Chemical Targeting of Multi-Protein Complexes

点击翻译按钮获取中文摘要

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.