Small Molecules from Arthropods

1) Scientific background: Arthropods are chemists par excellence: most species possess exocrine glands in which they produce chemical protectants against predators and microorganisms. Such secretions comprise a stunning diversity of small lipid molecules, representing a rich, but largely unexplored source of bioactive compounds. In soil - a moist, dirty, and microorganism-rich habitat - a large diversity of chemically protected arthropod taxa is found: these soil-dwellers belong to groups such as moss mites, harvestmen, springtails, and millipedes, and represent taxa that look back to an evolution of several 100 of millions of years. Such taxa have literally had enough time to diversify and perfect their chemical defenses. Interestingly, most therapeutics are small lipid molecules that meet Lipinskis rule of five, a concept to preliminarily evaluate the druglikeliness of a compound. Small molecules from arthropods (SMARTs) perfectly fit into this category. 2) Hypotheses & aims: Only very little research has been done with respect to the chemical ecology of soil-dwelling arthropod taxa, but has already led to the elucidation of several unusual, bioactive components that exclusively occur in these animals. We hypothesize that particularly soil-arthropods being equipped with a large number of exocrine glands to survive in their dirty, microorganism-rich habitat - possess a diversity of bioactive compounds far beyond the current state of knowledge. Objectives are i) to locate promising soil-arthropod taxa for a chemical screening of their exocrine secretions, including a complete chemical analysis of compounds involved; ii) to test these secretion compounds for bioactivity in bioassays. 3) New scientific ground: The proposed concept will result in the first representative screening of a new, unusual, and currently still neglected source of promising bio-pharmaceuticals. 4) Methods: will include: i) Collection of selected taxa of all major groups of soil arthropods in Austria, the Czech Republic, Poland, Germany, Italy, France, Spain, and all over the Balkans, relying on a unique network of specialists for different groups of soil Arthropoda. ii) Extraction and chemical analysis of exocrine secretions by modern mass spectrometric and nuclear magnetic resonance spectroscopic methods, followed by the synthesis of relevant compounds. iii) Evaluation of the bioactivity of extracts and their single (purified or synthesized) compounds in bioassays, mainly focusing on their antimicrobial activity but also measuring cellular responses of cultivated cells subsequent to component exposure. 5) Sientists involved: Günther Raspotnig (zoology, chemical ecology), Hans-Jörg Leis (chemistry/medicine/pharmacology), Slobodan Makarov (chemical ecology, bioactivity screening)

Exocrine glands of soil-dwelling arthropods represent ancient organ systems in which protective bioactive compounds evolved over several hundred million years. These small molecules are highly adapted for predator defense and to suppress microorganisms in the microbially rich soil environment and represent a largely unexplored potential source of novel antibiotics and bio-pharmaceuticals. This project focused on such small exocrine molecules from arthropods (SMARTs), which to date have remained poorly characterized. Using a phylogenetic prospecting approach, we identified promising taxa of chemically defended soil arthropods within specific lineages of millipedes (mainly Colobognatha), harvestmen (Insidiatores, Cyphophthalmi, troguloid Dyspnoi, sclerosomatid Eupnoi), and mites (primarily Oribatida). These groups possess large, sac-like exocrine glands that represent homologous systems within each taxon. We hypothesize that the presence of ancient homologous glands promotes the evolution of a high diversity of bioactive compounds. Particularly promising systems were the opisthonotal glands of Oribatida, prosomal scent glands of Opiliones, and serial defensive glands of Diplopoda. More than 100 compounds were extracted and chemically characterized. Most of them conform to Lipinski's rule of five, indicating favorable properties for potential pharmaceutical application. The compounds were compiled into comprehensive databases: so far, one covering exocrine compounds of harvestmen and one focusing on oil gland compounds of Oribatida. These databases represent the most complete compilations currently available for these groups, including analytical data, taxonomic distribution, and biological functions for detected compounds. Recurring chemical motifs were identified across taxa, including nitrogen-containing motifs, as well as spirocyclic- (Colobognatha), ethyl ketone- (Opiliones), aromatic- (Opiliones, Oribatida), and terpene-structures (Oribatida). Many compounds proved to be highly unusual or new to science. Notable examples include spirocyclic compounds from colobognathan millipedes and the "hermanniellines", a novel class of multi-ringed oil gland compounds from certain Oribatida (brachypyline Hermanniellidae). These compounds show pronounced species specificity and structural complexity, including multiple stereocenters. Bioassays revealed promising antibacterial activities for several compounds against a broad spectrum of bacteria. Other compounds, particularly nitrogen-containing substances, appeared to be neurotoxic and act as modulators of nervous system receptors. Stereochemistry proved critical, as different isomers often displayed markedly different biological activities. New scientific ground: The project identifies soil-dwelling arthropods as a highly promising and largely untapped source of bioactive natural products. By linking phylogeny, gland morphology, chemistry, and bioactivity, it establishes a novel framework for targeted natural product discovery and highlights ancient arthropod exocrine systems as a valuable resource for future bio-pharmaceutical research. Publications: Eight peer-reviewed publications resulted directly from this FWF project, complemented by three conference contributions. Six additional manuscripts are currently in press, under review, or in preparation.