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Chemical Targeting of Multi-Protein Complexes

多蛋白复合物的化学靶向

基本信息

批准号：
10014712
负责人：
Federico Bernal
金额：
$ 9.53万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014712
关键词：
Active Sites Affinity Amino Acid Sequence Amino Acids Animal Model Area B-Lymphocytes Behavior Binding Binding Sites Biochemical Biological Biological Assay Biological Process C-terminal Calorimetry Cells Cellular Assay Chemicals Chemistry Circular Dichroism Clinical Co-Immunoprecipitations Collaborations Collection Complex Crystallization DNA Data Development Dimensions Elements Entropy Epitopes Fluorescence Polarization Generations Genetic Transcription Glycine Goals Hydrocarbons Hydrophobicity Kinetics Laboratories Lead Malignant Neoplasms Mediating Methodology Mind Modification Multiprotein Complexes Mutation N-terminal Nuclear Nuclear Localization Signal Nucleic Acids Oncogenic Pathway interactions Peptides Permeability Phosphorylation Phosphotransferases Play Positioning Attribute Proline Property Proteins Recombinants Reporting Research Roentgen Rays Role Secondary Protein Structure Series Shapes Side Signal Transduction Specificity Structure Surface System Testing Therapeutic Thermodynamics Time Titrations Transcriptional Activation Transcriptional Regulation UBA Domain Ubiquitin Universities Work alpha helix base biophysical properties cancer therapy chemotherapeutic agent crosslink cytotoxic design flexibility in vivo Model inhibitor/antagonist knock-down large cell Diffuse non-Hodgkin&apos s lymphoma novel protein complex scaffold sharpin side effect small hairpin RNA small molecule targeted treatment transcription factor

项目摘要

The activated B cell-like (ABC) subclass of diffuse large B cell lymphoma (DLBCL) depends on the constitutive activation of the nuclear factor (NF)-kappaB signaling pathway109. Transcriptional activation of the NF-kappaB pathway relies on the degradation of the inhibitors of kappaB (IkappaB) which occlude a nuclear localization signal within NF-kappaB. Phosphorylation of IkappaB by the IkappaB kinase (IKK) leads to its subsequent ubiquitylation and degradation, allowing NF-kappaB to perform its transcriptional functions. The proteasomal degradation of these components is driven by an E3 complex dubbed the linear ubiquitin-chain assembly complex, or LUBAC112. This complex is composed of three proteins: RNF31, RBCK1, and Sharpin. In concert, these three components play a significant role in the constitutive activation of NF-kappaB in ABC DLBCL. The proper functioning of LUBAC depends mostly on the ability of RNF31 and RBCK1 to interact. Recently, an X-ray crystal structure of the complex was reported, and the data show that the interaction is mediated by the "Ubiquitin-like domain" (UBL) of RBCK1 and the "Ubiquitin-associated domain" (UBA) of RNF31. The interaction is mediated by two seemingly discrete alpha helices of the RNF31 UBA domain. The laboratory of Dr. Louis Staudt has shown by shRNA knockdowns that functional LUBAC is essential for the constitutive activation of NF-kappaB, and without it, the viability of ABC DLBCL cells is compromised. The unique structure and function make inhibition of this enzymatic complex an attractive drug target for the treatment of ABC DLBCL. The interaction between the LUBAC components RBCK1 and RNF31 is governed by a continuous, but bent, alpha helix. Using the structure of its macrohelix as a scaffold, our goal is to design and synthesize singly- and doubly-stapled RNF31 peptides as intracellular inhibitors of the LUBAC complex. Peptides with mutations at strategic positions will be designed to modify the physical, and thus the biological, properties of each compound. Fluoresceinated derivatives will be developed for fluorescence polarization binding assays as well as cell permeability assays. Unfunctionalized derivatives will be used for isothermal titration calorimetry studies in order to determine the thermodynamic parameters that govern the binding interaction. Data from biological assays performed in the Staudt Laboratory (e.g. NF-kappaB inhibition assays, competition co-immunoprecipitation of LUBAC components) along with results from biochemical studies performed in our laboratory will be used to select lead compounds for use in animal models. The RNF31 macrohelix is responsible for providing the contacts necessary for the proper function of LUBAC. The bend in the macrohelix is caused by the presence of a proline residue in the sequence. Because proline residues are notorious helix-breakers, the folds of the helices on each side of the bend are, in effect, independent of each other. Based on the sequence of RNF31, we first designed a set of four compounds. We synthesized a compound with a hydrocarbon staple on the N-terminal helix of RNF31 (RNF31-N), one with the staple on the C-terminal helix (RNF31-C), and one with cross-links on both helices (RNF31-NC). A wild type control peptide without any hydrocarbon cross-links was also synthesized. In biological assays, the Staudt laboratory determined that both RNF31-N and RNF31-NC disrupted the LUBAC complex in cells. These data suggest that the binding of RNF31 to RBCK1 is favored by preorganization of the N-terminal helix rather than the C-terminal helix. After successfully synthesizing stapled peptides capable of dissociating RNF31 from RBCK1 in LUBAC, the chemistry-based work will be split into two tasks. The first one consists of optimizing both the synthesis and the properties of the compounds through the design of second generation RNF31 peptides containing sequence modifications and different helix pairings. The second one entails the complete biochemical characterization of the binding of stapled RNF31 peptides to recombinant RBCK1. Given the better biological activity of N-stapled RNF31 peptides over the C-terminal counterparts, we designed and synthesized a new set of N-stapled RNF31 compounds containing several sequence modifications which alter their biophysical properties and behavior. The compounds will be subjected to all of the biochemical and cellular assays that were carried out with the first generation compounds. Optimized leads will be selected for use in in vivo models. Using a combination of circular dichroism and isothermal titration calorimetry, our goal is to determine the thermodynamic parameters that govern the binding of RNF31 to RBCK1 in the LUBAC complex. The biological evidence shows greater ABC DLBCL cytotoxic activity when N-stapled RNF31 peptides are used, suggesting that the binding of the macrohelix is sequential. Circular dichroism using intact stapled RNF31 peptides alone or in conjunction with recombinant RBCK1 will help determine the kinetics of helix nucleation upon binding. Isothermal titration calorimetry will be used to obtain the thermodynamic values (e.g., deltaG, deltaH, and deltaS) to establish the mechanism of the binding interaction. In collaboration with the laboratory of Kazuhiro Iwai at Kyoto University, we have also developed inhibitors targeting the interaction between RBCK1 and SHARPIN. We have shown that SHARPIN stapled peptides are better at disrupting the LUBAC complex than any of the compounds we had tested before. The work on this project concluded in April 2019

弥漫性大B细胞淋巴瘤(DLBCL)中活化的B细胞样(ABC)亚类依赖于核因子(NF)-kappaB信号通路109的结构性激活。NF-kappaB途径的转录激活依赖于kappaB抑制物(IkappaB)的降解，IkappaB阻断了NF-kappaB内的核定位信号。IkappaB激酶(IKK)使IkappaB磷酸化，导致其泛素化和降解，使核因子-kappaB发挥其转录功能。这些成分的蛋白酶体降解是由被称为线性泛素链组装复合体或LUBAC112的E3复合体驱动的。该复合体由三种蛋白质组成：RNF31、RBCK1和Sharpin。总之，这三个组分在ABC DLBCL中NF-kappaB的结构性激活中发挥着重要作用。LUBAC的正常功能主要取决于RNF31和RBCK1的相互作用能力。最近，该配合物的X射线晶体结构被报道，数据表明，相互作用是由RBCK1的泛素样域(UBL)和RNF31的泛素相关域(UBA)介导的。这种相互作用是由RNF31 UBA结构域的两个看似离散的α螺旋介导的。Louis Staudt博士的实验室已经通过shRNA敲除表明，功能性LUBAC对于NF-kappaB的结构性激活是必不可少的，如果没有它，ABC DLBCL细胞的生存能力就会受到影响。这种独特的结构和功能使得抑制这种酶复合体成为治疗ABC DLBCL的一个有吸引力的药物靶点。LUBAC组分RBCK1和RNF31之间的相互作用受连续但弯曲的α螺旋控制。利用其大螺旋结构作为支架，我们的目标是设计和合成单链和双链RNF31多肽作为LUBAC复合体的细胞内抑制剂。在关键位置发生突变的多肽将被设计成改变每种化合物的物理性质，从而改变其生物学性质。荧光化的衍生物将被开发用于荧光偏振结合分析以及细胞通透性分析。非官能化的衍生物将用于等温滴定量热法研究，以确定支配结合作用的热力学参数。STATUT实验室进行的生物分析数据(例如，核因子-kappaB抑制试验、LUBAC成分的竞争免疫共沉淀)以及我们实验室进行的生物化学研究的结果将被用于选择用于动物模型的先导化合物。RNF31巨螺旋负责为LUBAC的正常运行提供必要的接触。大螺旋中的弯曲是由序列中存在的脯氨酸残基引起的。因为脯氨酸残基是臭名昭著的螺旋断裂，弯曲两边的螺旋折叠实际上是相互独立的。根据RNF31的序列，我们首先设计了一套四个化合物。我们合成了一个在RNF31的N-末端螺旋上带有碳氢链的化合物(RNF31-N)，一个在C-末端螺旋上带有碳氢链的化合物(RNF31-C)，以及一个在两个螺旋上都带有交联链的化合物(RNF31-NC)。还合成了不含碳氢化合物交联物的野生型对照多肽。在生物检测中，STATUT实验室确定RNF31-N和RNF31-NC都破坏了细胞中的LUBAC复合体。这些数据表明，RNF31与RBCK1的结合有利于N端螺旋的预组织，而不是C端螺旋的预组织。在LUBAC中成功合成能够将RNF31与RBCK1解离的装订多肽后，这项基于化学的工作将分为两项任务。第一个是通过设计包含序列修饰和不同螺旋配对的第二代RNF31多肽来优化化合物的合成和性质。第二个是RNF31多肽与重组RBCK1结合的完整生化特征。鉴于N-末端RNF31比C-末端具有更好的生物活性，我们设计并合成了一组新的N-末端RNF31化合物，其中包含了几种改变其生物物理性质和行为的序列修饰。这些化合物将接受与第一代化合物相同的所有生化和细胞分析。优化的导联将被选择用于活体模型。使用圆二色谱和等温滴定量热法相结合，我们的目标是确定控制RNF31与LUBAC络合物中RBCK1结合的热力学参数。生物学证据表明，当使用N-链RNF31肽时，ABC DLBCL具有更强的细胞毒活性，这表明大螺旋的结合是顺序的。使用完整的装订RNF31多肽单独或与重组RBCK1结合使用圆二色谱将有助于确定结合时螺旋成核的动力学。等温滴定量热法将被用来获得热力学数值(例如，增量G、增量H和增量)，以确定结合作用的机理。与京都大学岩井一弘的实验室合作，我们还开发了针对RBCK1和夏平之间相互作用的抑制剂。我们已经证明，夏平装订的多肽比我们以前测试过的任何化合物都更能破坏LUBAC复合体。该项目的工作于2019年4月结束