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.

抽象的 对表观遗传信号传导蛋白MLL1的基因编码的染色体易位是经常发生的事件 急性髓样白血病（AML）和急性淋巴细胞性白血病（ALL）。这些Mll1融合白血病是 始终不良的预后，强调了对新型治疗方法的需求。我们已经证明了 保留在驱动融合蛋白的白血病中的MLL1的CXXC结构域特异性结合 非甲基化的CpG元素并保护它们免受甲基化。此外，引入点突变 进入MLL-AF9融合蛋白的上下文中破坏DNA结合的MLL1 CXXC结构域 废除了融合蛋白在体内引起白血病的能力。此数据验证了MLL1 CXXC域 作为治疗MLL融合白血病的药物开发的靶标。 我们已经开发了MLL1 CXXC结构域与DNA结合的荧光极化测定。我们 使用此测定法筛选了两个片段库，并通过15N-1H的化学移动扰动确认了36个命中 CXXC域和化合物的HSQC光谱。我们的结构数据表明，暴露了一个溶剂 Cys残基位于DNA结合界面。基于此，我们还筛选了一个Cys库反应性 分子并鉴定出6次命中。我们建议将活动片段与得出的化合物共价链接 从Cys反应性文库，该文库与蛋白质上的分离位点结合以产生MLL1的有效抑制剂 CXXC域。一旦产生有效的抑制剂，我们将介绍其对MLL1融合生长的影响 白血病细胞系与非MLL1融合白血病细胞系以建立作用的选择性。此外，我们 将评估对良好特征靶基因的DNA甲基化和基因表达的影响。 基于此，我们提出了2个目标： 目标1：开发MLL1 CXXC域的有效抑制剂。 我们将通过共价连接片段和一个 从Cys反应性文库得出的化合物，该文库与蛋白质上的分离位点结合。 AIM 2：MLL1 CXXC结构域抑制剂的靶向活性验证。 我们将通过测试对MLL融合白血病细胞系的影响来验证MLL1 CXXC结构域抑制剂 增殖，靶基因的DNA甲基化以及靶基因的基因表达。