Optimization of Small-Molecule Inhibitors of Shiga and Ricin Toxins

志贺毒素和蓖麻毒素小分子抑制剂的优化

基本信息

批准号：
7645354
负责人：
YUAN-PING PANG
金额：
$ 80.28万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-06 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7645354
关键词：
Abrin Active Sites Adenine Advanced Development Affinity Animals Biochemical Biochemistry Biological Assay Categories Cell membrane Cells Clinic Clip Cytoprotection Data Development Development Plans Disease Doctor of Philosophy Escherichia coli Escherichia coli EHEC Event FDA approved Free Energy Generations Goals Immune Immunity Immunization Immunology Injury Instruction Intoxication Knowledge Measures Mediating Methods Molecular Molecular Biology Molecular Conformation Morbidity - disease rate Mus Oligonucleotides Outcome Permeability Pharmaceutical Chemistry Pharmaceutical Preparations Plants Principal Investigator Protein Biosynthesis Proteins Public Health RNA Binding Recombinants Reporting Research Research Institute Research Personnel Ribosomal RNA Ribosomes Ricin Ricin Vaccine Ricinus communis Screening procedure Shiga Toxin Shiga-Like Toxins Structure Surface Plasmon Resonance System Targeted Toxins Testing Therapeutic Tissues Toxin Triage Universities Vaccinated Vaccine Therapy Vaccines Work X-Ray Crystallography analog base biodefense combinatorial cytotoxicity design drug candidate effective therapy extracellular foodborne outbreak improved in vivo inhibitor/antagonist luminescence mortality multidisciplinary novel strategies pathogen prevent protective efficacy protein function small molecule structural biology translation assay vaccine candidate vaccine efficacy

项目摘要

DESCRIPTION (provided by applicant): This proposal brings together investigators from Mayo Clinic, Rutgers University, and Walter Reed Army Institute of Research with expertise in computational and medicinal chemistry, structural biology, biochemistry, molecular biology and immunology to develop broad-spectrum therapy against ribosome inactivating proteins. Our primary goal is to develop second-generation small-molecule inhibitors of Shiga-like toxin 2 (Stx2) and ricin. Both toxins are category B agents that can cause significant morbidity and mortality. Currently, there is no FDA-approved vaccine or therapeutics that can protect against either toxin. Traditional approaches to ricin/Shiga toxin inhibitors use either a brute-force screening method to identify leads or a structure-based method to identify analogs of substrate adenine. The toxin-transport-system-targeting inhibitors with IC5O values of 25-50 ?M identified from the screening method suffer from the challenge in optimizing the leads into nanomolar inhibitors because of the lack of the structural information on the transport system. All adenine-like inhibitors reported to date are either small molecules with submillimolar potencies or oligonucleotides that can neutralize the extracellular toxin to prevent further intoxication but cannot effectively cross the plasma membrane to rescue intoxicated cells. This application innovatively uses the molecular "clip" approach that identifies small molecules able to lock TyrSo of ricin (or its equivalent Tyryy of Stx2) into the conformation that blocks the function of the protein. Such inhibitors do not have to be adenine-like for direct competition with RNA for binding to the toxin, thereby avoiding the permeability problem of the known ricin/Shiga inhibitors. This novel approach has already culminated in small molecules that inhibited both ricin and Stx2 at a drug concentration of 10 nM and showed ~io% cell protection against ricin and 20% cell protection against Stx2 at a drug concentration of 300 nM. In this context, we propose to optimize our inhibitor leads to improve their affinity for the active site of Stx2/ricin and their cell permeability, measure the potency of the inhibitors in antagonizing Stx2/ricin in cell free and cell based assays and examine the inhibitors in combination with a ricin vaccine under development for synergistic efficacy to prevent or alleviate ricin intoxication. The significant outcomes of this project are (i) small molecules that inhibit both Shiga and ricin at nanomolar drug concentrations in vivo, enabling a therapy effective against multiple toxins; (2) knowledge useful for identification and optimization of other small-molecule inhibitors of toxins that target ribosomes; (3) a proof-of- concept for use of small-molecule inhibitors as adjuncts to immunization to explore the synergy of small-molecule and vaccine therapies. Ultimately the work proposed here will result in the development several highly potent and specific inhibitors of toxin-mediated injury that are potential drug candidates. RELEVANCE (See instructions): The research is most relevant to public health and biodefense.

描述（由申请人提供）：该提案汇集了来自梅奥诊所、罗格斯大学和沃尔特·里德陆军研究所的研究人员，他们拥有计算和药物化学、结构生物学、生物化学、分子生物学和免疫学方面的专业知识，以开发针对该疾病的广谱疗法。核糖体失活蛋白。我们的主要目标是开发志贺样毒素 2 (Stx2) 和蓖麻毒素的第二代小分子抑制剂。这两种毒素都是 B 类毒剂，可导致严重的发病率和死亡率。目前，尚无 FDA 批准的疫苗或疗法可以预防这两种毒素。蓖麻毒素/志贺毒素抑制剂的传统方法使用强力筛选方法来鉴定先导化合物，或使用基于结构的方法来鉴定底物腺嘌呤的类似物。通过筛选方法鉴定出的IC50值为25-50μM的毒素转运系统靶向抑制剂在将先导化合物优化为纳摩尔抑制剂方面面临着挑战，因为缺乏转运系统的结构信息。迄今为止报道的所有腺嘌呤样抑制剂要么是具有亚毫摩尔效力的小分子，要么是寡核苷酸，它们可以中和细胞外毒素以防止进一步中毒，但不能有效穿过质膜来拯救中毒细胞。该应用创新性地使用分子“夹子”方法，识别能够将蓖麻毒素的 TyrSo（或其等效的 Stx2 的 Tyryy）锁定为阻断蛋白质功能的构象的小分子。此类抑制剂不必是腺嘌呤样的以与RNA直接竞争结合毒素，从而避免了已知的蓖麻毒素/志贺抑制剂的渗透性问题。这种新颖的方法已经最终产生了在10nM的药物浓度下抑制蓖麻毒素和Stx2的小分子，并且在300nM的药物浓度下显示出～10%的针对蓖麻毒素的细胞保护和20%的针对Stx2的细胞保护。在这种情况下，我们建议优化我们的抑制剂引线，以提高它们对 Stx2/蓖麻毒素活性位点的亲和力及其细胞渗透性，测量抑制剂在无细胞和基于细胞的测定中拮抗 Stx2/蓖麻毒素的效力，并检查抑制剂与正在开发的蓖麻毒素疫苗结合使用，可发挥协同功效，预防或减轻蓖麻毒素中毒。该项目的重要成果是（i）小分子能够在体内以纳摩尔药物浓度抑制志贺菌和蓖麻毒素，从而能够有效对抗多种毒素； (2) 对于识别和优化其他针对核糖体的毒素小分子抑制剂有用的知识； (3) 使用小分子抑制剂作为免疫辅助手段的概念验证，以探索小分子和疫苗疗法的协同作用。最终，这里提出的工作将导致开发出几种高效且特异性的毒素介导损伤抑制剂，这些抑制剂是潜在的候选药物。相关性（参见说明）：该研究与公共卫生和生物防御最相关。