Synthesis of Structural and Fluorescently Labelled Peptidic Ligands for Optical Imaging of G Protein-Coupled Receptors
Creators: name: Mengjie Liu
Publisher: name: Figshare
Year: 2017
Subjects: Uncategorized

description: Fluorescence
imaging is capable of facilitating highly specific and non-invasive
investigation of cellular and molecular events both <i>in vitro</i> and <i>in vivo</i>.
Fluorescently labelled peptidic ligands are useful in studying the location,
distribution, trafficking and functions of G protein-coupled receptors (GPCRs).
Although GPCRs are popular targets in modern target-oriented drug design, many
of them have not been therapeutically exploited. An effective strategy in
preparing such ligands is to conjugate fluorophores to high-affinity and
selective peptide analogues that are derived from the endogenous peptides. This
thesis describes the synthesis and pharmacological properties of structural and
fluorescent peptide analogues that target the GPCRs of interest. Their
usefulness as receptor optical imaging agents has been verified in selected
analogues. <br>
    <br>
   Chapter 1 describes the advantages of using fluorescently
labelled peptides as optical imaging agents in studying GPCRs, and provides an
overview of the key steps and considerations in designing such peptides. To
support our ideas, a table containing representative examples of
literature-documented fluorescently labelled peptides that target GPCRs is
included. <br>
    <br>
   Chapter 2 demonstrates the application of various synthesis
strategies in preparing structural and fluorescent peptide analogues derived
from the two endogenous neuropeptides: ghrelin and kisspeptin. Specifically, we
have verified the effectiveness of standard Fmoc-based solid phase synthesis,
use of orthogonal protecting groups and fluorophore conjugation in both solid
and solution phase, which have resulted in fluorescently labelled ghrelin and
kisspeptin analogues useful in visualising their corresponding receptors. <br>
    <br>
   Chapter 3 and 4 describe utility of the verified synthesis
strategies in preparing fluorescently labelled peptides that target
neuropeptide Y (NPY) receptors. Chapter 3 presents synthesis and
pharmacological evaluation of ligands derived from the modified NPY C-terminal
9-amino acid fragment BVD-15 scaffold
(Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-NH<sub>2</sub>). Fluorescence labelling was attempted
at the 3-position via propargyloxyproline, and the 2- and 4-position via Lys or
Lys(azide). We have found that the 2-position labelled analogue [Lys(sCy5)<sup>2</sup>,
Arg<sup>4</sup>]BVD-15 exhibited Y<sub>1</sub>R antagonism and Y<sub>4</sub>R agonism, and it represents a novel
ligand useful in imaging studies of these receptors. Chapter 4 presents
synthesis and pharmacological evaluation of Y<sub>4</sub> receptor ligands derived from
the Y<sub>4</sub>R agonist BVD-74D, a dimeric peptide comprised of two
Tyr-Arg-Leu-Arg-Tyr-NH<sub>2</sub> monomers cross-linked by a 2,7-diaminosuberoyl group.
We have shown the synthesis strategies towards the two optically pure BVD-74D
stereoisomers and their structural and mono-labelled fluorescent analogues, by
exploiting cross metathesis between suitably protected allylglycine residues with
the desired stereo configuration. We have found that the (R,R)-stereoisomer
exhibited stronger Y<sub>4</sub>R affinity and agonism. Importantly, the fluorescent
analogue mono-sCy5-(R,R)-BVD-74D retained the pharmacological profiles of the
unlabelled parent compound, and represents a novel ligand useful in imaging
studies of Y<sub>4</sub>R. <br>
    <br>
   Chapter 5 summarises the achievement presented in this
thesis, and provides future directions in the relevant areas.