Cyclotides are head-to-tail cyclized plant derived peptides of 28 to 35 amino acids in size. They are well known for their circular cystine-knot motif and resulting stability. Hence they appear promising natural products for pharmacological screening to discover bioactive peptide leads or for targeted drug development as engineered peptide probes. To date the therapeutic application of non-grafted cyclotides is limited due to the lack of specific cellular target proteins to modulate cellular signaling. Recently, we reported that cyclotides are able to inhibit the proliferation of activated T-lymphocytes in vitro without any cytotoxic effects [1]. The anti-proliferative activity of kalata B1 is mediated through an IL2-dependent mechanism [2]. Since no molecular targets involved in this promising effect on human T-cells has been elucidated, yet, we are aiming to discover a cyclotide-specific interaction partner using a chemical proteomics approach.
The cyclotide analogue [T20K]kalata B1 has been derivatized with biotin for application in affinity chromatography using lymphocyte lysates in target deconvolution experiments to reveal molecular binding partners. [T20K]kB1 was further equipped with light-inducible crosslinking residues to enable photo-affinity labeling during pull down experiments. These crosslinker probes were applied for target identification using lysates from activated mouse spleenocytes of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. The identified target proteins were validated by competition pull down assays to account for non-specific interactions. Co-precipitation experiments provided further evidence for an interaction of the cyclotide and the cellular protein target. Finally, we focused to study the interaction on a molecular level and revealed insight into cellular signaling events upon cyclotide administration in the EAE mouse model. All native and modified cyclotides were characterized for their cytotoxicity, serum stability, cell penetration ability and anti-proliferative activity to provide evidence of their unaltered activity profile. In conclusion, we have developed a target deconvolution strategy for cyclotides using an interdisciplinary approach combining peptide chemistry, proteomics and cell biology. Potentially our results will aid development of new peptide-based drugs for therapeutic application in autoimmune diseases.
References:
[1] C. Gruendemann, et al., J Nat Prot., 75(2), 2012
[2] C. Gruendemann, et al., PLoS One, 8(6): e68016, 2013.