Emergence of drug resistance in prion populations

朊病毒群体中出现耐药性

• 批准号: 8259850
• 负责人: ROY G SMITH
• 金额: $ 42.45万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259850
• 关键词: Amino Acid Sequence; Bacteria; Biological Assay; Brain; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chemicals; Chronic; Complex; Cultured Cells; Discrimination; Drug resistance; Drug-sensitive; Environment; Exhibits; Genetic Materials; Heterogeneity; Hydrochloride Salt; Infection; Investigation; Isomerism; Laboratories; Molecular Conformation; Mus; Mutation; Neuroblastoma; Nucleic Acids; Pharmaceutical Preparations; Population; PrPC Proteins; PrPSc Proteins; Prion Diseases; Prions; Process; Property; Proteins; RNA Phages; RNA Viruses; Relative (related person); Resistance; Resistance development; Scrapie; Temperature; Therapeutic; Time; Variant; Virus; Work; abstracting; base; brain cell; conformer; design; glycosylation; guanidinium; inhibitor/antagonist; innovation; mutant; pathogen; prevent; protein misfolding cyclic amplification; research study; treatment strategy

Abstract Prions, the pathogens causing transmissible spongiform encephalopathies, are unique in that they consist mainly, if not entirely of multimers of PrPSc, a conformational isomer of the host- encoded protein PrPC. Prions occur in the form of diverse strains, which differ in various phenotypic properties but whose PrPSc has the same amino acid sequence. It is believed that strain identity is encoded by the conformation of PrPSc and that there are as many distinct conformations as there are strains. We have observed that a prion strain, 22L, when transferred from brain to cell culture, changed its characteristics, but gradually regained them when re-introduced into brain. Moreover, when propagated in cell culture in the presence of an inhibitory drug, swainsonine, the prion population acquired resistance against the drug, but lost the resistance when propagated for prolonged periods in its absence. This is quite remarkable, considering that bacteria and viruses acquire drug resistance as a consequence of random mutations in their genetic material, whereas prions do not contain informational nucleic acids. Our working hypothesis is that a prion strain population is comprised not of PrPSc with a unique conformation but of a multitude of species ("sub-strains") with related conformations. This is analogous to the concept of "quasispecies" we first established for RNA phages three decades ago. We postulate that the replication rates of sub-strains may vary depending on the host cell. In a particular environment one of these species may represent the major component and out- replicate other conformers, but at the same time, variant conformers are continuously generated at some low rate (because the activation energy barriers for interconversion are low). Upon transfer to a different host or a change in the environment a different conformer may become the major constituent of the population. We wish to determine whether PrPSc associated with drug resistant and drug sensitive prions show physico-chemical differences that may reflect different conformations, and whether the two types of prions replicate at different rates in host cells. We will investigate whether prions can develop resistance against other inhibitory drugs. Importantly, we will determine whether prion populations that have never been exposed to swainsonine already comprise resistant variants, as would be expected from the "quasispecies" hypothesis. We intend to biologically clone a drug-sensitive prion and determine whether swainsonine-resistant variants arise during propagation in the absence of the drug, and if so, at what rate. Finally, we will ascertain whether it is possible to select prion "sub-strains" that are more resistant to chaotropic agents or to temperature, which would be further evidence in favor of heterogeneity of prion populations. The major technical innovation making this investigation possible is the Cell Panel Assay (CPA), which is based on the Standard Scrapie Cell Assay (SSCA), both of which were developed in our laboratories and which allow the rapid discrimination between prion strains. The finding that prions can acquire drug resistance impacts on the strategies envisaged for therapeutic approaches to prion disease.

摘要 Prion是引起传染性海绵状脑病的病原体，其独特之处在于 它们主要由PrPSc的多聚体组成，即使不完全是PrPSc的多聚体，也是宿主的构象异构体- 编码蛋白PrPC。Prion以不同的菌株的形式存在，它们在不同的 表型特性，但其PrPSc具有相同的氨基酸序列。据信， 菌株的同一性是由PrPSc的构象编码的，有许多不同的 因为有菌株的构象。 我们已经观察到，当从大脑转移到细胞培养物中时，一种名为22L的普恩病毒株改变了它的 特征，但当重新引入大脑时逐渐恢复这些特征。此外，当 在抑制药物苦马豆素存在的情况下，在细胞培养中繁殖，Pron群体 获得了对药物的抗药性，但在长期繁殖时失去了抗药性 在它不在的情况下，句号。考虑到细菌和病毒获得 由于遗传物质的随机突变而产生的耐药性，而普恩 不包含信息性核酸。 我们的工作假设是，PrPSc不是由一个唯一的PrPSc组成的 而是具有相关构象的多种物种(“亚种”)的构象。这是 类似于我们三十年来首次为rna噬菌体建立的“准种”概念。 那是以前的事了。我们推测，根据宿主细胞的不同，亚株的复制速度可能会有所不同。在……里面 这些物种中的一种可能代表着一个特定的环境， 复制其他构象，但同时，不断产生变异构象 在一些低速率下(因为相互转化的激活能垒很低)。vt.在.的基础上 转移到不同的主机或环境中的变化不同的整合体可能会成为 人口的主要组成部分。 我们希望确定PrPSc是否与耐药和药物敏感的Prion有关 显示可能反映不同构象的物理化学差异，以及 两种类型的普恩在宿主细胞中以不同的速度复制。我们将调查普恩病毒是否 会对其他抑制药物产生抗药性。重要的是，我们将确定是否 从未接触过苦马豆素的Pron种群已经包含了抗性 变种，正如“准物种”假说所预期的那样。我们打算在生物学上 克隆一个药物敏感的普里子，并确定在 在没有药物的情况下传播，如果是这样，传播的速度是多少。最后，我们将确定是否 有可能选择对杂色剂更具抵抗力或对 温度，这将是有利于Pron种群异质性的进一步证据。 使这项调查成为可能的主要技术创新是细胞板分析(CPA)， 它基于标准瘙痒细胞检测(SSCA)，这两种方法都是在 我们的实验室，并允许快速区分普恩病毒菌株。 Prion可以获得抗药性的发现对设想的战略产生了影响 Pron病的治疗方法。