛋白家族的分子调节剂，可用作探针和先导物 治疗前药物。