The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally com-parable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclo-tides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bio-insecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phos-pholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previ-ously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Automated summary

This study evaluated the insecticidal activity of acyclotides from Rinorea bengalensis on Drosophila melanogaster and found that ribe 31 showed the most potent toxicity. Acyclotides were found to have lower toxicity towards human red blood cells compared to insect cells, indicating their selectivity and bio-insecticide potential. The cytotoxicity of ribe 31 is likely mediated via membrane disruption and it preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. Overall, with further optimization, ribe 31 could be used as an insecticide due to its potent in vivo activity against Drosophila melanogaster.