Chemical Targeting of Multi-Protein Complexes

多蛋白复合物的化学靶向

• 批准号: 9343966
• 负责人: Federico Bernal
• 金额: $ 15.88万
• 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/9343966
• 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; 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; knock-down; 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) 亚类取决于核因子 (NF)-kappaB 信号通路的组成型激活109。 NF-kappaB 通路的转录激活依赖于 kappaB 抑制剂 (IkappaB) 的降解，该抑制剂会阻断 NF-kappaB 内的核定位信号。 IkappaB 激酶 (IKK) 磷酸化 IkappaB 导致其随后泛素化和降解，从而使 NF-kappaB 能够发挥其转录功能。这些成分的蛋白酶体降解是由称为线性泛素链组装复合物或 LUBAC112 的 E3 复合物驱动的。该复合物由三种蛋白质组成：RNF31、RBCK1 和 Sharpin。综上所述，这三种成分在 ABC DLBCL 中 NF-kappaB 的组成型激活中发挥着重要作用。 LUBAC 的正常功能主要取决于 RNF31 和 RBCK1 相互作用的能力。最近，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 博士的实验室通过 shRNA 敲低表明，功能性 LUBAC 对于 NF-kappaB 的组成型激活至关重要，如果没有它，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 DLBCL 的有吸引力的药物靶点。 LUBAC 成分 RBCK1 和 RNF31 之间的相互作用由连续但弯曲的 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钉和双钉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肽将被设计来改变每种化合物的物理特性，从而改变其生物学特性。荧光素衍生物将被开发用于荧光偏振结合测定以及细胞渗透性测定。未官能化的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。 Staudt 实验室进行的生物测定数据（例如 NF-kappaB 抑制测定、LUBAC 成分的竞争性共免疫沉淀）以及我们实验室进行的生化研究结果将用于选择用于动物模型的先导化合物。 RNF31 宏螺旋负责提供 LUBAC 正常功能所需的接触。大螺旋中的弯曲是由序列中脯氨酸残基的存在引起的。由于脯氨酸残基是臭名昭著的螺旋破坏者，因此弯曲两侧的螺旋折叠实际上是彼此独立的。基于RNF31的序列，我们首先设计了一组四种化合物。我们合成了一种在 RNF31 的 N 端螺旋上具有烃钉的化合物 (RNF31-N)，一种在 C 端螺旋上具有钉的化合物 (RNF31-C)，以及一种在两个螺旋上都有交联的化合物 (RNF31-NC)。还合成了没有任何烃交联的野生型对照肽。在生物测定中，Staudt 实验室确定 RNF31-N 和 RNF31-NC 都会破坏细胞中的 LUBAC 复合物。这些数据表明 N 端螺旋而不是 C 端螺旋的预组织有利于 RNF31 与 RBCK1 的结合。在 LUBAC 中成功合成能够将 RNF31 与 RBCK1 分离的钉合肽后，基于化学的工作将分为两项任务。第一个包括通过设计包含序列修饰和不同螺旋配对的第二代 RNF31 肽来优化化合物的合成和特性。第二个需要对钉合的 RNF31 肽与重组 RBCK1 的结合进行完整的生化表征。鉴于 N-stapled RNF31 肽比 C-末端对应物具有更好的生物活性，我们设计并合成了一组新的 N-stapled RNF31 化合物，其中包含几个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。将选择优化的引线用于体内模型。结合使用圆二色性和等温滴定量热法，我们的目标是确定控制 LUBAC 复合物中 RNF31 与 RBCK1 结合的热力学参数。生物学证据显示，当使用 N-stapled RNF31 肽时，ABC DLBCL 细胞毒性活性更高，这表明大螺旋的结合是连续的。单独使用完整钉合的 RNF31 肽或与重组 RBCK1 结合使用的圆二色性将有助于确定结合后螺旋成核的动力学。等温滴定量热法将用于获得热力学值（例如 deltaG、deltaH 和 deltaS），以建立结合相互作用的机制。