Protocols for measuring the stability and cytotoxicity of cyclotides

Cyclotides are plant host-defense peptides that have a wide range of biological activities and have diverse potential applications in medicine and agriculture. These 27-37 amino acid peptides have a head-to-tail cyclic backbone and are built around a cystine knot core, which makes them exceptionally stable. This stability and their amenability to sequence modifications has made cyclotides attractive scaffolds in drug design, and many synthetic cyclotides have now been designed and synthesized to test their efficacy as leads for a wide range of diseases, including infectious disease, cancer, pain and multiple sclerosis. Additionally, some natural cyclotides are selectively toxic to certain cancer cell lines, opening their potential as anticancer agents, and others have insecticidal activity, with applications in crop protection. With these applications in mind, there is a need to be able to measure cyclotides in pharmaceutical or agrichemical formulations and in biological media such as blood serum, as well as to assess their potential persistence in the environment when used as agrichemical agents. This chapter describes protocols for quantifying cyclotides in biological fluids, measuring their stability, and assessing their relative cytotoxicity on various types of cells.