Anti-inflammatory glycosides from plants: Identification of cellular targets and examination of structure–function relationships

植物糖苷的独特多样性蕴藏着相当大的潜力来衍生天然药物。在过去，我们专注于将糖基化三萜类化合物作为靶向肿瘤治疗中的内体逃逸促进剂。我们从防风中鉴定出一种特殊的皂苷SO1861，并在细胞培养中证明了肿瘤靶向毒素的细胞杀伤效率可以提高到2500,000倍，这导致小鼠的治疗窗口扩大了10倍到500倍。在所有的小鼠研究中，肿瘤消退率约为90%，所需剂量仅为不使用SO1861的治疗剂量的2%。剂量越低，副作用越少，免疫原性越低。我们证明，这种增强作用依赖于毒素部分通过内吞泡膜进入胞浆的增强转移。需要强调的是，SO1861的这一特定产物的浓度远远低于引发一般皂素效应(如溶血)所需的浓度，并且基于苷元和糖链的特殊和独特的化学结构。抗炎皂苷的研究日益引起人们的兴趣。超过25%的关于这一主题的科学论文是在过去4年内发表的。毫无疑问，某些皂苷具有抗炎作用，然而，大多数研究应用半纯化的皂苷混合物或总皂苷，结果是描述性的，缺乏关于结构-功能关系的详细知识。到目前为止，还没有证明细胞内的哪些分子是皂苷的目标。有人假设，皂苷的各种抗炎作用是基于特定的相互作用，这种相互作用只能归因于具有特定化学功能的皂苷的一个亚群。我们将首先选择已知结构和抗炎作用的商业可用皂苷，以确定主要的细胞靶点，并揭示结构与功能的关系。特别是，我们想要展示皂苷和靶点之间的相互作用如何导致细胞因子设备的变化。在确认单一皂苷在细胞培养中的抗炎作用后，我们将继续进行标记研究、结合分析、定位研究、抑制剂实验、膜分析和生物路径追踪。具有相似行为的新皂苷的鉴定和结构阐明将被用于提炼和概括所观察到的分子机制。我们将通过MALDI和核磁共振技术来确定重要的结构特征(苷元、糖残基的组成、糖键、立体异构体构型)。如果成功，计划在接下来的一段时间内研究其体内抗炎作用的分子机制。

The unique diversity of plant glycosides harbors a considerable potential to derive a natural drug. In the past, we focused on the identification of glycosylated triterpenoids as endosomal escape enhancers in targeted tumor therapies. We identified a particular saponin, SO1861, from Saponaria officinalis L. and demonstrated in cell culture that tumor-targeted toxins can become enhanced in their cell killing efficacy up to 2,500,000-fold, which resulted in a broadening of the therapeutic window in mice between 10-fold to 500-fold. The tumor regression across all mouse studies was about 90% and the required dose was only 2% of the dose used for a treatment without SO1861. The lower dose resulted in decreased side effects and reduced immunogenicity. We demonstrated that the augmentative effect relies on an enhanced transfer of the toxin moiety across the membrane of the endocytic vesicle into the cytosol. It is important to emphasize that this specific outcome of SO1861 occurs at concentrations that are far below the concentration required to trigger general saponin effects (such as hemolysis) and is based on the particular and unique chemical structure of the aglycone and sugar chains.Investigations of anti-inflammatory saponins are of increasing interest. More than 25% of scientific papers on this topic are published within the last 4 years. There is no doubt that certain saponins have an anti-inflammatory potential, however, most investigations applied semi-purified saponin mixtures or total saponins, the results are descriptive and detailed knowledge on structure–function relationships is missing. It has not been shown to date, which molecules inside the cell are the targets of the saponins. It is hypothesized that the various anti-inflammatory effects of saponins are based on specific interactions that can be attributed to only a defined subgroup of saponins with particular chemical functionalities. We will first select commercially available saponins with known structure and described anti-inflammatory effects to identify primary cellular targets and to reveal structure–function relationships. In particular, we want to demonstrate how interactions between saponins and targets result in changes in the cytokine equipment. After confirming the anti-inflammatory effects of single saponins in cell culture, we will continue with labeling studies, binding assays, localization studies, inhibitor experiments, membrane assays and tracking of biological pathways. Identification and structural elucidation of new saponins with similar behavior will then be used to refine and generalize the observed molecular mechanisms. We will determine important structural characteristics (aglycone, composition of sugar residues, sugar linkage, stereoisomer configurations) by MALDI and NMR techniques. If successful, it is planned in a following period to investigate the molecular mechanism of the anti-inflammatory effects in vivo.