Native Metabolomics Unveils Suomilide Analogs with Potent Trypsin Inhibitory Activity.

Cyanobacteria are well-known for their ability to produce a diverse array of serine protease inhibitors and thus are a promising source of pharmaceutical lead structures. Here, native metabolomics was employed with trypsin to rapidly screen whole cyanobacterial metabolomes and uncover new potent inhibitors. In combination with subsequent correlative LC-HR-MS/MS-based analysis, this led to the fast discovery of the well-characterized natural product family of suomilides from Nostoc sp. KVJ20 as highly potent trypsin inhibitors. Notably, two of the six putative binders were recognized as potential unknown analogs and thus were chosen for isolation and structural elucidation by means of 1D/2D NMR spectroscopy, Marfey's analysis, and bioinformatic investigations. This resulted in the identification of new suomilides G and H (1/2), which were isolated together with the known congeners suomilide B (3) and D (4). We also demonstrated that all four isolated suomilides inhibit trypsin at low nanomolar to high picomolar concentrations in a competitive bioassay, confirming the exceptional potency of this growing compound family as protease inhibitors.