Chemical Targeting of Multi-Protein Complexes

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）-κ B信号通路的组成性活化109。NF-κ B通路的转录激活依赖于κ B抑制剂（IkappaB）的降解，所述抑制剂封闭NF-κ B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，从而允许NF-κ B执行其转录功能。这些组分的蛋白酶体降解是由E3复合物驱动的，该复合物被称为线性泛素链组装复合物或LUBAC 112。该复合物由三种蛋白质组成：RNF31，RBCK1和Sharpin。一致地，这三种组分在ABC DLBCL中NF-κ B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，数据显示该相互作用是由RBCK 1的"泛素样结构域"（UBL）和RNF31的"泛素相关结构域"（乌巴）介导的。这种相互作用由RNF31乌巴结构域的两个看似离散的α螺旋介导。Louis施陶德博士的实验室已经通过shRNA敲除表明，功能性LUBAC对于NF-κ B的组成性激活是必不可少的，并且没有它，ABC DLBCL细胞的活力受到损害。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标。LUBAC组分RBCK1和RNF31之间的相互作用由连续但弯曲的α螺旋控制。使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。在关键位置具有突变的肽将被设计成修饰每种化合物的物理性质，从而修饰每种化合物的生物性质。将开发用于荧光偏振结合测定以及细胞渗透性测定的双孢霉素衍生物。未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。施陶德实验室进行的生物学试验数据（例如NF-κ B抑制试验、LUBAC组分的竞争性免疫共沉淀）沿着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大螺旋负责提供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。因为脯氨酸残基是众所周知的螺旋破坏者，所以弯曲两侧的螺旋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四个化合物。我们合成了在RNF31的N-末端螺旋上具有烃钉合物（RNF31-N）的化合物，在C-末端螺旋上具有钉合物（RNF31-C）的化合物，以及在两个螺旋上具有交联的化合物（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，施陶德实验室确定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-末端螺旋而不是C-末端螺旋的预组织的支持。在LUBAC中成功合成能够将RNF31与RBCK1解离的钉合肽后，基于化学的工作将分为两项任务。第一个包括通过设计含有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性质。第二个需要钉合RNF31肽与重组RBCK1结合的完整生物化学表征。鉴于N-钉合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-钉合RNF31化合物，其含有几种改变其生物物理性质和行为的序列修饰。将对化合物进行使用第一代化合物进行的所有生化和细胞试验。将选择优化的电极导线用于体内模型。使用圆二色性和等温滴定量热法的组合，我们的目标是确定的热力学参数，管理的结合RNF31 RBCK1的LUBAC复合物。生物学证据显示，当使用N-钉合的RNF31肽时，ABC DLBCL细胞毒性活性更大，表明大螺旋的结合是顺序的。单独使用完整的钉合RNF31肽或与重组RBCK1结合使用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。

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.