Supramolecular Tandem Membrane Assays

有机分子的膜转运在小分子药物的发现和传递以及抗菌肽和细胞穿透肽的作用机制中起着至关重要的作用。然而，测量这些化合物在脂质双层膜上的实际转运仍然是一个主要的实验挑战。我们最近已经证明，脂质体包裹的超分子报告对(由荧光染料和大环受体分子组成)有资格通过一种简单的基于荧光的方法来明确地检测有机分子和多肽的膜转运。在这个方案中，我们将全面建立这种方法的实用性和适用性。具体来说，我们将在不同大小和膜组成的脂质体中包裹超分子报告对，以吸收光谱和荧光光谱方法研究选定的有机分子和多肽的膜转运。这包括光谱表征、时间分辨测量，包括用于快速动力学的停流技术，以及巨大单层囊泡(GUV)和活细胞的荧光显微镜。本文将开发的超分子串联膜分析将用于跟踪小的、未标记的分子和CPP的膜运输。这将为小分子有机分子的膜传输提供膜渗透系数和活化能作为实验基准，例如用于计算研究。对平行人工膜转运分析(PAMPA)的适应将展示未来在制药行业高通量筛选中的潜在应用。我们还将建立脂质体包裹的超分子报告对作为先进的超分子传感系统的关键组件，其中膜传输过程提高了超分子传感器系统的检测极限和选择性，从而实现了数量级的灵敏度增强和多肽序列的区分。

Membrane transport of organic molecules plays a crucial role in the discovery and delivery of small-molecule drugs as well as in the mechanism of action of antimicrobial peptides and cell-penetrating peptides (CPPs). The measurement of the actual transport of these compounds across the lipid bilayer membrane remains nevertheless a major experimental challenge. We have recently shown that liposome-encapsulated supramolecular reporter pairs (consisting of a fluorescent dye and a macrocyclic receptor molecule) qualify to unambiguously detect membrane transport of organic molecules and peptides by a simple and fluorescence-based method. Within this proposal, we will comprehensively establish the utility and applicability of this method.In detail, we will encapsulate supramolecular reporter pairs in liposomes of varying size and membrane composition in order to investigate membrane transport of selected organic molecules and peptides by absorption and fluorescence spectroscopic methods. This includes spectroscopic characterizations, time-resolved measurements, including stopped-flow techniques for rapid kinetics, as well as fluorescence microscopy with giant unilamellar vesicles (GUVs) as well as live cells.The supramolecular tandem membrane assays to be developed herein will be utilized to follow membrane transport of small, unlabeled molecules and CPPs. This will afford membrane permeability coefficients and activation energies for membrane transport of small, organic molecules as experimental benchmarks, for example, for computational studies. The adaptation to parallel artificial membrane transport assays (PAMPA) will demonstrate potential future applications in high-throughput screening in the pharmaceutical industry. We will also establish liposome-encapsulated supramolecular reporter pairs as key components in advanced supramolecular sensing systems, in which the membrane transport process improves the detection limit and selectivity of supramolecular sensor systems, thereby enabling orders of magnitude sensitivity enhancements and the discrimination of peptide sequences.