Eliasen, Rasmus

I dette Pd.D.-projekt blev muligheden for at grafte melanocortin receptor bindende sekvenser i det inhibitor cystine knottede peptid gurmarin undersøgt. Målet med at designe disse analoger var, at producere stabile peptid agonister med potentiale for at behandle fedme.Inhibitor cystine knottede peptider er en gruppe af peptider, som findes i forskellige planteog dyrearter. De indeholder alle seks cysteiner, som danner tre disulfider, der bestemmer deres tertiære struktur og giver dem ekseptionel proteolytisk stabilitet. Det er blevet vist, at disse peptider kan blive brugt som molekylære platformer, i hvilke peptidsekvenser af farmaceutisk interesse kan blive indsat, hvormed meget stabile lægemidler kan blive fremstillet.Melanocortinreceptorerne er en familie af fem G-protein-koblede receptorer, der findes over hele kroppen. Melanocortin 4 receptoren findes i hjernen, hvor den regulerer appetit og energiomsætning. Melanocortinreceptorerne bliver aktiveret af peptider, der indeholder en His-Phe-Arg-Trp tetrapeptidsekvens. Dermed kan inhibitor cystine knottede peptider, der har denne sekvens graftet måske blive til nye appetitregulerende peptider til behandling af fedme.Først blev en stabil metode til at syntetisere gurmarin udviklet. Peptidet blev oxideret med en oxidationsmetode, der vilkårligt danner disulfiderne ved brug af en buffer indeholdende et glutathion/cystamin redox-par. Forskellige forsøg på at bekræfte disulfid-kombinationen i det vilkårligt oxiderede gurmarin blev afprøvet. Den native disulfid-kombination af det vilkårligt oxiderede gurmarin blev til sidst bekræftet med en syntetisk metode. Denne metode brugte en kombination af thermolysin-kløvning af det vilkårligt oxiderede peptid og udviklingen af en orthogonal oxidationsmetode. I denne metode blev disulfiderne i gurmarin oxideret i rækkefølge, ved at bruge tre forskellige par cystein-beskyttelsesgrupper der gjorde selektiv disulfiddannelse muligt. Derefter blev redox-buffer oxidationsmetoden brugt til at syntetisere seks melanocortinanaloger af gurmarin. Disse peptider blev designet på baggrund af en strukturel sammenligning af gurmarin og agouti-related protein, en endogen antagonist af melanocortin receptorerne. Den C-terminale del af agouti-related protein indeholder flere disulfider ligesom det inhibitor cystine knottede gurmarin. Derudover, blev to tetradisulfidanaloger af gurmarin designet og syntetiseret baseret på sammenligningen med agouti-related protein. Melanocortinanalogerne blev karakteriseret for deres binding til melanocortinreceptorerne. Generelt blev det vist, at det er muligt at syntetisere gurmarinanaloger, der binder til melanocortinreceptorerne. Analogen med højest affinitet for melanocortin 4 receptoren havde en bindingsaffinitet på 500 nM, hvilket er omkring ti gange lavere affinitet end den endogene agonist a-melanocyte stimulating hormone. Endeligt blev det vist, at det var muligt at syntetisere melanocortinanaloger af det cykliske cystine knottede peptid kalata B1, et plantepeptid hvis to termini er cykliserede. Det blev bekræftet med NMR spektroskopi, at disse analoger havde tredimensionelle strukturer lignende kalata B1. Det blev vist at én analog bandt til melanocortin 4 receptoren med en bindingsaffinitet på 29 nM, hvilket er højere affinitet end a-melanocortin stimulating hormone. Analogen var derimod mindre potent end a-melanocortin stimulating hormone til at aktivere melanocortin 4 receptoren. Derudover blev det også vist at analogerne var meget stabile overfor proteolyse sammenlignet med a-melanocyte stimulating hormone.Alt i alt blev det bekræftet, at det er muligt at grafte melanocortin bindende sekvenser ind i cystine knottede peptider ved brug af to forskellige peptider som molekylær platform. Disse melanocortin analogers evne til at danne native strukturer blev bekræftet. Derudover blev det vist at de syntetiserede peptide kan binde til og aktivere melanocortin 4 receptoren. Yderligere var peptiderne proteolytisk stabile. Denne nye melanocortin grafting-metode har produceret nye kandidater til udviklingen af lægemidler til behandling af fedme.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

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Design, synthesis and characterisation of gurmarin and melanocortin analogues of gurmarin

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Design, synthesis and characterisation of gurmarin and melanocortin analogues of gurmarin

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