A study on the interactions of synthetic IGF-II analogues with the type 1 IGF and insulin receptors.

Abstract

Insulin-like growth factor II (IGF-II) is a unique regulatory peptide containing 67 residues and three disulfide bonds. It binds with high affinity to three receptors, the insulin receptor (IR), the type 1 insulin-like growth factor receptor (IGF-1R) and the We 2 insulin-like growth factor receptor (IGF-2R). Binding of IGF-II to these receptors signals mitogenic responses, such as cell proliferation, differentiation and migration. The interactions of IGF-II with the IR and IGF-1R have recently been identified as potential therapeutic targets for the treatment of cancer. Thus, an increased understanding of the interactions of IGF-II with the IGF-1R and the IR-A is required for the improved design and development of potential anticancer therapeutics. A crystal structure of IGF-II bound to either the IGF-1R or the IR-A has not been reported. Thus, the precise location of IGF-II within the receptor binding pocket remains undefined. A fluorescence resonance energy transfer (FRET) approach was proposed to investigate the binding location and orientation of IGF-II within the IGF-1R. Two fluorescent IGF-II analogues, the Fl9Cou IGF-II and F28Cou IGF-II proteins, were synthesised for use in the desired FRET studies. These FRET experiments first required the synthesis of an appropriate coumarin-based probe for incorporation into IGF-II. The synthesis of a range of fluorescent coumaryl amino acids is described in Chapter 2, and an analysis of the spectroscopic properties of these coumaryl amino acids is also detailed. Site-specific incorporation of the coumarin-based probe into IGF-II was then undertaken. Three complementary methods were used for the preparation of the desired fluorescent IGFII analogues. Chapter 3 describes the use of the nonsense suppression methodology for the expression of the novel Fl9Cou IGF-II protein. This was followed by an improved chemical synthesis of the Fl9Cou IGF-II protein using a linear solid phase peptide synthesis (SPPS) approach and is detailed in Chapter 4. A robust native chemical ligation approach was developed in Chapter 5, which allowed for the facile incorporation of the coumarin-based probe at various locations within the IGF-II protein. Chapter 5 also details the synthesis of the native IGF-II, Fl9Cou IGF-II and F28Cou IGF-II proteins. The biological activity of the resultant IGF-II analogues was evaluated by competition binding assays. The fluorescent IGFII analogues bind with low nanomolar affinity to the IR and IGF-1R, and as such were deemed suitable for use in the desired FRET-based experiments. The FRET-based investigation into the binding interactions of the native IGF-II, Fl9Cou IGFII and F28Cou IGF-II proteins to the IGF-1R is described in Chapter 6. FRET interactions were observed for both the Fl9Cou IGF-II and F28Cou IGF-II proteins. The results show the fluorophore binds in close proximity to Trp residues within the IGF-1R receptor and suggest the location of IGF-II binding within the IGF-1R is consistent with what is proposed in the literature. These experiments provide a basis for further investigations for determining the precise binding location and orientation of IGF-II within the IGF-1R.