Establish RPN 13-Proteasome Association as a Novel Anticancer Target

 描述（由申请人提供）：26 S蛋白酶体是一种大型多亚基复合物，由中心核心颗粒和侧翼调节颗粒组成，中心核心颗粒降解可进入其埋藏的蛋白水解位点的任何蛋白质，侧翼调节颗粒识别并加工多聚泛素化蛋白质，以转位到核心颗粒中进行后续降解。26 S蛋白酶体是癌症治疗的既定靶标，核心颗粒蛋白水解位点的抑制剂被批准用于治疗多发性骨髓瘤和套细胞淋巴瘤。然而，这些药物的功效受到其毒性和可治疗的癌症范围的限制，主要是因为26 S蛋白酶体的蛋白水解在真核细胞中是必需的。最近，蛋白酶体调节颗粒亚基RPN 13已被报道是一个有前途的新的癌症靶点的研究与细胞培养和动物模型使用RA 190，亲电子小分子，共价结合到RPN 13。靶向RPN 13的吸引力在于，与大多数蛋白酶体亚基不同，它在酵母或小鼠中不是必需的，在酵母或小鼠中，缺失仅导致轻度表型，但似乎对至少几种类型的癌症很重要。基于我们未发表的生物化学和结构数据，我们提出了一种抑制RPN 13的替代方法，该方法将采用与RA 190正交的机制，并且似乎可能产生更少的副作用，因为：（1）它不会直接抑制RPN 13， 核心颗粒中的基本催化位点，（2）不会使用化学反应基团，（）不会抑制RPN 13的非蛋白酶体作用（RPN 13也与INO 80染色质重塑剂一起发挥作用）。我们的基础研究包括确定人RPN 13和RPN 2 C-末端残基之间的复合物的1.45 A分辨率晶体结构，我们的初步数据显示，这是RPN 13与蛋白酶体结合的唯一决定因素。我们还开发了SPR测定，实验确定RPN 13和RPN 2 C-末端之间的结合亲和力为10 nM，并将主要结合决定簇映射到RPN 2的两个相邻芳香族残基，其位于RPN 13上的深口袋中，仅包括240 A2的接触表面积。为我们的方法增加了进一步的信心，我们已经表明，从RPN 2中删除结合肽可以从纯化的蛋白酶体中取代RPN 13。在这些见解的基础上，该提案寻求建立RPN 13-蛋白酶体缔合的抑制作为新型癌症治疗的有吸引力的靶标。为了实现这一目标，我们正在开发方法来识别RPN 13-蛋白酶体缔合的小分子抑制剂（Aim 1），并开发细胞培养方法来确定在几种不同的癌细胞系中破坏RPN 13-蛋白酶体缔合的后果（Aim 2）。

 DESCRIPTION (provided by applicant): The 26S proteasome is a large multisubunit complex comprised of a central core particle, which degrades any protein that can access its buried proteolytic sites, and flanking regulatory particles, which recognize and process polyubiquitylated proteins for translocation into the core particle for subsequent degradation. The 26S proteasome is an established target for cancer therapeutics, with inhibitors of the core particle proteolytic sites approved for the treatment of multiple myeloma and mantle cell lymphoma. Efficacy of these drugs is limited, however, by their toxicity and range of cancers that can be treated, primarily because proteolysis by the 26S proteasome is essential in eukaryotic cells. Recently, the proteasome regulatory particle subunit RPN13 has been reported to be a promising new cancer target in studies with cell culture and animal models using RA190, an electrophilic small molecule that covalently binds to RPN13. An appeal of targeting RPN13 is that, unlike the majority of proteasome subunits, it is not essential in yeast or mice, where deletion elicits only a mild phenotype, but does seem to be important for at least several types of cancer. Based upon our unpublished biochemical and structural data, we propose an alternative approach to inhibiting RPN13 that will employ a mechanism orthogonal to that of RA190 and seems likely to generate fewer side effects because: (1) it will not directly inhibit the essential catalytic sites in the core particle, (2) will not use a chemically reactive group, and () will not inhibit non-proteasomal roles of RPN13 (which also functions with the INO80 chromatin remodeler). Our foundational studies include determining a 1.45 A resolution crystal structure of a complex between human RPN13 and the RPN2 C-terminal residues, which our preliminary data show serve as the sole determinant of RPN13 association with the proteasome. We have also developed an SPR assay, experimentally determined the binding affinity between RPN13 and the RPN2 C-terminus to be 10nM, and mapped the primary binding determinant to two adjacent aromatic residues of RPN2 that sit in a deep pocket on RPN13 that includes only 240 A2 of contact surface area. Adding further confidence to our approach, we have shown that deletion of the binding peptide from RPN2 can displace RPN13 from purified proteasomes. Building upon these insights, this proposal seeks to establish inhibition of RPN13-proteasome association as an attractive target for novel cancer therapeutics. Toward this goal we are developing approaches to identify small molecule inhibitors of RPN13- proteasome association (Aim 1) and developing cell culture approaches to establish the consequences of disrupting RPN13-proteasome association (Aim 2) in several different cancer cell lines.