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

项目摘要/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 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 主动抗菌素耐药性，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治疗失败的所有情况。 事实上，生物利用度和药代动力学是导致药物失败的第三大常见原因。 发展。然而，药代动力学研究主要集中在生物液体和组织药物上。 级别。对于细胞内的病原体来说，这是不够的，因为细胞内的药物积累是至关重要的。 以确保病原体清除。单细胞质谱学(SCMS)在抗癌药物领域的研究 研究表明，细胞内药物水平具有广泛的异质性。然而，到目前为止， 在细胞内病原体感染的情况下安全地执行这种分析的技术和技巧 一直都很缺乏。这是我们理解传染病治疗失败和 引导传染病药物开发。为了解决这一差距，提案MPI开发了一种生物安全- 与锥虫相关的细胞内药物和代谢物水平定量的SCMS兼容方法 克鲁兹寄生虫感染。这项提议的总体目标是证明这一点的广泛适用性 方法确定较低的细胞内药物水平与药物代谢和治疗失败的关系 清除细胞内病原体。这一提议的中心假设是异源细胞内药物 药物水平和细胞内药物代谢是抗菌药物治疗失败的关键因素，而这一点 利用新的SCMS方法可以揭示机理，具有广泛的适用性。我们将测试这个中心 假设使用三个相辅相成但相互独立的目标。目标1号将使用一种克氏锥虫寄生虫 与细胞间异种抗寄生虫药物水平相关的感染系统，与体外治疗克雷伯氏毛滴虫有关 失败了。目标2将使用相同的克氏锥虫感染系统来联系不同来源的抗寄生虫药物代谢 细胞间，对体外克氏锥虫的治疗失败。目标3将扩大这些SCMS的适用范围 技术，以量化特定的抗菌药和抗病毒药物，并将其水平与治疗失败在 细菌和病毒感染的背景。这项拟议的研究具有创新性，因为它将导致 开发一种新的生物分析技术来解决抗菌剂的关键生物学问题 治疗失败。这项拟议的研究具有重要意义，因为它将导致对 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.