Targeting protein-protein interactions of Bmi1 oncoprotein

项目摘要 BMI1(B细胞特异性Moloney鼠病毒插入位点1)基因已被鉴定为一种癌基因。 多种癌症，导致B细胞和T细胞白血病。BMI1是一种干细胞基因，它决定了细胞的增殖 正常和白血病干细胞的能力和自我更新。多项研究证实Bmi1蛋白可诱导 在多种体外和体内模型中进行致癌转化和促进肿瘤生长。因此， 靶向Bmi1活性是开发新疗法和 诱人的药物可以根除癌症干细胞。 BMI1蛋白是多梳抑制复合体1(PRC1)的一种成分，已被证明 作为INK4a/ARF基因座编码的转录抑制因子，在细胞周期调控中发挥关键作用 两个重要的肿瘤抑制基因p16INK4a和p14ARF。BMI1是一种相对较小的37 kDa蛋白质，由 具有E3泛素连接酶活性的N-末端环区和第二区(B2D，Bmi12 结构域)，代表蛋白质-蛋白质相互作用基序。B2D结构域的蛋白质-蛋白质相互作用 是Bmi1活性所必需的。已有研究表明，B2D参与了与多同源异体的相互作用 Prc1复合体中的蛋白质以及锌指转录因子E4F1、Zfp277和PLZF- RARA融合蛋白。 在这个项目中，我们建议开发针对B2D蛋白-蛋白质的小分子抑制剂 Bmi1相互作用结构域作为新的化学探针进一步研究Bmi1的B2D结构域和TO 发现新的潜在抗癌药物。我们已经刻画了B2D领域的相互作用 BMI1与PHC2结合，建立了适用于小分子筛选的高质量生化分析方法 图书馆。我们将在化学基因组学中心(CCG)进行高通量筛查 密歇根大学发现了这种蛋白质-蛋白质相互作用的小分子抑制剂。我们将聘用 结合生化分析、生物物理实验和结构生物学的跨学科方法 方法验证HTS HITS，鉴定高质量的Bmi1小分子抑制剂。最具活力的活动 有效的抑制剂将在一组基于细胞的实验中进行评估，以评估它们抑制 白血病细胞的自我更新特性。我们的项目代表了一种非常新颖和创新的目标方法 Bmi1通过阻断蛋白质间的相互作用，目前还没有Bmi1的抑制剂。总而言之， 我们希望鉴定出高度有价值的化合物，这些化合物可以作为化学探针，适用于机械学 研究并进一步开发成有效的体内致癌Bmi1抑制剂。

Project Summary Bmi1 (B-cell specific Moloney murine virus insertion site 1) gene has been identified as an oncogene in multiple cancers, inducing B- and T-cell leukemias. Bmi1 is a stem cell gene, which determines the proliferative capacity and self-renewal of normal and leukemic stem cells. Multiple studies identified Bmi1 protein to induce oncogenic transformation and promote tumor growth in a variety of in vitro and in vivo models. Therefore, targeting Bmi1 activity represents a very promising approach for development of novel therapeutics and attractive agents to eradicate cancer stem cells. Bmi1 protein is a component of the polycomb suppressive complex 1 (PRC1), and has been shown to play a critical role in cell cycle regulation by acting as a transcriptional repressor of INK4a/ARF locus encoding two important tumor suppressors p16Ink4a and p14Arf. Bmi1 is a relatively small, 37 kDa protein composed of N-terminal RING domain which has E3 ubiquitin ligase activity and the second domain (B2D, the Bmi1 2nd domain) which represents a protein-protein interaction motif. Protein-protein interactions of the B2D domain are essential for Bmi1 activity. It has been shown that B2D is involved in the interactions with polyhomeotic proteins within the PRC1 complex as well as with the zinc-finger transcription factors E4F1, Zfp277 and PLZF- RARA fusion protein. In this project we propose to develop small molecule inhibitors targeting the B2D protein-protein interaction domain of Bmi1 as new chemical probes to further study the role of B2D domain of Bmi1 and to identify new potential anti-cancer agents. We have already characterized the interaction of B2D domain of Bmi1 with PHC2 and developed high quality biochemical assays suitable for screening of small molecule libraries. We will carry out high-throughput screening at the Center for Chemical Genomics (CCG) at the University of Michigan to identify small molecule inhibitors of this protein-protein interaction. We will employ interdisciplinary approach combining biochemical assays, biophysical experiments and structural biology methods to validate HTS hits and identify high quality small molecule inhibitors of Bmi1. Activity of the most potent inhibitors will be evaluated in a panel of cell-based experiments to assess their capability to inhibit the self-renewal properties of leukemic cells. Our project represents a very novel and innovative approach to target Bmi1 through blocking the protein-protein interactions as currently there are no inhibitors of Bmi1. In summary, we expect to identify highly valuable compounds that can serve as chemical probes suitable for mechanistic studies and for further development into potent in vivo inhibitors of the oncogenic Bmi1.