Farmer, James
(2024)
Universal sensors for identifying use-dependent intracellular allosteric modulators of G protein coupled receptors.
PhD thesis, University of Nottingham.
Abstract
G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors targeted by therapeutics. Their diverse signalling capabilities lead to involvement in a wide range of physiological and disease responses. The vast majority of molecules currently available to target GPCRs are designed to act at the binding site (the orthosteric site) for endogenous ligands, either promoting receptor signalling as agonists, or inhibiting as antagonists. However, a continuing challenge within GPCR drug discovery is to develop compounds with sufficient selectivity across closely related targets, and more nuanced mechanisms of action than classical agonism or antagonism. Ultimately these may offer both improved therapeutic efficacy, and better minimise the occurrence of on- and off-target side effects.
This challenge has given rise to rapidly advancing understanding of alternative allosteric mechanisms for GPCRs, in which molecules bind in a separate location from the orthosteric ligand to influence receptor activity. One such class are the intracellular allosteric modulators that bind at the receptor-G protein interface, thereby inhibiting effector coupling and acting as non-competitive antagonists. These types of molecules have been identified for a few GPCR family members currently (e.g. chemokine receptors), and offer advantages in tackling receptors with large orthosteric binding sites, and in emergent properties such as use dependence. Currently broader development and understanding of intracellular allosteric modulators is hindered by a lack of suitable, high-throughput, screening methods, with the majority identified via bespoke receptor functional studies. There is a need for the development of a more universally applicable screening platform for the rapid identification of intracellular allosteric modulators at the GPCR superfamily.
This thesis describes the development of a novel approach for screening ligands able to bind the GPCR intracellular binding site, employing G protein derived peptidomimetics. These are 11 – 24 amino acid peptides derived from the C-terminal tail of G protein alpha-subunits previously identified to display G protein selective inhibition of effector signalling, with the potential to also act as positive modulators of agonist binding. This G protein-based selectivity is hypothesised to provide these peptidomimetics with the ability to couple to multiple GPCRs, thereby making them putative tools in developing more universally applicable probes for the GPCR intracellular binding site.
Initially, a range of these peptides were characterised, varying in length and amino acid sequence, based on their ability to alter orthosteric agonist binding at the Gs coupled β2-adrenoceptor and Gi coupled Neuropeptide Y1 receptor. This was achieved by the development of TR-FRET binding assays to determine orthosteric agonist affinity via competition analysis, in the absence and presence of Gα C terminal peptide. Peptides were evaluated based on their ability to positively modulate agonist affinity, on the hypothesis that their binding promotes the receptor active conformation.
Selected peptides with the greatest modulatory capacity were then engineered to contain a tetramethyl rhodamine fluorophore at their N terminus and evaluated for GPCR binding directly using NanoBRET technology. NanoBRET binding studies confirmed that fluorescent Gα C peptides bound to the β2-adrenoceptor and Y1 receptor in an orthosteric agonist dependent binding mechanism, preferentially coupling to the GPCR active conformation. These studies (for example for the Gs receptor tracer TMR-Gαs19cha18) demonstrated both the use of these probes as tracers in competition binding studies to identify competitive unlabelled ligands binding at the intracellular modulator site, and also as activation sensors for the receptors to rank the potency and efficacy of orthosteric agonists. Further, proof of concept data indicated that TMR-Gαs19cha18 is a probe that can be used with advanced bioimaging techniques in conjunction with solubilised receptor lipid particles, including fluorescence correlation spectroscopy (FCS), to provide information about both binding and stoichiometry of the bound peptide-receptor complexes. These studies provided future directions for further development of labelled G protein peptidomimetic tracers to be used in FCS analysis systems.
Finally, TMR-Gαs19cha18 was employed in a small-scale screening assay to identify novel intracellular modulators of the Gs prostaglandin EP2 receptor. Utilising NanoBRET competition assays and the TMR-Gαs19cha18 probe, unlabelled small-molecule modulators were screened, allowing accurate derivation of their binding affinity at the EP2 receptor intracellular site and subsequent evaluation of ligand structure activity relationships. CD006 was identified as a novel small molecule inhibitor of the EP2 receptor, validated through employment of NanoBiT complementation assay to evaluate CD006’s functional effect on receptor--arrestin2 recruitment by PGE2.
Together, the data presented in this thesis identify G protein C-terminus mimetic peptides to be effective tools for the development of novel, broadly applicable, tracers for GPCR intracellular allosteric binding sites.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Holliday, Nicholas
Mistry, Shailesh
Laughton, Charles |
| Keywords: |
G protein-coupled receptors; Allosteric mechanisms; Allosteric modulators; G protein derived peptidomimetics |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
77576 |
| Depositing User: |
Farmer, James
|
| Date Deposited: |
16 Jul 2024 04:40 |
| Last Modified: |
16 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/77576 |
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