Establish RPN 13-Proteasome Association as a Novel Anticancer Target

建立 RPN 13-蛋白酶体协会作为新的抗癌靶点

基本信息

批准号：
8985672
负责人：
CHRISTOPHER P. HILL
金额：
$ 16.2万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-11 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8985672
关键词：
26S proteasome Active Sites Adverse effects Affinity Anchorage-Independent Growth Animal Model Animals Apoptosis Area Binding Biochemical Biological Biological Assay Bortezomib C-terminal Cancer cell line Catalytic Domain Cell Culture Techniques Clinical Collaborations Complex Computer Analysis Cultured Cells Data Development Epitopes Eukaryotic Cell Fluorescence Polarization Goals Health Human Investigation Libraries Malignant Neoplasms Mantle Cell Lymphoma Maps Multiple Myeloma Mus Nucleosome Core Particle Pathway interactions Peptides Pharmaceutical Chemistry Pharmaceutical Preparations Phenotype Preclinical Drug Evaluation Process Proteasome Inhibition Protein translocation Proteins Proteolysis Reporting Resolution Role Site Structure Surface Testing Therapeutic Toxic effect Yeasts base cancer type chromatin remodeling drug efficacy inhibitor/antagonist insight multicatalytic endopeptidase complex novel particle response small molecule small molecule inhibitor

项目摘要

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.

描述（由适用提供）：26S蛋白酶体是由中央核心粒子组成的大型多生育络合物，它可以降解任何可以访问其内置蛋白水解位点的蛋白质以及侧翼调节位点，该蛋白质识别并处理多偶联性蛋白，以转移到核心粒子中，以降解核心粒子。 26S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这些药物的疗效受到限制，但是，由于26S蛋白酶体的蛋白水解在真实性细胞中必不可少，因此可以治疗可治疗的癌症和癌症范围。最近，据报道，蛋白酶体调节颗粒亚基RPN13是使用RA190的细胞培养和动物模型的研究中有希望的新癌症靶标，RA190是一种与RPN13共价结合的亲电的小分子。靶向RPN13的外观是，与大多数蛋白酶体亚基不同，它在酵母或小鼠中并不是必需的，在酵母或小鼠中，缺失仅引起温和的表型，但对于至少几种类型的癌症来说似乎确实很重要。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的机制正交机制，并且似乎喜欢产生更少的副作用，因为：（1）它不会直接抑制它的副作用。核心颗粒中的必需催化位点（2）将不使用化学反应性基团，并且（）不会抑制RPN13的非头膜角色（也可与INO80染色质重塑剂一起起作用）。我们的基础研究包括确定人类RPN13和RPN2 C末端保留之间复合物的分辨率晶体结构，我们的初步数据表明，这是RPN13与蛋白酶体的关联的唯一确定性。我们还开发了一种SPR分析，在实验上确定了RPN13和RPN2 C端之间的结合亲和力为10nm，并将主要结合确定性绘制在RPN2的两个相邻芳族芳族残留物中，该残留物位于RPN13上的深袋中，其中仅包括240个A2 A2 A2 A2 A2 A2 A2 a2。为我们的方法增加了置信度，我们已经表明，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，该提案旨在建立对RPN13-蛋白酶体关联的抑制作用，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，以鉴定RPN13-蛋白酶体关联（AIM 1）的小分子抑制剂（AIM 1）和开发细胞培养方法，以确定破坏RPN13-蛋白酶体关联的后果（AIM 2）在几种不同的癌细胞系中。