Design, synthesis and biological evaluation of hepcidin analogues

Student thesis: Doctoral ThesisDoctor of Philosophy


Hepcidin is a peptide hormone involved in the control of iron homeostasis. It has 25 amino acids with an antiparallel beta-sheet structure stabilized by four disulfide bonds. Hepcidin binds to the sole known iron exporter, ferroportin, leading to its internalization and degradation by a mechanism not fully understood. Hepcidin has an important role in iron metabolism disorders, such as hemochromatosis and anaemia. However, hepcidin analogues are currently not available for clinical use. The aims of this project are twofold:
- to design and synthesise a fluorescent hepcidin analogue that can be used as a biological tool to further investigate hepcidin-ferroportin interactions;
- to design and synthesise small peptide-like hepcidin analogues which can bind and internalize ferroportin.
At the end of the thesis there is a pullout which summarises the structures of the peptides synthesised in this project.
Chapter 2 describes the synthesis of eleven linear peptides, synthesised to facilitate a structure-activity relationship study on the N-terminus of hepcidin, which is the most active part of the peptide. Chapter 3 describes the synthesis of two hepcidin analogues (15 and 19) containing intramolecular disulfides. Peptide 15 represents the N-terminus of hepcidin constrained by one intramolecular disulfide. Peptide 19 sequence contains amino acids from the N-terminus of hepcidin and from the C-terminus, which we considered to be also relevant for the binding to ferroportin. The cysteines in peptide 19 were oxidised to disulfides. Chapter 4 focuses on the synthesis of two hepcidin analogues (27 and 35) with intermolecular disulfides: peptide 27 containing one intermolecular disulfide and peptide 35 containing two. Intermolecular disulfide formation is more challenging to achieve, as oxidation of the cysteines needs to be selective. A successful approach was developed by carefully selecting the protecting groups for the thiol group of the cysteines, which were removed stepwise in order to achieve selectivity. Chapter 5 discusses the synthesis and folding of [Lys21] 6-carboxy tetramethylrhodamine (TMR) labelled hepcidin and N13 6-carboxy uorescein (CF) labelled hepcidin 20. Hepcidin sequence presents a methionine in position 21, near the C-terminus, which was replaced, in the synthesis of [Lys21] TMR hepcidin, with a lysine protected at the N with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde). This approach provides a site where TMR can be selectively attached. In N13 CF hepcidin 20 the last ve amino acids of hepcidin were not included in the synthesis and the peptide was labelled at the lysine in position 13. This peptide is not active and was synthesised as a negative control for biological evaluation purposes. Chapter 6 is divided in two sections. The first section describes the structural analysis by circular dichroism of the analogues synthesised in this project. The second section illustrates the biological evaluation results. Biological assays were performed at Vifor Pharma laboratories in Zurich. The [Lys21] TMR hepcidin was found to possess appreciable biological activity, being able to bind and internalize ferroportin with a potency only 4 fold lower than that of synthetic hepcidin.
The structure-activity relationship study, conducted with peptides mimicking the N-terminus of hepcidin, suggests that a disul de exchange may be involved in the binding between the N-terminus and ferroportin. Furthermore, between these analogues, peptides 5 and 8 were found to be able to bind ferroportin without leading to its internalisation, suggesting an interesting antagonist activity. Peptides 19 and 27 show some activity, being 189 and 13 fold less active than hepcidin 25, respectively.
Date of Award2015
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorRobert Hider (Supervisor) & Sukhi Bansal (Supervisor)

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