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，创造了新的药物系统设计的新方法的需要。两性霉素B（AmB）是一种抗真菌药物，临床应用已有50多年，仅报告了有限的临床耐药病例。由于AmB副作用的毒性，已经开发了替代抗真菌药物，但是尽管使用时间短得多，但使用这些替代药物的治疗已经产生了越来越多的耐药菌株。这导致了对AmB研究的新兴趣，因为更好地了解其活性可以为如何设计更有效的抗菌剂提供信息，同时降低耐药性的发生。广泛的调查已经产生了三个，非常不同的，提出的AmB的作用机制的模型。我们提出了一系列的实验的基础上，瞬态吸收显微镜将图像无标记的AmB，因为它与生活和模型系统相互作用。我们的假设是：（1）我们提出的方法将能够区分不同的AmB活性模型，以及（2）AmB与膜相互作用的作用机制作为麦角固醇螯合、离子通道形成和氧化损伤模型之间的环境驱动相互作用而存在。在本项目的第一个具体目标中，我们将监测AmB在巨单层囊泡和S。cerevisiae和C.白色念珠菌细胞第二个具体目标将阐明聚集在AmB相互作用机制中的作用。这种聚集取决于环境条件，使其成为AmB采用的作用模型的可能驱动因素，同时也对药物的递送具有重要影响。总的来说，这些创新研究符合NIGMS的使命，即“为疾病治疗的进步奠定基础”，并将为四名学生（包括本科生和研究生）提供尖端成像仪器的实践培训和教育。

 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.