Design and synthesis of novel fluorescent allosteric modulators for the prostaglandin EP2 receptor

The prostaglandin E2 receptor (EP2R) is an important target involved in chronic inflammation and cancer and belongs to the family of G protein-coupled receptors (GPCRs). GPCRs are complex and flexible membrane-bound proteins comprising ~800 family members. The EP2R is activated by endogenous lipid hormones, called prostanoids through their binding to the orthosteric binding site on the receptor. However, recently, for a handful of GPCRs - most recently the EP2R, a topographically distinct 'intracellular allosteric' binding site has been discovered (Jiang et al, 2020).

To date, only a single small molecule has been reported which is predicted to bind to this site and which exhibits intriguing pharmacology. Whilst an agonist ligand such as prostaglandin E2 is required to enable binding of the allosteric ligand, the consequence is a reduction in the efficacy of the orthosteric agonist. Thus the ligand is a negative allosteric modulator (NAM), whose effects may have therapeutic benefits in controlling inflammatory responses.

There is limited information about how this class of allosteric ligands interacts with GPCRs and influences signalling to functionally different effectors (G proteins or arrestins). For example, we now know that binding of G proteins and arrestins to the GPCR intracellular domain exploits different molecular footprints, with the intracellular modulator binding sites positioned at this GPCR-effector interface. Better structure activity relationships (SARs) at this position thus have potential to reveal new ligand classes - for example "biased" modulators that selectively regulate G protein or arrestin interaction to tune the functional prostanoid response. However, the development of good allosteric SAR is often limited by the poor range of compound analogues and the lack of suitable probe ligands to monitor binding at the target site directly.

To explore allosteric SAR at the EP2R, the student will therefore develop fluorescent high affinity ligands, and use them to monitor ligand binding and kinetics by our established methods (Sykes et al 2017). In conjunction with receptor modelling studies this will advance our understanding of the nature of GPCR allostery at this site, and how allosteric ligands influence both prostanoid messenger and effector protein binding in the three-partner activated GPCR complex. In due course, these data will also inform future drug discovery projects focused on EP2R allosteric ligands.

Specific aims of the project will be:
1. To design, synthesis and characterise novel fluorescent allosteric ligands for the EP2R
2. To develop TR-FRET based competition binding kinetic assays using these fluorescent modulators to probe EP2R modulator SAR directly (Sykes et al 2017).
3. To develop models of modulator- EP2R interaction and use these to understand the allosteric regulation of prostanoid and effector binding.
4. To pharmacologically characterise novel compound pharmacology using EP2R signalling assays, and the effects of receptor mutants as informed by modelling studies

This chemical biology-focused project will span the disciplines of synthetic chemistry, modelling and pharmacology to increase our understanding of EP2R biology. With increasing numbers of allosteric ligands being discovered for the wider GPCR family, the results will be of direct relevance to many other important receptors.