Selective Allosteric Inhibitors of SENP8

描述（由申请人提供）：Neddylation途径最近被验证为癌症靶点。SENP 8蛋白酶处理Nedd 8的前体，并且对于其活化是必需的。基于结合到SENP 8的Nedd 8的高分辨率晶体结构，底物具有两个基本的相互作用位点：在催化中心中的Nedd 8的C-末端，以及与覆盖蛋白酶的实质性表面的Nedd 8蛋白质的大部分的相互作用-外部位点。SENP 8结构的分析揭示了在外部位点中的空腔，表明存在推定的变构结合位点。我们使用基于五肽的测定法在约330，500 MLSMR小分子文库的uHTS上对该SENP的初步结果鉴定了与催化中心结合的抑制剂，并且对其他SENP没有选择性。为了找到选择性SENP 8配体并探索这种额外的变构结合位点，我们开发了一种新的荧光强度uHTS测定，其利用生理蛋白底物Nedd 8底物。为了研究这种在治疗上重要的酶的功能，我们建议使用这种新开发的测定来筛选选择性的机械新颖的SENP 8抑制剂。基于现有文献，这将是第一次使用全长底物在仅靶向催化位点的uHTS筛选后询问靶向泛素样蛋白的代表性蛋白酶。我们的申请解决了鉴定SENP的特异性调节剂的重要的未满足的需求。我们将利用该项目中鉴定的化合物来表征这类酶在维持正常细胞稳态中的生理参与及其在癌症中的作用。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。 公共卫生相关性：Nedd 8是一种泛素样蛋白，它通过一种称为neddylation的过程与靶蛋白结合，对特定的细胞途径至关重要。以前的工作表明，用小分子靶向neddylation循环可以减少肿瘤负担，这种小分子现在正在临床试验中。为了研究这种在治疗上重要的酶的功能，我们建议部署一种新的荧光强度uHTS测定法，该测定法利用全长Nedd 8蛋白底物来发现有效的和SENP 8选择性抑制剂，该抑制剂可以使用这种新开发的测定法证明是变构的。

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.