Mining for Halogenated Metabolites of Aetokthonos hydrillicola, the “Eagle Killer” Cyanobacterium

The cyanobacterium Aetokthonos hydrillicola has recently become famous as the “eagle killer”, producing the biindole alkaloid aetokthonotoxin (AETX), a pentabrominated neurotoxin causing the wildlife disease vacuolar myelinopathy. HPLC-HRMS2 analysis of extracts from environmental samples of the cyanobacterium revealed the presence of AETX derivatives and biosynthetic intermediates of the cyanobacterial neurotoxin. Mass spectrometry-based molecular networking and other advanced computational data mining techniques were employed to explore the chemical space of natural AETX derivatives. We identified a total of 43 biosynthetic intermediates and derivatives of AETX, including several iodinated derivatives, a rare halogenation in specialized metabolites of freshwater organisms. Structural characterization of these metabolites showed that most of them are AETX derivatives with varying substitution patterns of the bromo or iodo substituents, but also, AETX biosynthetic intermediates and other biindole derivatives were detected. Cytotoxicity assays of two isolated derivatives and AETX showed that they differ markedly in their activity.