Disrupting the AF4-AF9 protein complex in MLL leukemias.

项目描述（由申请人提供）：该项目的最终目标是开发有前景的治疗以特异性t（4;11）染色体易位为特征的急性淋巴细胞白血病（ALL）的新药。t（4;11）白血病是一种相对常见的ALL亚型，对细胞毒性化疗具有不同寻常的耐药性。考虑到目前可用治疗方法的局限性，分离、验证和优化治疗t（4;11）白血病的新化合物非常重要。我们已经确定了一种蛋白质：蛋白质相互作用似乎对t（4;11）白血病的生存至关重要。更重要的是，破坏这两种蛋白（AF4和AF9）相互作用的小肽可诱导t（4;11）白血病细胞的程序性细胞死亡。此外，这些肽不会抑制正常造血祖细胞的生长和分化。这些数据表明，AF4-AF9蛋白复合物是一个有希望的药物开发靶点。NIH分子文库筛选中心网络提供了一个独特的机会来筛选广泛的分子文库，以确定阻断AF4-AF9结合的其他化合物。在这里，我们建议开发一种适合于高通量筛选AF4-AF9结合抑制剂的检测系统。荧光偏振（FP）是一种测量均匀溶液中蛋白质结合的强大技术。该技术特别适合于测量AF4-AF9的结合，因为分子复合物可以通过结合到AF9的c端结构域的肽“探针”来模拟。此外，可以很容易地修改FP测定来分析蛋白质结合抑制剂。FP竞争分析已成功地用于高通量筛选其他蛋白质的抑制剂：蛋白质相互作用。一旦优化，该检测方法简单、快速、廉价。除了初级FP筛选试验外，我们还描述了分析“命中”的二级试验。这些二级分析包括分析铅化合物的抗白血病活性，并已得到验证。预计，经过改进，通过这种筛选过程鉴定和验证的化合物将提供有用的新的抗白血病治疗方法。尽管白血病的治疗方法有所改进，但某些形式的疾病仍然对治疗有抗药性。在这里，我们描述了一个系统，以快速识别化学物质，可能作为新药治疗最耐药的急性白血病之一。我们建议开发和完善这个测试系统，以便它可以用来从NIH开发的大量化学物质中分离出有希望的候选药物。

DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop promising new agents for the treatment of acute lymphoblastic leukemia (ALL) characterized by a specific t(4;11) chromosome translocation. t(4;11) leukemia is a relatively common subtype of ALL and is unusually resistant to cytotoxic chemotherapy. The isolation, validation, and optimization of new compounds to treat t(4;11) leukemia is important given the limitations of currently available treatments. We have identified a protein:protein interaction that appears to be critical for the survival of t(4;11) leukemia. More important, small peptides that disrupt the interaction of these two proteins, AF4 and AF9, induce programmed cell death in t(4;11) leukemia cells. Additionally, these peptides do not inhibit the growth and differentiation of normal hematopoietic progenitor cells. These data indicate that the AF4-AF9 protein complex is a promising target for drug development. The NIH Molecular Libraries Screening Center Network offers a unique opportunity to screen an extensive molecular library to identify additional compounds that block AF4-AF9 binding. Here, we propose to develop an assay system that is well suited for high throughput screening of inhibitors of AF4-AF9 binding. Fluorescence polarization (FP) is a powerful technique to measure protein binding in a homogeneous solution. The technique is particularly well suited to measuring AF4-AF9 binding as the molecular complex can be mimicked by a peptide "probe" bound to the C-terminal domain of AF9. Furthermore, FP assays can be easily modified to analyze inhibitors of protein binding. FP competition assays have been used successfully for high throughput screening of inhibitors of other protein:protein interactions. Once optimized, the assay is simple, rapid, and inexpensive. In addition to a primary FP screening assay, we describe secondary assays to analyze "hits". These secondary assays include analyses of the anti-leukemic activity of lead compounds and have already been validated. It is anticipated that, following refinement, compounds identified and verified by this screening process will provide useful new anti-leukemic therapies. Despite improvements in the treatment of leukemia, some forms of the disease remain resistant to therapy. Here, we describe a system to rapidly identify chemicals that may serve as new drugs for the treatment of one of the most resistant forms of acute leukemia. We propose to develop and refine this testing system so that it may be used to isolate promising drug candidates from a large collection of chemicals developed by NIH.