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

项目摘要/摘要 这项工作的目标是表征一种独特的核蛋白TOX小分子探针 (胸腺细胞选择相关HMG盒蛋白)，以帮助理解该蛋白是如何调节的 免疫系统的发展和功能。这些研究还将提供重要的证据-- 小分子可以靶向蛋白质和DNA小沟的界面的原理。TOX毒素 小鼠和人类的氨基酸序列有94%的同源性，表明它们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在 毒素缺乏的小鼠，包括CD4T淋巴细胞和整个固有淋巴样细胞谱系，由于 胸腺和骨髓中的特定祖细胞不能继续分化。我们 这里重点介绍TOX的DNA结合HMG-box结构域及其在基因表达调控中的作用。这是 蛋白质在进化上高度保守的区域，与其他三个成员共享 属于TOX蛋白质亚家族。因此，我们已经确定的小分子探测器有可能具有 在许多生物学背景下具有广泛的实用性。 我们已经确定了TOX的HMG-box结构域的晶体结构，并使用这些数据和 分子对接以确定我们预测会结合TOX和ALTER的小分子--神经达嗪 蛋白质与DNA的相互作用。Neurodazine是一种咪唑类细胞通透性小分子 这是通过它改变与神经细胞相关的基因表达的能力来确定的，但还没有得到证实 研究得很好。在这里，我们将讨论这个小分子是否作为TOX活性的调节器是活跃的。 我们开发的检测TOX的新方法表明，神经达嗪可以影响TOX的活性 与DNA结合，并允许轻松读出TOX介导的基因表达调控。我们建议 为了表征神经达嗪与TOX的结合，包括晶体结构的测定 复杂，并确定神经达嗪是否特异性地抑制TOX诱导的基因调控。多数 有趣的是，神经达嗪可以增强TOX与DNA的结合，但似乎扰乱了TOX与DNA的功能 蛋白。这导致了一种假设，即神经达嗪可能稳定染色质上的TOX，改变其 核动力学。这将在活细胞中进行测试，使用光漂白后的荧光恢复。 最后，两个不同的细胞系统将被用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样细胞和TOX- 皮肤T细胞淋巴瘤细胞生长依赖。总而言之，这些研究将为 这种蛋白质的结构-功能关系，并形成了未来发展的基础额外的小 TOX蛋白家族的分子调节剂，可用作探针和引线 治疗前学。

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.