Specialized metabolites have played a central role since ancient times in medicine, culture, and science, from early herbal remedies to groundbreaking antibiotics, and have always been an extraordinary source of new drug leads, shaping modern drug research. Cyanobacterial specialized metabolites possess chemically diverse structures and a broad range of biological activities, which not only affect aquatic ecosystems and human health but also provide promising opportunities to discover new drug leads. This dissertation presents two case studies demonstrating how high-resolution mass spectrometry, enhanced by advanced computational tools, accelerates and streamlines the discovery and characterization of novel halogenated cyanobacterial specialized metabolites by efficiently mining complex datasets. The first study focuses on new analogues of the anhydro congener of the known herbicide cyanobacterin, all isolated from Tolypothrix sp. PCC 9009. Using a mass spectrometry-based chemical-guided screening, 15 new cyanobacterin analogues were isolated and characterized. Based on the known furanolide core assembly and the findings of this study, a biosynthetic pathway is proposed that may explain the tailoring enzyme reactions leading to cyanobacterin formation, with the newly discovered structures incorporated into the pathway. The second study investigates the pentabrominated biindole alkaloid aetokthonotoxin from Aetokthonos hydrillicola causing Vacuolar Myelinopathy. Based on analyses of environmental samples and supplementation studies, brominated, iodinated, and mixed halogenated aetokthonotoxin congeners and biosynthetic intermediates were discovered, highlighting the remarkable substrate flexibility of the involved halogenases and expanding the previously known biosynthetic pathway. Moreover, cytotoxicity assays of the isolated congeners showed that they differ markedly in their cytotoxicity. The third study, a mode-of-action investigation, reveals that the primary targets of aetokthonotoxin intoxication are mitochondria, where it functions as a weakly acidic uncoupler of mitochondrial respiration via its protonophore activity. The thesis demonstrates the diversity of cyanobacterial halogenated specialized metabolites, the indisputable advantages of the mass spectrometry-based tools mentioned, and has developed adaptable workflows to efficiently mine for these compounds.
