Small Molecule Inhibitors of a Reader of DNA Methylation

摘要 表观信号蛋白MLL1编码基因的染色体易位在 急性髓系白血病(AML)和急性淋巴细胞白血病(ALL)。这些MLL1融合白血病是 预后一直很差，突出表明需要新的治疗方法。我们已经证明了 MLL1的CXXC结构域保留在白血病驱动融合蛋白中，与 非甲基化的CpG元件，保护它们免受甲基化。此外，引入点突变 进入MLL1CXXC结构域，完全破坏MLL-AF9融合蛋白中的DNA结合 取消融合蛋白在体内引起白血病的能力。该数据验证MLL1 CXXC域 作为治疗MLL融合白血病的药物开发的靶点。 我们建立了MLL1 CXXC结构域与DNA结合的荧光偏振分析方法。我们 利用该方法筛选了两个片段文库，并通过15N-1H中的化学位移扰动确认了36个片段 CXXC结构域加化合物的HSQC谱。我们的结构数据显示有一种溶剂暴露出来 半胱氨酸残基位于DNA结合界面。在此基础上，我们还筛选了半胱氨酸反应性文库 分子，并确定了6个匹配。我们建议将活性片段与衍生的化合物共价连接 来自Cys反应文库，它与蛋白质上的不同位置结合，产生MLL1的有效抑制物 CXXC域。一旦产生了有效的抑制剂，我们将分析它对MLL1融合生长的影响 建立白血病细胞系与非MLL1融合白血病细胞系的作用选择性。此外，我们 将评估对DNA甲基化和特征明确的目标基因的基因表达的影响。 基于此，我们提出了两个目标： 目的1：开发一种有效和特异的MLL1 CXXC结构域的抑制物。 我们将开发一种有效和特异的MLL1 CXXC结构域的抑制剂，通过将一个片段和一个 来源于半胱氨酸反应文库的化合物，结合到蛋白质上的不同位置。 目的2：验证MLL1 CXXC结构域抑制剂的靶向活性。 我们将通过测试对MLL融合白血病细胞系的作用来验证MLL1 CXXC结构域抑制物 增殖、靶基因的DNA甲基化和靶基因的基因表达。

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.