Dash Rachita, Liu Zichen, Lepori Irene, Chordia Mahendra D, Ocius Karl, Holsinger Kadie, Zhang Han, Kenyon Ryan, Im Wonpil, Siegrist M Sloan, Pires Marcos M
bioRxiv. 2025 Mar 12:2025.01.17.633618. doi: 10.1101/2025.01.17.633618.
Understanding the factors that influence the accumulation of molecules beyond the mycomembrane of ( ) - the main barrier to accumulation - is essential for developing effective antimycobacterial agents. In this study, we investigated two design principles commonly observed in natural products and mammalian cell-permeable peptides: backbone -alkylation and macrocyclization. To assess how these structural edits impact molecule accumulation beyond the mycomembrane, we utilized our recently developed Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings provide the first empirical evidence that peptide macrocyclization generally enhances accumulation in mycobacteria, while -alkylation influences accumulation in a context-dependent manner. We examined these design principles in the context of two peptide antibiotics, tridecaptin A1 and griselimycin, which revealed the roles of -alkylation and macrocyclization in improving both accumulation and antimicrobial activity against mycobacteria in specific contexts. Together, we present a working model for strategic structural modifications aimed at enhancing the accumulation of molecules past the mycomembrane. More broadly, our results also challenge the prevailing belief in the field that large and hydrophilic molecules, such as peptides, cannot readily traverse the mycomembrane.
了解影响分子在（）的霉菌膜之外积累的因素至关重要，因为霉菌膜是积累的主要障碍，这对于开发有效的抗分枝杆菌药物至关重要。在本研究中，我们研究了天然产物和哺乳动物细胞可渗透肽中常见的两种设计原则：主链N-烷基化和大环化。为了评估这些结构修饰如何影响分子在霉菌膜之外的积累，我们利用我们最近开发的肽聚糖可及性点击介导评估（PAC-MAN）测定法进行活细胞分析。我们的研究结果提供了首个实证证据，即肽大环化通常会增强在分枝杆菌中的积累，而N-烷基化则以上下文依赖的方式影响积累。我们在两种肽类抗生素tridecaptin A1和griselimycin的背景下研究了这些设计原则，这揭示了N-烷基化和大环化在特定背景下改善对分枝杆菌的积累和抗菌活性方面的作用。我们共同提出了一个旨在增强分子在霉菌膜之外积累的战略结构修饰的工作模型。更广泛地说，我们的结果也挑战了该领域中普遍存在的观念，即诸如肽之类的大分子和亲水分子不易穿过霉菌膜。
