Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R.

J M Cottam Jones,B E Forbes,P W R Harris,A D Abell,D B Scanlon,M A Brimble

doi:10.1039/c5ob02110c

Abstract

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Highlights

Insulin-like growth factor II (IGF-II) is a 67-residue regulatory peptide that binds with high affinity to three receptors; the insulin receptor (IR), type 1 insulin-like growth factor receptor (IGF-1R), and type 2 insulin-like growth factor receptor (IGF-2R)
The six Cys residues (Cys[9], Cys[21], Cys[46], Cys[47], Cys[51] and Cys60) of the IGF’s provide suitable sites for a native chemical ligation (NCL) approach to the synthesis.[48]. The viability of such an approach to IGF-II and its analogues was first investigated with a two fragment-based synthesis of native IGF-II. This began with the synthesis of a C-terminal IGF-II (47–67) fragment by standard Fmoc-SPPS and the N-terminal IGF-II (1–46) thioester using an in situ neutralisation Boc-SPPS protocol.[49]
The fluorescence emission at 455 nm for F19Cou IGF-II in complex with the soluble form of the IGF-1R (sIGF-1R) was identical in intensity to the emission for the F19Cou IGF-II alone. These results demonstrate that fluorescence emission at 455 nm is enhanced upon F19Cou IGF-II binding to the receptor, and direct excitation (Ex 320 nm) of the coumarin residue is unaffected after ligand binding

Summary

Introduction

Insulin-like growth factor II (IGF-II) is a 67-residue regulatory peptide that binds with high affinity to three receptors; the insulin receptor (IR), type 1 insulin-like growth factor receptor (IGF-1R), and type 2 insulin-like growth factor receptor (IGF-2R). Excitation at 280 nm of F19Cou IGF-II (acceptor) in the presence of the sIGF-1R (donor) (Fig. 5A: black solid line) resulted in fluorescence emissions at 332 nm and 455 nm.

Results

Conclusion