Inhibitors targeting ribosome interactions of ricin

这项提案汇集了来自罗格斯大学(Tumer，Kimball，Augery)的研究人员， 阿尔伯特·爱因斯坦医学院(施拉姆、阿尔莫和卡梅隆)和沃兹沃斯中心 (螳螂)具有生物化学、结构生物学、药物化学和毒理学方面的专业知识 确定针对核糖体相互作用和蓖麻毒素催化活性的抑制剂。目前，有 对于蓖麻毒素中毒或相关志贺毒素产生的感染，是否没有经过证实的安全治疗方法？ 志贺氏菌或大肠杆菌。两种临床试验中的蓖麻毒素疫苗不能产生强大的毒素中和作用 活动。这项提议的目标是通过识别多肽和小分子来填补这一空白 与蓖麻毒素A链(RTA)上的关键口袋结合并抑制其活性的片段。在.期间 在之前的资助期间，我们确定了蓖麻毒素的宿主靶点是保守的C-末端11-聚体 (P11)核糖体P蛋白茎。我们发现RTA的核糖体结合面是 在活性中心的对面裂解，并提出了一个模型，其中结合到 核糖体茎通过重定向RTA的活性部位来刺激核糖体去嘌呤 SRL。这些研究建立了一种新的通便机制和 确定毒素/核糖体相互作用是发现抑制剂的新靶点。我们最近 发现了一种新的疏水口袋，由核糖体关键的必需精氨酸锚定 RTA的相互作用。我们的总体假设是，我们可以抑制催化活性和 蓖麻毒素干扰RTA核糖体相互作用的毒性。我们会找出钥匙 RTA核糖体结合面上的相互作用残基作为抑制剂的起点 发现号。使用核糖体靶标的多肽阵列，我们将识别结合的多肽类似物 与核糖体结合表面结合，阻断RTA的核糖体相互作用。我们将使用 基于碎片的铅发现(FBLD)和表面等离子激元共振(SPR)识别 能与核糖体结合口袋、活性部位裂解或以前结合的片段 并抑制RTA的排便活性和毒性。核糖体结合 活性位点突变体将用于确定结合位点的选择性和X-射线晶体 结构分析将用于阐明多肽和片段的结合模式。 片段命中将在基于细胞的分析中和在小鼠模型中进行优化和评估 蓖麻毒素中毒。有希望的线索将被用作搭建脚手架的起点。我们的 创新的方法、严谨的方法论和对结构功能分析的深刻见解 将为分子识别的基本机制提供新的知识。 Stack，并将有助于确定可用作未来治疗设计的新线索的抑制剂。

This proposal brings together investigators from Rutgers University (Tumer, Kimball, Augery), Albert Einstein College of Medicine (Schramm, Almo and Cameron) and Wadsworth Center (Mantis) with expertise in biochemistry, structural biology, medicinal chemistry, and toxicology to identify inhibitors that target ribosome interactions and catalytic activity of ricin. Currently, there is no proven, safe treatment for ricin intoxication or infection by related Shiga toxin producing Shigella or E. coli. Two ricin vaccines in clinical trials do not elicit robust toxin neutralizing activity. The goal of this proposal is to fill this gap by by identifying peptides and small molecule fragments that bind to key pockets on ricin A chain (RTA) and inhibit its activity. During the previous funding period we identified the host target of ricin as the conserved C-terminal 11-mer (P11) of the ribosomal P-protein stalk. We showed that the ribosome binding surface of RTA is on the opposite face of the active site cleft and proposed a model where binding to the ribosomal stalk stimulates ribosome depurination by reorienting the active site of RTA towards the SRL. These studies established a new paradigm for the mechanism of depurination and identified toxin/ribosome interactions as a new target for inhibitor discovery. We recently discovered a new hydrophobic pocket anchored by an essential arginine critical for ribosome interactions of RTA. Our overall hypothesis is that we can inhibit the catalytic activity and the toxicity of ricin by interfering with ribosome interactions of RTA. We will identify the key interacting residues at the ribosome binding surface of RTA as a starting point in inhibitor discovery. Using peptide arrays of the ribosomal target we will identify peptide analogs that bind to the ribosome binding surface and block the ribosome interactions of RTA. We will use fragment based lead discovery (FBLD) with surface plasmon resonance (SPR) to identify fragments that can bind to the ribosome binding pocket, the active site cleft or previously unidentified pockets and inhibit the depurination activity and toxicity of RTA. Ribosome binding and active site mutants will be used to determine the binding site selectivity and X-ray crystal structure analysis will be used to elucidate the binding mode of the peptides and the fragments. The fragment hits will be optimized and evaluated in cell-based assays and in a mouse model of ricin intoxication. The promising leads will be used as a starting point to build a scaffold. Our innovative approach, rigorous methodology and deep insight into the structure function analysis of RTA will provide new knowledge about the basic mechanism for molecular recognition of the stalk and will help identify inhibitors that can be used as new leads for future therapeutic design.