Chemical Targeting of Multi-Protein Complexes

多蛋白复合物的化学靶向

• 批准号: 9153960
• 负责人: Federico Bernal
• 金额: $ 36.65万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/9153960
• 关键词: Active Sites; Adverse effects; 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; Collection; Complex; DNA; Data; Data Reporting; Development; Drug Targeting; Elements; Entropy; Epitopes; Fluorescence Polarization; Generations; Genetic Transcription; Glycine; Goals; Hydrocarbons; Kinetics; Laboratories; Lead; Malignant Neoplasms; Mediating; Methodology; Mind; Modification; Mutation; N-terminal; Nuclear; Nuclear Localization Signal; Nucleic Acids; Oncogenic; Pathway interactions; Peptides; Permeability; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Proline; Property; Proteins; Recombinants; Research; Roentgen Rays; Role; Secondary Protein Structure; Series; Shapes; Side; Signal Transduction; Specificity; Structure; Surface; System; Thermodynamics; Time; Titrations; Transcriptional Activation; Transcriptional Regulation; UBA Domain; Ubiquitin; Work; alpha helix; base; biophysical properties; cancer therapy; chemotherapeutic agent; crosslink; cytotoxic; design; flexibility; in vivo Model; inhibitor/antagonist; large cell Diffuse non-Hodgkin' s lymphoma; novel; protein complex; scaffold; sharpin; small hairpin RNA; small molecule; targeted treatment; transcription factor

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.

弥漫性大B细胞淋巴瘤（DLBCL）的活化B细胞样（ABC）亚类依赖于核因子(NF)-kappaB信号通路的组成性活化109。NF-kappaB通路的转录激活依赖于kappaB抑制剂（IkappaB）的降解，IkappaB阻断NF-kappaB内的核定位信号。IkappaB激酶（IKK）对IkappaB的磷酸化导致其随后的泛素化和降解，使NF-kappaB能够发挥其转录功能。这些成分的蛋白酶体降解是由一种称为线性泛素链组装复合物（LUBAC112）的E3复合物驱动的。该复合体由三种蛋白质组成：RNF31、RBCK1和Sharpin。协同作用，这三个成分在ABC DLBCL中NF-kappaB的组成性激活中起重要作用。LUBAC的正常功能主要取决于RNF31和RBCK1的相互作用能力。最近，研究人员报道了该复合物的x射线晶体结构，数据表明，这种相互作用是由RBCK1的“泛素样结构域”（UBL）和RNF31的“泛素相关结构域”（UBA）介导的。这种相互作用由RNF31 UBA结构域的两个看似离散的α螺旋介导。Louis Staudt博士的实验室通过shRNA敲低表明，功能性LUBAC对NF-kappaB的组成性激活至关重要，没有它，ABC DLBCL细胞的活力就会受到损害。独特的结构和功能使得抑制这种酶复合物成为治疗ABC型DLBCL的一个有吸引力的药物靶点。LUBAC组分RBCK1和RNF31之间的相互作用是由一个连续但弯曲的α螺旋控制的。利用其大螺旋结构作为支架，我们的目标是设计和合成单钉和双钉的RNF31肽作为LUBAC复合物的细胞内抑制剂。在战略位置发生突变的多肽将被设计用来改变每种化合物的物理性质，从而改变其生物学性质。荧光衍生物将被开发用于荧光偏振结合分析以及细胞渗透性分析。非功能化衍生物将用于等温滴定量热研究，以确定控制结合相互作用的热力学参数。在Staudt实验室进行的生物测定（例如NF-kappaB抑制测定，LUBAC组分的竞争共免疫沉淀）的数据以及在我们实验室进行的生化研究的结果将用于选择用于动物模型的先导化合物。RNF31巨螺旋负责提供LUBAC正常功能所需的接触。大螺旋的弯曲是由序列中脯氨酸残基的存在引起的。因为脯氨酸残基是臭名昭著的螺旋破坏者，所以弯曲两侧螺旋的折叠实际上是相互独立的。基于RNF31的序列，我们首先设计了一组4个化合物。我们合成了一个在RNF31的n端螺旋上有碳氢短链的化合物（RNF31- n），一个在c端螺旋上有短链的化合物（RNF31- c），一个在两个螺旋上都有交联的化合物（RNF31- nc）。同时合成了一种不含烃交联的野生型控制肽。在生物学分析中，Staudt实验室确定RNF31-N和RNF31-NC都破坏了细胞中的LUBAC复合物。这些数据表明RNF31与RBCK1的结合更倾向于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分离的钉状肽后，基于化学的工作将分为两个任务。第一部分是通过设计含有序列修饰和不同螺旋对的第二代RNF31肽来优化化合物的合成和性质。第二步需要完成钉接RNF31肽与重组RBCK1结合的完整生化表征。鉴于n -钉RNF31肽的生物活性优于c -末端对应物，我们设计并合成了一组新的n -钉RNF31化合物，其中包含几个序列修饰，可以改变其生物物理性质和行为。这些化合物将接受与第一代化合物一起进行的所有生化和细胞分析。优化引线将被选择用于体内模型。利用圆二色性和等温滴定量热法的结合，我们的目标是确定控制LUBAC复合体中RNF31与RBCK1结合的热力学参数。生物学证据表明，当使用n -钉RNF31肽时，ABC DLBCL的细胞毒活性更大，这表明大螺旋的结合是顺序的。单独使用完整的钉接RNF31肽或与重组RBCK1结合使用圆形二色性将有助于确定结合后螺旋成核的动力学。采用等温滴定量热法获得热力学值（如deltaG、deltaH和deltaS），建立结合相互作用的机理。