Establish RPN 13-Proteasome Association as a Novel Anticancer Target

 描述(申请人提供)：26S蛋白酶体是一个大型的多亚单位复合体，由一个中央核心颗粒和侧翼调节颗粒组成，核心颗粒降解任何可以进入其埋藏的蛋白水解点的蛋白质，调节颗粒识别和处理多泛素化的蛋白质，转位到核心颗粒中进行后续降解。26S蛋白酶体是癌症治疗的既定靶点，核心颗粒蛋白水解点的抑制剂已被批准用于治疗多发性骨髓瘤和套细胞淋巴瘤。然而，这些药物的疗效受到它们的毒性和可治疗的癌症范围的限制，主要是因为26S蛋白酶体的蛋白分解在真核细胞中是必不可少的。最近，在使用RA190的细胞培养和动物模型研究中，蛋白酶体调节粒子亚单位RPN13被报道为一个有前途的新的癌症靶点。RA190是一种与RPN13共价结合的亲电小分子。靶向RPN13的吸引力在于，与大多数蛋白酶体亚单位不同，RPN13在酵母或小鼠中不是必需的，因为缺失只会引起轻微的表型，但似乎对至少几种类型的癌症确实重要。基于我们未发表的生化和结构数据，我们提出了一种抑制RPN13的替代方法，该方法将采用与RA190垂直的机制，并且似乎可能产生较少的副作用，因为：(1)它不会直接抑制RPN13 (2)不会使用化学反应基团，()不会抑制RPN13的非蛋白酶体作用(它也与INO80染色质重构体一起起作用)。我们的基础研究包括确定人RPN13和RPN2C末端残基之间复合物的1.45A分辨率晶体结构，初步数据表明这是RPN13与蛋白酶体结合的唯一决定因素。我们还建立了SPR实验，实验测定了RPN13与RPN2C-末端的结合亲和力为10 nm，并将主要结合决定簇映射到RPN13上仅包括240 A2接触表面积的深口袋中的两个相邻的RPN2芳香族残基。为了进一步增强我们的方法的信心，我们已经证明了从RPN2中删除结合肽可以从纯化的蛋白酶体中取代RPN13。在这些见解的基础上，这项提案试图将抑制RPN13-蛋白酶体关联作为新的癌症治疗方法的一个有吸引力的靶点。为了实现这一目标，我们正在开发识别RPN13-蛋白酶体结合的小分子抑制剂(AIM 1)的方法，并开发细胞培养方法以确定在几种不同的癌细胞系中破坏RPN13-蛋白酶体结合(AIM 2)的后果。

 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 Based upon our unpublished biochemical and structural data, we propose an alternative approach to inhibiting RPN13 that will employ a mechanism orthogonal to that of RA190 and seems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.