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.

项目摘要/摘要 这项工作的目的是表征独特的核蛋白毒素的小分子探针 （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 免疫系统的开发和功能。这些研究还将提供重要的证明 小分子可以靶向蛋白质和DNA小凹槽的界面的原理。毒品 小鼠和人类之间的氨基酸序列相同94％，表明具有高度保守的功能。 我们的实验室以前已经证明，免疫系统的许多方面都无法发展 由于A 特定祖细胞分别在胸骨和骨髓中继续分化。我们 将这里的重点放在托克斯的DNA结合HMG-box结构域及其在调节基因表达中的作用。这是 蛋白质的高度进化保守区域，另一个与其他三个成员共享的区域 蛋白质的托克斯亚家族。因此，我们确定的小分子探针具有 在许多生物学环境中的广泛效用。 我们已经确定了托克斯的HMG-box域的晶体结构，并使用了这些数据和 分子对接以识别我们预测的小分子神经阵阵将结合和改变 蛋白质与DNA的相互作用。 Neurodazine是一种基于咪唑的细胞渗透小分子 它通过改变与神经元细胞相关的基因表达的能力来确定，但尚未 良好的研究。在这里，我们将解决这个小分子是否作为托克斯活性调节剂的活性。 神经阿阿嗪可以通过我们开发的新的测定方法来影响托克斯活性。 与DNA结合，并可以轻松读取托克斯介导的基因表达调节。我们建议 在这里表征神经阿嗪与托克斯的结合，包括晶体结构的确定 复合物，并确定神经阿嗪是否特异性抑制了托克斯诱导的基因调节。最多 有趣的是，神经阿嗪可以增强毒TNA的结合，但似乎破坏了 蛋白质。这导致了以下假设：神经阿嗪可能会稳定毒质的毒品，从而改变其 核动力学。光漂白后，将使用荧光回收在活细胞中进行测试。 最后，将使用两个不同的细胞系统来确定神经嗪抑制托克斯活性的潜力 在免疫细胞的背景下；骨髓祖细胞细胞分化与先天淋巴样细胞和托克斯 依赖性皮肤T细胞淋巴瘤细胞生长。这些研究将共同​​提供关键的见解 该蛋白质的结构 - 功能关系，构成了未来开发的基础 蛋白质的毒素的分子调节剂，可以用作探针和引导 预花者。