Fragment-Based Ligand Discovery in Proteomes

描述（由申请人提供）：人类蛋白质组的很大一部分（大于50%）仍然是未表征的，这些蛋白质的更大一部分缺乏共价的，用于功能分析的选择性探针。高通量筛选（HTS）和基于片段的配体发现（FBLD）在开发新的探针和抑制剂方面都取得了成功，但受到生物学洞察力和目标结构信息的先决条件的挑战。为了解决全球鉴定蛋白质调节新位点的需求，我们已经开始开发基于片段的蛋白质组学配体发现（FBLDiP）的化学蛋白质组学平台。我们建议使用这个平台来发现新的配体结合位点和片段，以大规模并行的方式调节这些位点上的蛋白质活性，从而进入传统的“不可药物”靶点。FBLDiP同时报告了蛋白质组内数千种半胱氨酸片段结合的标记位点和结构活性关系（SAR）。我们已经开始实施FBLDiP平台，并在许多癌症相关蛋白中发现了新的配体结合位点，包括表观遗传酶PRMT1、转录因子NF-？B，致癌接头蛋白CRKL，癌症代谢酶IDH1和特征不明显的激酶ZAK。半胱氨酸反应片段文库的合成正在进行中，我们预计将生产100多个化合物文库。配体结合对选定的已鉴定蛋白质的生物学评价已经开始，初步数据支持FBLDiP方法对于发现新的配体结合位点和这些位点的探针是有效的。在对接和结构生物学的指导下，我们将在癌症相关靶点的新配体结合位点上产生有效的选择性抑制剂。抑制剂的合成将以FBLDiP筛选和二级凝胶蛋白分析的感兴趣目标的SAR谱为指导。我们预计FBLDiP方法将发现广泛的蛋白质调节新位点和靶向这些位点的先导抑制剂。总的来说，这项研究提供了一种令人兴奋的新方法来识别配体结合位点，并指导合成重要的和非靶向的生物相关蛋白质的抑制剂。

DESCRIPTION (provided by applicant): A significant portion of the human proteome (greater than 50%) remains uncharacterized and an even greater fraction of these proteins lack covalent, selective probes for functional analysis. High-Throughput Screening (HTS) and Fragment-Based Ligand Discovery (FBLD) have both had success in the development of new probes and inhibitors but are challenged by prerequisites for biological insight and structural information about a target. To address the need for global identification of new sites for protein modulation, we have begun to develop a chemoproteomic platform for Fragment-Based Ligand Discovery in Proteomes (FBLDiP). We propose to use this platform to discover novel ligand-binding sites and fragments that modulate protein activity at these sites in a massively parallel fashion that will access traditionally 'undruggable' targets. FBLDiP simultaneously reports the site of labeling and the structure activity relationship (SAR) of fragment binding at thousands of cysteines within the proteome. We have already begun to implement the FBLDiP platform and have uncovered novel ligand-binding sites in many cancer relevant proteins, including the epigenetic enzyme PRMT1, the transcription factor NF-?B, the oncogenic adaptor protein CRKL, cancer metabolism enzyme IDH1 and the poorly characterized kinase ZAK. The synthesis of a cysteine-reactive fragment library is ongoing and we anticipate the production of a 100+ compound library. Biological evaluation of the effects of ligand-binding on selected identified proteins has begun and preliminary data supports that the FBLDiP methodology is effective for discovery of novel ligand-binding sites and probes for these sites. Guided by docking and structural biology, we will generate potent, selective inhibitors at novel ligand-binding sites in cancer relevant targets. Inhibitor synthesis will be guided by SAR profiles for targets of interest from FBLDiP screening and secondary gel-based protein profiling. We anticipate that the FBLDiP methodology will uncover a wide range of new sites for protein modulation and lead inhibitors to target these sites. As a whole, this research offers an exciting new means to identify ligand-binding sites and to guide the synthesis of inhibitors for important and untargeted, biologically-relevant proteins.