Design, synthesis and pharmacological evaluation of novel fluorescent ligands for the mu-opioid receptor

Marshall, William (2021) Design, synthesis and pharmacological evaluation of novel fluorescent ligands for the mu-opioid receptor. PhD thesis, University of Nottingham.

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The mu opioid receptor (MOR) belongs to the superfamily of G-protein coupled receptors (GPCRs) and remains an important target in the management of pain and addiction, with an emerging role in promoting tumour growth. Understanding of the complex role of the MOR in these signalling pathways would be aided by further study of receptor-ligand interactions. Development of fluorescent ligands to target the MOR may provide the necessary tools to study such receptor pharmacology and localisation in healthy and diseased tissue.

Previously described high affinity fluorescent MOR ligands have been unsuitable for confocal imaging studies due to high levels of non-specific interactions with the cellular membrane. The introduction of amino acid-based linker moieties to separate the orthostere and fluorophore of fluorescent ligands has been reported to improve receptor binding affinity, receptor subtype selectivity and the confocal imaging properties of fluorescent ligands for various GPCRs.

This thesis describes the development of novel fluorescent MOR ligands based upon the opioid antagonists naltrexone and alvimopan which also contain amino acid-based linkers. Evaluation of the reported SAR of small molecule opioid receptor ligands was used to inform orthostere selection and location of linker attachment. Different amino acid linker compositions were investigated through the synthesis and evaluation of MOR binding affinity of non-fluorescent congeners in a series of TR-FRET competition binding assays. Coupling of the optimised congeners to red-emitting fluorophores (BODIPY 630/650 or sulfo-Cy5) afforded nine amino acid-linked fluorescent ligands for MOR. Assessment of MOR binding affinity of the fluorescent ligands was achieved in TR-FRET saturation binding assays.

Investigation of the linker composition of β-naltrexamine-based ligands did not identify any significant differences in MOR binding affinity between non-fluorescent congeners containing different amino acid linkers. However, a subsequent series of BODIPY 630/650-containing fluorescent ligands were identified to possess sub-nanomolar MOR binding affinities (pKD = 9.20-9.58).

Non-fluorescent derivatives of (3R,4R)-3,4-dimethyl-4-(3-hydroxyphenyl) piperidine displayed improved MOR binding affinity when a phenylalanine moiety was bound via an N-propanoate, but further elaboration of the linker was found not to improve binding further. High affinity fluorescent ligands for the MOR containing the BODIPY 630/650 fluorophore were once more identified utilising this design approach (pKD = 8.14-8.47).

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Kellam, Barrie
Mistry, Shailesh
Briddon, Stephen
Scammells, Peter
Keywords: Design, Synthesis, Pharmacological evaluation, Novel fluorescent ligands, mu-opioid receptor
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 66118
Depositing User: Marshall, William
Date Deposited: 31 Dec 2021 04:40
Last Modified: 31 Dec 2021 04:40

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