Imaging Amphotericin B's Mechanism of Action with Transient Absorption Microscopy

 描述(由申请人提供)：项目摘要抗菌素耐药性的急剧上升导致了在设计新的药物系统时需要新的方法。两性霉素B(Amb)是一种临床应用超过50年的抗真菌药物，临床耐药病例报道较少。由于Amb副作用的毒性性质，已经开发了替代抗真菌药物，但使用这些替代药物治疗已经产生了越来越多的耐药菌株，尽管使用期要短得多。这使人们对Amb的研究重新产生了兴趣，因为对其活性的更好了解可以为如何设计更有效、耐药发生率更低的抗菌药提供信息。广泛的调查导致了三种截然不同的AMB作用机制模型的提出。我们提出了一系列基于瞬变吸收显微镜的实验，当AMB与生物和模型系统相互作用时，它将成像无标记AMB。我们的假设是：(1)我们提出的方法学将能够区分AMB活性的不同建议模型和(2)AMB与膜相互作用的作用机制存在于所建议的麦角固醇隔离、离子通道形成和氧化损伤模型之间的环境驱动的相互作用。在这个项目的第一个具体目标中，我们将监测Amb在巨大的单层囊泡以及酿酒酵母和白色念珠菌细胞中的分布和定位。第二个特定目的将阐明聚集在AMB相互作用机制中的作用。这种聚集依赖于环境条件，这使得它可能是AMB所采用的行动模式的驱动因素，同时也对药物的输送具有重要影响。总体而言，这些创新研究非常符合NIGMS的使命，即“为疾病治疗的进步奠定基础”，并将为四名学生提供实践培训和教育，使用尖端成像仪器，包括本科生和研究生。

 DESCRIPTION (provided by applicant): Project Summary The dramatic rise of antimicrobial resistance has created the need for new approaches in the design of novel drug systems. Amphotericin B (AmB) is an antifungal drug that has been used clinically for over 50 years with only limited cases of clinical resistance reported. Due to the toxic nature of AmB's side effects, alternative antifungals have been developed, but treatment with these alternatives is already generating increasing numbers of resistant strains despite a much shorter period of use. This has led to a renewed interest in the study of AmB as a better understanding of its activity could inform on how to design more effective antimicrobials with lower occurrences of resistance. A wide range of investigations has resulted in three, very different, proposed models for AmB's mechanism of action. We are proposing a series of experiments based on transient absorption microscopy which will image label-free AmB as it interacts with both living and model systems. Our hypotheses are (1) that our proposed methodology will be able to distinguish between the different proposed models of AmB activity and (2) that the mechanism of action by which AmB interacts with a membrane exists as an environmentally driven interplay between the proposed models of ergosterol sequestration, ion channel formation, and oxidative damage. In the first specific aim of this project we will monitor the distribution and orientation of AmB in giant unilamellar vesicles and S. cerevisiae and C. albicans cells. The second specific aim will elucidate the role of aggregation in the mechanism of AmB's interaction. This aggregation is dependent on the environmental conditions making it a likely driver for which model of action AmB employs while also having important implications for the delivery of the drug. Overall, these innovative studies fit well within NIGMS's mission to "lay the foundation for advancements in disease treatment" and will provide hands-on training and education with cutting-edge imaging instrumentation for four students, including those at both the undergraduate and graduate levels.