Fragment-Based Ligand Discovery in Proteomes

描述(申请人提供)：人类蛋白质组的很大一部分(超过50%)仍未确定特征，其中更大一部分蛋白质缺乏用于功能分析的共价、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，但受到有关靶点的生物学洞察和结构信息的先决条件的挑战。为了满足在全球范围内识别新的蛋白质调控位点的需要，我们已经开始开发一个基于片段的蛋白质组配体发现化学蛋白质组平台(FBLDiP)。我们建议使用这个平台来发现新的配体结合位点和片段，这些位点和片段以一种大规模平行的方式调节这些位点的蛋白质活性，从而访问传统上“不可用药”的靶点。FBLDiP同时报道了蛋白质组中数千个半胱氨酸结合的片段的标记位置和构效关系(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-βB、致癌适配蛋白CRKL、癌症代谢酶IDH1和功能不佳的激酶Zak。半胱氨酸反应片段文库的合成正在进行中，我们预计将产生100+个化合物文库。配基结合对选定识别蛋白质的影响的生物学评估已经开始，初步数据支持FBLDiP方法在发现新的配基结合位点和这些位点的探针方面是有效的。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部位产生有效的、选择性的抑制剂。抑制剂的合成将由FBLDiP筛选和基于二次凝胶的蛋白质图谱中感兴趣的目标的SAR图谱来指导。我们预计FBLDiP方法将发现一系列新的蛋白质调控位点，并以铅抑制剂为靶点。总体而言，这项研究提供了一种令人兴奋的新方法来识别配基结合部位，并指导重要的和非靶向的、生物相关蛋白质的抑制剂的合成。

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.