Synthesis and structural studies of cyclic bioactive cysteine rich peptides

Abstract

Naturally occurring cyclic peptides have great therapeutic potential. Cyclotides are circular proteins found in the plants of the Rubiaceae, Violaceae and Cucurbitaceae families. They have an exceptional stability against chemical, enzymatic and thermal conditions due to their cyclic cystine knot (CCK) motif, which is the combination of a cyclic backbone and multiple disulfide bonds. In this thesis, kalata B5, a cyclotide from the bracelet subfamily, was synthesized and oxidized. The outcome was a misfolded product with different disulfide connectivity than the native kalata B5. The structure of this misfolded peptide was determined. It will potentially give useful information regarding factors that cause the misfolding of bracelet cyclotides. Members of the bracelet subfamily consist of up to 70% of the cyclotides. As these peptides have great potential as drug scaffolds, knowledge about the conditions that fold these cyclotides into their native fold will be essential. Cyclotides display a wide range of bioactivities, but act as a host defense against insects and microorganisms in the plants that produce them. Two cyclotides from the subfamily Möbius, named kalata B1 and kalata B2, have already been investigated for their insecticidal activity. In this report the native cyclotide kalata B5 from the bracelet subfamily was investigated for its insecticidal activity. Also presented in this thesis is the investigation of the cyclization of three analogues of conotoxin PVIIA. Conotoxins are tightly folded peptide toxins from the venom of cone snails. These peptides are small and disulfide rich and have shown to target ion channels and receptors with high specificity in the human brain and nervous system. These are important therapeutic targets and make the cyclotides very interesting as pharmacological tools, potential drugs or drug leads. However, as they are peptides they suffer from proteolytic degradation and have poor bioavailability. Cyclization of the backbone via a linker of amino acids would potentially increase their stability and hence their potential as therapeutics. A number of strategies were pursued to produce a cyclic analogue of PVIIA and a potentially successful approach was identified, but a cyclized PVIIA was not achieved yet. However, the cyclization of conotoxins has been successfully done with other conotoxins, therefore PVIIA has the potential to be cyclized. This will require some time to optimize the folding and cyclization conditions.