Novel single-cell mass spectrometry methods to assess the role of intracellular drug concentration and metabolism in antimicrobial treatment failure

项目总结/摘要 传染病治疗失败是一个严重的健康问题。据预测，它将成为导致 到2050年，死亡率将取代癌症。治疗失败可能是由多种因素引起的，包括 主动抗微生物药物抗性，例如通过外排泵，以及病原体休眠、耐受性和 持久性机制然而，这些因素不能单独解释所有治疗失败的病例。 事实上，生物利用度和药代动力学是药物治疗失败的第三大最常见原因。 发展然而，药代动力学研究主要集中在生物流体和组织药物 程度.这在胞内病原体的情况下是不够的，其中胞内药物积累是关键的 确保病原体清除单细胞质谱（SCMS）在肿瘤药物研究中的应用 发展揭示了细胞内药物水平的广泛异质性。然而，到目前为止， 在细胞内病原体感染的情况下安全地进行这种分析的技术和方法 一直缺乏。这是我们理解传染病治疗失败的能力和 指导传染病药物开发。为了弥补这一差距，建议多边投资机构制定了一项生物安全- 一种用于定量锥虫体内细胞内药物和代谢物水平的兼容SCMS方法 克氏寄生虫感染本提案的总体目标是证明这一建议的广泛适用性， 确定较低的细胞内药物水平和药物代谢与未能 清除细胞内病原体。这一建议的中心假设是， 水平和细胞内药物代谢是抗菌治疗失败的关键因素， 机制可以揭示使用新的SCMS方法具有广泛的适用性。我们将测试这个中央 假设使用三个互补但独立的目标。目标1将使用克氏锥虫寄生虫 感染系统与细胞间异质性抗寄生虫药物水平相关，与体外T.克氏疗法 失败目标2将使用相同的T。与异源抗寄生虫药物代谢有关的克氏感染系统 细胞之间，到体外T. cruzi治疗失败。目标3将扩大这些供应链管理系统的适用范围 技术，以量化特定的抗菌药物和抗病毒药，并将其水平与治疗失败， 细菌和病毒感染的背景。拟议的研究是创新的，因为它将导致 开发一种新的生物分析技术，以解决抗菌药物的关键生物学问题 治疗失败。拟议的研究是重要的，因为它将导致扩大了解的 传染病治疗失败的机制。

Project Summary/Abstract Infectious disease treatment failure is a critical health issue. It is predicted to become the leading cause of mortality by 2050, superseding cancer. Treatment failure can be caused by a combination of factors, including active antimicrobial drug resistance, for example via efflux pumps, and pathogen dormancy, tolerance and persistence mechanisms. However, these factors cannot alone account for all cases of treatment failure. Indeed, bioavailability and pharmacokinetics are the 3rd most common cause of failure during drug development. Pharmacokinetic studies, however, have predominantly focused on biofluid and tissue drug levels. This is insufficient in the case of intracellular pathogens, where intracellular drug accumulation is critical to ensure pathogen clearance. Single-cell mass spectrometry (SCMS) studies in the field of cancer drug development have revealed extensive heterogeneity in intracellular drug levels. However, until now, the technology and techniques to safely perform such analyses in the context of intracellular pathogen infection have been lacking. This is a critical gap in our ability to understand infectious disease treatment failure and to guide infectious disease drug development. To address this gap, proposal MPIs have developed a biosafety- compatible SCMS method to quantify intracellular drug and metabolite levels in the context of Trypanosoma cruzi parasite infection. The overall objective of this proposal is to demonstrate the broad applicability of this method to determine the relationship between lower intracellular drug levels and drug metabolism vs failure to clear intracellular pathogens. The central hypothesis of this proposal is that heterogenous intracellular drug levels and intracellular drug metabolism is a key contributor to antimicrobial treatment failure, and that this mechanism can be revealed using novel SCMS approaches with broad applicability. We will test this central hypothesis using three complementary yet independent aims. Aim 1 will use a Trypanosoma cruzi parasite infection system to relate heterogenous antiparasitic drug levels between cells, to in vitro T. cruzi treatment failure. Aim 2 will use the same T. cruzi infection system to relate heterogenous antiparasitic drug metabolism between cells, to in vitro T. cruzi treatment failure. Aim 3 will broaden the applicability of these SCMS techniques to quantify specific antibacterials and antivirals, and to relate their levels to treatment failure in the context of bacterial and viral infection. The proposed research is innovative because it will lead to the development of a new bioanalytical technology to address the critical biological problem of antimicrobial treatment failure. The proposed research is significant because it will lead to an expanded understanding of the mechanisms of treatment failure in infectious diseases.