Interfering with protein-protein interactions in the spliceosme

Pre-mRNA剪接是由一种精细的核糖核蛋白（RNP）酶进行的，剪接体通过snrnp和多个非snrnp剪接因子的逐步相互作用在每个Pre-mRNA内含子上重新组装。在其成熟为具有催化活性的RNP的过程中，剪接体被反复重塑，经历了许多组成和构象的变化。剪接体的成熟和催化伴随着剪接体RNA和RNP网络的深刻重排以及超过100种蛋白质的动态交换。目前，我们对单个蛋白质或蛋白质复合物的作用以及剪接体成熟和催化过程中蛋白质交换的顺序知之甚少。最有可能的是，除了迄今为止确定的有限数量的中间体之外，还存在大量的组装中间体。为了获得新的和组成均匀的中间体，我们计划开发和表征干扰特定剪接体蛋白-蛋白相互作用的小分子抑制剂。抑制剂将通过三种策略确定：(1)基于蛋白质复合物的晶体结构进行合理设计；(2)基于分离蛋白对的靶向筛选；(3)基于蛋白募集试验的靶向筛选。在蛋白质与抑制剂复合物的共晶结构的辅助下，合成优化将提高初始命中率。所获得的结果不仅有助于阐明剪接体组装和催化的构象动力学，而且可能为开发治疗性剪接体抑制剂提供有用的线索。

Pre-mRNA splicing is carried out by an elaborate ribonucleoprotein (RNP) enzyme, the spliceosome, which is assembled anew on each pre-mRNA intron by the stepwise interaction of snRNPs and multiple non-snRNP splicing factors. During its maturation into a catalytically active RNP, the spliceosome is repeatedly remodeled, undergoing numerous compositional and conformational changes. Spliceosome maturation and catalysis are accompanied by profound rearrangements in the spliceosomal RNA and RNP networks and by the dynamic exchanges of more than 100 proteins. At present we have incomplete knowledge of the role of individual proteins or protein complexes and of the sequence of protein exchanges during spliceosome maturation and catalysis. Most likely, a large number of assembly intermediates exist in addition to the limited number of intermediates identified to date. In order to access novel and compositionally homogenous intermediates, we plan to develop and characterize small molecule inhibitors that interfere with particular spliceosomal protein-protein interactions. Inhibitors will be identified by three strategies: (1) Rational design based on crystal structures of protein complexes; (2) Targeted screening based on isolated protein pairs; (3) Targeted screening based on a protein recruitment assay. Initial hits will be improved by synthetic optimization aided by co-crystal structures of proteins in complex with inhibitors. The results obtained will not only help to elucidate the conformational dynamics of spliceosome assembly and catalysis but may also provide useful leads for the development of therapeutic spliceosome inhibitors.