Cyclotides are plant-derived miniproteins with a macrocyclic backbone of around 30 amino acids, including six conserved cysteine residues that form a cystine knot motif [1]. Their exceptional stability and amenability to chemical synthesis via solid-phase peptide synthesis and engineering of individual intercysteine loops has attracted interest in using the cyclotide scaffold for molecular grafting studies. Indeed, cyclotide grafts were shown to potently engage with a range of molecular targets, for example G-protein coupled receptors [2].
All synthesis approaches described to date rely on efficient oxidative folding as the last step to yield the desired products. However, grafted sequences often do not fold and thus there is a need for novel synthetic strategies to fully unlock the potential of cyclotides for bioactive epitope grafting.
Here we present our recent efforts to overcome cyclotide folding by applying a modular synthesis approach. We exploit the ability of cyclotides that have individual loops removed to adapt native cyclotide-like folds and demonstrate the potential to introduce bioactive epitopes post-folding of the scaffold peptide.
As an example, we show grafting of the κ-opioid receptor ligand, the 14 amino acid long dynorphin-A onto the cyclotide kalata B1. This peptide was unable to be synthesized using traditional solid-phase peptide synthesis approaches. Using solution NMR, we confirmed the grafted peptide to adopt a native cyclotide-like fold. Pharmacological characterization revealed binding to and activation of its cognate receptor with values in the low nanomolar range. We further show the ability to apply this approach for several different intercysteine loops and different bioactive peptide epitopes.
We envisage this approach to be a valuable tool to complement the available synthetic strategies to generate cyclotide grafts. It allows access to peptides that do not readily adopt a native cyclotide-like fold and will facilitate grafting applications of ‘difficult-to-synthesize’ sequences.