Molecular determinants of cellular penetration and permeability

拟议的研究计划主要旨在确定，在设计的分子的帮助下，细胞内化和细胞渗透性的基本原理。它由两个主题组成。 在主题I中，我们将设计和合成模块化的富含胍的转运蛋白（GRT），旨在探索和理解由细胞膜定义的化学空间。事实上，后者是分子在被细胞内化之前遇到的第一个环境。然而，它的结构和与分子的相互作用的特点很差。因此，我们将改变细胞渗透载体的结构来探测这种环境，以改进有效的、稳定的细胞渗透载体。我们将使用荧光激活细胞分选研究细胞内化机制，并使用共聚焦显微镜研究其细胞内分布。我们还将研究它们的细胞选择性性质，目的是了解结构-选择性关系。这一知识对于随后设计能够特异性地将货物（药物、成像剂）特异性地递送到感兴趣部位的分子是重要的。 在主题二中，我们将研究半肽大环化合物的结构-渗透性关系。与纯肽大环化合物相反，半肽大环化合物的渗透性尚未研究。我们的初步结果表明，微妙的结构变化，通过简单的甲基化在选定的位置，深刻影响细胞的渗透性。应该记住的是，大环化合物是作为调节蛋白质-蛋白质相互作用的特权结构出现的，这是一个由数十万个潜在靶点组成的整体，小分子的表现非常差。了解它们的渗透性以及如何优化它对于这些分子对这些细胞内靶点有效至关重要。 这两个主题都处于结构-渗透性连续体的极端，并将提供关键知识，有助于更好地开发实用的解决方案，以优化更智能的药物。

The proposed research program broadly aims to identify, with the help of designed molecules, the fundamentals of cellular internalization and cellular permeability. It is composed of 2 themes. In Theme I, we will design and synthesize modular guanidine-rich transporters (GRT) aimed to probe and understand the chemical space defined by the cellular membrane. Indeed, the latter is the first environment that molecules meet prior to be internalized by cells. However, its structure and interactions with molecules is poorly characterized. Thus, we will vary the structure of cellular penetration vectors to probe this environment, in order to refine efficacious, stable cellular penetration vectors. We will study cellular internalization mechanims using Fluorescence-Activated Cell Sorting, and their intracellular distribution using confocal microscopy. We will also study their cellular selectivity properties, with the goal to understand structure-selectivity relationship. This knowledge is important to subsequently engineer molecules able to specifically deliver cargos (drugs, imaging agents) specifically to a site of interest. In Theme II, we will study the structure-permeability relationship of semi-peptidic macrocycles. As opposed to purely peptidic macrocycles, semi-peptidic ones have not been investigated for their permeability properties. Our preliminary results indicate that subtle structural variations, via simple methylation at selected positions, deeply influence cellular permeability. It should be kept in mind that macrocycles emerge as privileged structures for the modulation of protein-protein interactions, an ensemble of several hundred thousand potential targets for which small molecules have performed very poorly. Understanding their permeability and how to optimize it is critical for these molecules to be efficacious on these intracellular targets. Both themes are at the extremes of the structure-permeability continuum, and will provide critical knowledge that will contribute to better develop practical solutions to optimize smarter drugs.