In this Ph.D. project the possibility of grafting melanocortin receptor binding sequences into the inhibitor cystine knotted peptide gurmarin was investigated. The goal of designing these analogues was to produce highly stable peptide agonists with potential for obesity treatment Inhibitor cystine knotted peptides are a group of peptides found in a variety of plant and animal species. They all contain six cysteines forming three disulphides which determine their tertiary structure and provide them with exceptional proteolytic stability. It has been demonstrated that these peptides can be used as molecular scaffolds into which peptide sequences of pharmaceutical interest can be incorporated, thus providing highly stable drug candidates. The melanocortin receptors are a family of five G protein-coupled receptors distributed throughout the body. The melanocortin 4 receptor is found in the brain where it regulates appetite and energy expenditure. The melanocortin receptors are activated by peptides containing a His-Phe-Arg-Trp tetrapeptide sequence. Consequently, inhibitor cystine knotted peptides in which this tetrapeptide are grafted could provide novel appetite regulating peptides for the treatment of obesity. First, a stable method to synthesise gurmarin was developed. The peptide was oxidised by a random oxidation method using a buffer containing a glutathione/cystamine redox pair. Various attempts were made to confirm the disulphide connectivity of the randomly oxidised gurmarin. The native disulphide connectivity of the randomly oxidised gurmarin was ultimately confirmed by a synthetic approach. This approach used a combination of thermolysin cleavage of the randomly oxidised peptide and the development of an orthogonal oxidation method. In this method, the disulphides in gurmarin were oxidised sequentially using three different pairs of cysteine side chain protection groups, which allowed selective disulphide formation. Secondly, the redox buffer oxidation was used to synthesise six melanocortin analogues of gurmarin. These peptides were designed based on a structural alignment of gurmarin and agouti-related protein, an endogenous antagonist of the melanocortin receptors. The C-terminal part of agouti-related protein contains multiple disulphides similar to the inhibitor cystine knot of gurmarin. In addition, two tetra-disulphide analogues of gurmarin was designed and synthesised based on the alignment with agoutirelated protein. The melanocortin analogues were characterised for their binding to the melanocortin receptors. In general, it was proved that it is possible to synthesise gurmarin analogues which bind to the melanocortin receptors. The analogue with highest affinity to the melanocortin 4 receptor had a binding affinity of 500 nM which is around ten-fold lower affinity than the endogenous agonist a-melanocyte stimulating hormone. Finally, it was demonstrated that it was possible to synthesise melanocortin analogues of the cyclic cystine knotted peptide kalata B1, a plant peptide which two termini are cyclised. These analogues were shown by NMR spectroscopy to adopt similar threedimensional folds as kalata B1. One analogue was found to bind to the melanocortin 4 receptor with a binding affinity of 29 nM, which is higher affinity than a-melanocortin stimulating hormone. However, the analogue was less potent than a-melanocortin stimulating hormone at activating the melanocortin 4 receptor. In addition, the analogues were shown to be highly resistant to proteolysis compared to a-melanocyte stimulating hormone. In summary, the possibility of grafting melanocortin binding sequences into cystine knotted peptides was confirmed using two different peptides as molecular scaffolds. The ability of these melanocortin analogues to adopt native folds was confirmed. In addition, it was demonstrated that the synthesised peptides in fact are able to bind to and activate the melanocortin 4 receptor. Moreover, the peptides were resistant to proteolysis. This novel melanocortin grafting approach provides new candidates for the development of drugs treating obesity.