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信号通路的本构激活109。 NF-KAPPAB途径的转录激活取决于Kappab（Ikappab）抑制剂的降解，该抑制剂阻塞了NF-kappab中的核定位信号。 Ikappab激酶（IKK）对Ikappab的磷酸化导致其随后的泛素化和降解，从而允许NF-kappab执行其转录功能。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络合物驱动的。该复合物由三种蛋白质组成：RNF31，RBCK1和Sharpin。在协同中，这三个组件在ABC DLBCL中NF-kappab的组成型激活中起着重要作用。 LUBAC的正确功能主要取决于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，数据表明该相互作用是由RBCK1的“泛素样结构域”（UBL）和RNF31的“ Ubiquitin相关结构域（UBA）介导的”。该相互作用是由RNF31 UBA域的两个看似离散的α螺旋介导的。 Louis Staudt博士的实验室表明，功能性LUBAC对于NF-Kappab的本构激活至关重要，没有它，ABC DLBCL细胞的生存能力就会损害。独特的结构和功能使该酶复合物成为治疗ABC DLBCL的有吸引力的药物靶标。 LUBAC组件RBCK1和RNF31之间的相互作用由连续但弯曲的α螺旋支配。我们的目标是将其大赫利克斯的结构作为脚手架，是设计和合成单一和双粘的RNF31肽作为Lubac复合物的细胞内抑制剂。具有战略位置突变的肽将旨在修饰每种化合物的物理和生物学特性。将开发荧光衍生物用于荧光极化结合测定以及细胞渗透性测定。不官能化的衍生物将用于等温滴定量热法研究，以确定控制结合相互作用的热力学参数。来自Staudt实验室进行的生物测定的数据（例如NF-kappab抑制测定，竞争LUBAC组件的竞争共免疫沉淀）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中的铅化合物。 RNF31宏观螺旋体负责提供LUBAC正确功能所需的联系。宏观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断裂器，因此弯曲每一侧的螺旋褶皱实际上是彼此独立的。根据RNF31的序列，我们首先设计了一组四种化合物。我们在RNF31（RNF31-N）的N末端螺旋上合成了一个具有烃类固有的化合物，其中一个在C端螺旋（RNF31-C）上具有钉子，一个在两个螺旋上都具有交联（RNF31-NC）。还合成了没有任何烃交联的野生型控制肽。在生物学测定中，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的结合受到N末端螺旋的预组织而不是C末端螺旋的结合。成功合成能够从LUBAC中的RBCK1分离RNF31的固定肽后，基于化学的工作将分为两个任务。第一个是通过设计含有序列修饰和不同螺旋配对的第二代RNF31肽的设计来优化化合物的合成和性能。第二个需要钉钉RNF31肽与重组RBCK1的结合的完整生化表征。鉴于N-固定的RNF31肽比C末端对应物具有更好的生物学活性，因此我们设计和合成了一组新的N stapled RNF31化合物，其中包含几种序列修饰，这些序列修饰会改变其生物物理特性和行为。这些化合物将受到第一代化合物进行的所有生化和细胞测定。优化的导线将被选择用于体内模型。我们的目标结合了圆形二色性和等温滴定量热法，我们的目标是确定控制RNF31与RBCK1在Lubac复合物中的结合的热力学参数。生物学证据表明，当使用N stapled RNF31肽时，ABC DLBCL细胞毒性活性较大，这表明宏观黑质的结合是顺序的。单独或与重组RBCK1结合使用完整的RNF31肽的圆二色性将有助于确定结合时螺旋成核的动力学。等温滴定量热法将用于获得热力学值（例如三角洲，三角洲和三角洲）来建立结合相互作用的机理。