Selective Allosteric Inhibitors of SENP8

描述（由申请人提供）：类泛素化通路最近被证实是一种癌症靶点。SENP8蛋白酶处理Nedd8的前体，对其激活至关重要。根据与SENP8结合的Nedd8的高分辨率晶体结构，底物有两个基本的相互作用位点：在催化中心的Nedd8的c端，以及与覆盖在蛋白酶表面的大部分Nedd8蛋白-外源体相互作用。对SENP8结构的分析揭示了外源物中的空腔，表明存在假定的变构结合位点。我们使用基于五肽的实验对约330,500个MLSMR小分子文库的uHTS进行了初步研究，发现了与催化中心结合的抑制剂，并且对其他SENP没有选择性。为了寻找选择性SENP8配体并探索这种额外的变容结合位点，我们开发了一种利用生理蛋白底物Nedd8底物的新型荧光强度uHTS试验。为了研究这种具有重要治疗意义的酶的功能，我们建议使用这种新开发的检测方法筛选选择性机制新颖的SENP8抑制剂。根据现有文献，这将是第一次使用全长底物在仅靶向催化位点的uHTS筛选后对靶向泛素样蛋白的代表性蛋白酶进行询问。我们的应用程序解决了识别特定senp调制器的重要未满足需求。我们将利用项目中确定的化合物来表征这类酶在维持正常细胞内稳态及其在癌症中的作用方面的生理参与。这些化合物将提供给其他研究实验室，从而加速SENP领域的研究。

DESCRIPTION (provided by applicant): The Neddylation pathway was recently validated as a cancer target. The SENP8 protease processes the precursor of Nedd8 and is essential for its activation. Based on the high resolution crystal structure of Nedd8 bound to SENP8 the substrate has two fundamental interaction sites: at the C-terminus of Nedd8 in the catalytic center, and interactions with the bulk of the Nedd8 protein covering a substantial surface of the protease - the exosite. Analysis of the SENP8 structure reveals cavities in the exosite, indicating the presence of a putative allosteric binding site. Our preliminary results for this SENP on uHTS of the ~ 330,500 MLSMR small molecule libraries using a penta-peptide based assay identified inhibitors binding to the catalytic center and not selective against other SENPs. In order to find selective SENP8 ligands and to explore this additional allosteric binding site, we developed a novel Fluorescent Intensity uHTS assay that utilizes a physiological protein substrate Nedd8 substrate. To study the function of this therapeutically important enzyme, we propose to screen for selective mechanistically novel SENP8 inhibitors using this newly developed assay. Based on available literature, this would be the first time that a representative protease targeting ubiquitin-like proteins is interrogated using a full-length substrate following an uHTS screen that targets only the catalytic site. Our application addresses an important unmet need for identification of specific modulators of SENPs. We will utilize the compounds identified in the project for characterization of physiological involvement of this class of enzymes in maintaining homeostasis of normal cells and their role in cancer. These compounds will be made available to other research labs, permitting acceleration of research in the SENP field. PUBLIC HEALTH RELEVANCE: Nedd8 is an ubiquitin-like protein, and its attachment to target proteins, by a process known as neddylation, is essential for specific cellular pathways. Previous work has shown that targeting the neddylation cycle with a small molecule can decrease tumor burdens, and this small molecule is now in clinical trials. To study the function of this therapeutically important enzyme, we propose to deploy a novel Fluorescent Intensity uHTS assay that utilizes a full length Nedd8 protein substrate to discover potent and SENP8 selective inhibitors that may prove to be allosteric using this newly developed assay.