Untargeted LC–MS Metabolomics-Driven Discovery of Unique Peptides Cyanogristatins A–E from Sponge-Associated Actinoalloteichus cyanogriseus LHW52806 and Anti-Inflammatory Activity Assessment

This study established LC-MS-based metabolomics integrated with multivariate statistical analysis (UMMMS) to identify differential metabolites in Actinoalloteichus cyanogriseus under solid versus liquid culture conditions. Using this approach, 48 molecules exhibiting significant abundance differences between solid and liquid media were rapidly screened out. Among these, 8 were preliminarily identified as peptides potentially containing the unique β-Me-Leu residue by targeted MS/MS analysis. Guided by these screening results, 5 previously undiscovered peptides, designated as cyanogristatins A–E (1–5), were isolated. Their structures were elucidated using 1D/2D NMR spectroscopy, HR-MS/MS, and the advanced Marfey’s method. Furthermore, 5 effectively reduced the number of migrating inflammatory cell aggregates in a zebrafish model of inflammatory bowel disease at a concentration of 20 μM. In subsequent experiments using an in vitro organoid model, 5 did not exert direct epithelial protective effects; however, ELISA further revealed that it decreased the level of production of TNF-α in intestinal macrophages. This finding suggests that the anti-inflammatory effect of 5 is most likely mediated by the regulation of the cytokine TNF-α. In summary, this study demonstrates the potential of mass spectrometry technology for the comprehensive analysis of natural products, facilitating the rapid discovery of novel compounds and enhancing the exploration of microbial secondary metabolites.本研究建立了结合多元统计分析的液相色谱-质谱联用代谢组学方法（UMMMS），用于分析青灰放线异孢菌（Actinoalloteichus cyanogriseus）在固体与液体培养条件下的差异代谢物。借助该方法，研究人员快速筛选出48种在固体与液体培养基中丰度存在显著差异的分子。其中8种经靶向质谱/质谱（MS/MS）分析，初步鉴定为可能含有独特β-甲基亮氨酸（β-Me-Leu）残基的肽类。基于上述筛选结果，研究人员分离得到5种此前未被发现的肽类，将其命名为cyanogristatins A-E（1~5）。研究人员通过一维/二维核磁共振波谱（1D/2D NMR）、高分辨质谱/质谱（HR-MS/MS）以及改良Marfey法解析了这些肽类的结构。此外，在20μM浓度下，化合物5可有效降低炎症性肠病斑马鱼模型中迁移性炎症细胞聚集体的数量。在后续的体外类器官模型实验中，化合物5未表现出直接的上皮保护作用；但酶联免疫吸附实验（ELISA）进一步显示，其可降低肠道巨噬细胞中肿瘤坏死因子-α（TNF-α）的产生水平。该结果表明，化合物5的抗炎作用极有可能通过调控细胞因子TNF-α实现。综上，本研究证明了质谱技术在天然产物综合分析中的应用潜力，可助力新型化合物的快速发现，并推动微生物次级代谢产物的研究与探索。