Inhibitors of MLL-Menin Interaction

描述(由申请人提供)：MLL(混合血统白血病)基因编码一种组蛋白甲基转移酶，它作为一种转录表观遗传调节因子，对具有发育和致癌重要性的各种从属基因起作用。它是通过染色体易位而突变的，导致嵌合融合蛋白的产生，其功能的获得与急性白血病预后不良亚群的发病机制密切相关。最近，MLL癌蛋白被证明与MEN1肿瘤抑制基因的产物Menin有关。遗传分析表明，MLL介导的转录和转化需要薄荷素，从而展示了肿瘤抑制蛋白作为癌蛋白在细胞转化中的关键辅助因子的不同寻常和前所未有的作用。MLL-Menin的相互作用严重依赖于一个5个氨基酸的高亲和力Menin结合基序，该基序在所有致癌形式的MLL中都保留着，因此构成了MLL功能的药物调控和可能的治疗干预的潜在靶点。本申请中提出的研究的总体目标是建立分离小分子的方法，这些小分子专门抑制MLL-薄荷素的相互作用。在第一个特定目标中，将开发一种能够定量测量与MLL共识结合序列相对应的荧光标记多肽的溶液中重组薄荷素结合的荧光偏振分析方法。在第二个特定目标中，MLL-薄荷素阻断分析将被配置和/或优化以用于高通量筛选。生化和细胞分析也将被设计和优化，以确定MLL-薄荷素相互作用的小分子抑制剂的特异性和有效性。通过1,280种化合物(Lopac、Sigma)的小分子库以定量高通量格式进行初步筛选将有助于识别技术问题，并确认更大规模高通量筛选的可行性。能够特异性干扰MLL-薄荷素相互作用的化合物将为研究这些蛋白在转录表观遗传调控和肿瘤发生中的异常功能协同作用提供有价值的试剂，并为治疗预后不良的白血病提供潜在的合理药物设计的先导化合物。急性白血病是发病率和死亡率的重要原因，其影响尤其显著，因为它们经常影响儿童和年轻人。尽管近年来在治疗和预后方面有所改善，但大多数白血病患者的长期生存预后仍然很差(1-4)。因此，迫切需要以合理的药物设计为基础的针对白血病发病机制的分子异常的新的治疗方式。

DESCRIPTION (provided by applicant): The MLL (Mixed Lineage Leukemia) gene codes for a histone methyltransferase, which functions as a transcriptional epigenetic regulatory factor for diverse subordinate genes of developmental and oncogenic importance. It is mutated by chromosomal translocations that result in the creation of chimeric fusion proteins whose gain of function is critically involved in the pathogenesis of a poor prognosis subset of acute leukemia. Recently, MLL oncoproteins have been shown to associate with menin, a product of the MEN1 tumor suppressor gene. Genetic analyses revealed that menin is required for MLL-mediated transcription and transformation, thus demonstrating an unusual and unprecedented role for a tumor suppressor protein serving as an essential cofactor for an oncoprotein in cellular transformation. The MLL- menin interaction is critically dependent on a 5 amino acid high affinity menin-binding motif, which is retained in all oncogenic forms of MLL, and therefore constitutes a potential target for pharmacologic manipulation of MLL function and possible therapeutic intervention. The overall objectives of the studies proposed in this application are to establish methodologies to isolate small molecules that specifically inhibit the MLL-menin interaction. In the first specific aim, a fluorescence polarization assay will be developed that is capable of quantitatively measuring recombinant menin binding in solution to a fluorescently labeled peptide corresponding to the MLL consensus binding sequence. In the second specific aim, the MLL-menin blocking assay will be configured and/or optimized for high throughput screening. Biochemical and cellular assays will also be devised and optimized for establishing the specificity and efficacy of small molecule inhibitors of MLL-menin interaction. A preliminary screen through a small molecule library of 1,280 compounds (LOPAC, Sigma) in a quantitative high throughput format will help identify technical problems and confirm the feasibility of a larger scale high throughput screen. Compounds that are capable of specifically interfering with MLL-menin interaction will provide valuable reagents for studying the unusual functional cooperative roles of these proteins in transcriptional epigenetic regulation and oncogenesis, and provide lead compounds for potential rational drug design in poor prognosis leukemia. Acute leukemias are an important cause of morbidity and mortality, and their effects are particularly significant because they often affect children and young adults. Despite improvements in therapy and outcome in recent years, the prognosis for long-term survival in most leukemia patients remains poor (1-4). Thus, there is an urgent need for novel therapeutic modalities based on rational drug design targeting the molecular abnormalities that underlie leukemia pathogenesis.