Small Molecule Inhibitors of a Reader of DNA Methylation

DNA 甲基化读取器的小分子抑制剂

• 批准号: 9808362
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808362
• 关键词: Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Automobile Driving; Binding; Binding Proteins; Biological Assay; Cell Line; Cell fusion; Chemicals; Chimeric Proteins; Chromosomal translocation; Code; DNA; DNA Binding; DNA Methylation; Data; Development; Drug Targeting; Elements; Epigenetic Process; Event; Fluorescence Polarization; Gene Expression; Genes; Goals; Growth; HRX protein; Histones; Length; Leukemic Cell; Libraries; Link; MLL gene; MLL-AF9; MLLT3 gene; Methylation; Nature; New Agents; Nucleosomes; Pharmaceutical Preparations; Pharmacotherapy; Point Mutation; Proteins; Reader; Signal Transduction; Signaling Protein; Site; Solvents; Specificity; Structure; Testing; Validation; base; bisulfite sequencing; expectation; gene translocation; in vivo; inhibitor/antagonist; leukemia; novel strategies; outcome forecast; small molecule inhibitor; therapy development

Abstract Chromosomal translocations of the gene coding for the epigenetic signaling protein MLL1 are frequent events in acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). These MLL1 fusion leukemias are consistently poor prognosis, highlighting the need for novel approaches to treatment. We have shown that the CXXC domain of MLL1, which is retained in the leukemia driving fusion proteins, binds specifically to nonmethylated CpG elements and protects them from methylation. Furthermore, introduction of point mutations into the MLL1 CXXC domain which disrupt DNA binding in the context of an MLL-AF9 fusion protein completely abrogates the ability of the fusion protein to cause leukemia in vivo. This data validates the MLL1 CXXC domain as a target for drug development for the treatment of MLL fusion leukemia. We have developed fluorescence polarization assays for the binding of the MLL1 CXXC domain to DNA. We screened two fragment libraries using this assay and confirmed 36 hits by chemical shift perturbations in 15N-1H HSQC spectra of the CXXC domain plus compounds. Our structural data showed there is one solvent exposed Cys residue located at the DNA binding interface. Based on this, we have also screened a library of Cys reactive molecules and identified 6 hits. We are proposing to covalently link an active fragment with a compound derived from the Cys reactive library which bind to separate sites on the protein to generate a potent inhibitor of the MLL1 CXXC domain. Once an effective inhibitor is generated, we will profile its effects on the growth of MLL1 fusion leukemia cell lines versus non-MLL1 fusion leukemia cell lines to establish selectivity of action. Furthermore, we will assess effects on DNA methylation and gene expression of well-characterized target genes. Based on this, we are proposing 2 aims: Aim 1: Development of a potent and specific inhibitor of the MLL1 CXXC domain. We will develop a potent and specific inhibitor of the MLL1 CXXC domain by covalently linking a fragment and a compound derived from the Cys reactive library which bind to separate sites on the protein. Aim 2: Validation of on-target activity of MLL1 CXXC domain inhibitor. We will validate the MLL1 CXXC domain inhibitor by testing effects on MLL fusion leukemia cell line proliferation, DNA methylation at target genes, and gene expression at target genes.

摘要 编码表观遗传信号蛋白MLL 1的基因的染色体易位是遗传学中的常见事件。 急性髓性白血病（AML）和急性淋巴细胞白血病（ALL）。这些MLL 1融合白血病是 持续的不良预后，突出了对新的治疗方法的需要。我们已经证明， 保留在白血病驱动融合蛋白中的MLL 1的CXXC结构域特异性结合至 非甲基化的CpG元件并保护它们免受甲基化。此外，点突变的引入 进入MLL 1 CXXC结构域，完全破坏MLL-AF 9融合蛋白背景下的DNA结合 消除了融合蛋白在体内引起白血病的能力。此数据验证了MLL 1 CXXC域 作为治疗MLL融合白血病的药物开发的靶点。 我们已经开发了用于MLL 1 CXXC结构域与DNA结合的荧光偏振测定法。我们 使用该测定筛选了两个片段文库，并通过15 N-1H中的化学位移扰动确认了36个命中 CXXC结构域加化合物的HSQC光谱。我们的结构数据显示有一种溶剂 Cys残基位于DNA结合界面。在此基础上，我们还筛选了一个Cys反应性文库， 分子并鉴定了6个命中物。我们建议将活性片段与衍生的化合物共价连接， 从Cys反应性文库中分离，其与蛋白质上的不同位点结合以产生MLL 1的有效抑制剂 CXXC域。一旦产生有效的抑制剂，我们将分析其对MLL 1融合蛋白生长的影响。 白血病细胞系与非MLL 1融合白血病细胞系的比较以建立作用选择性。而且我们 将评估对DNA甲基化和基因表达的影响，以及表征的靶基因。 基于此，我们提出两个目标： 目的1：开发MLL 1 CXXC结构域的有效和特异性抑制剂。 我们将通过共价连接MLL 1 CXXC结构域的一个片段和一个 衍生自Cys反应性文库的化合物，其结合蛋白质上的单独位点。 目的2：验证MLL 1 CXXC结构域抑制剂的中靶活性。 我们将通过测试对MLL融合白血病细胞系的作用来验证MLL 1 CXXC结构域抑制剂 增殖、靶基因处的DNA甲基化和靶基因处的基因表达。