T20K, an analogue of the prototypic moebius cyclotide kalata B1, effectively inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Recent work in our laboratories have explored the effect of partial or complete reduction of the cystine knot as well as the importance of the cyclic backbone upon loss of function in proliferation experiments. Cellular uptake studies have demonstrated that only peptide containing the native cyclic cystine knot motif are able to penetrate cell membranes and transfer to the cytosol of human immune cells.
Here we use NMR analysis to evaluate the three-dimensional structures of both cyclic and linear native folded peptides to elucidate structure-activity relationships. Acyclic T20K has a less rigid backbone and considerable structural changes in critical loops compared to the native cyclic T20K. Evidence is presented that the structural motif of the cyclic cystine knot governs bioactivity, transport across biological membranes, and the structural integrity of these peptides.