Design, synthesis and characterisation of novel allosteric modulators for the prostaglandin EP 2 receptor

Dalton, Constance

Design, synthesis and characterisation of novel allosteric modulators for the prostaglandin EP 2 receptor

Publication Date

July 29, 2026

Creator

Dalton, Constance

Abstract

The prostaglandin E2 (PGE2) receptor subtype 2 (EP2), a G protein-coupled receptor, is a key mediator of inflammatory response regulating pro-inflammatory cytokine expression and modulation of Th17 cell function. The EP2 receptor is reportedly upregulated in chronic inflammation and as a result its inhibition is a potential strategy for the treatment of cancer and chronic inflammatory neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Anti-inflammatory therapeutics, such as non-steroidal anti-inflammatory drugs (NSAIDs), inhibit cyclooxygenase (COX) enzymes upstream of the prostanoid synthesis pathway but are not approved by the Food and Drug Administration (FDA) for treatment of the above diseases due to numerous adverse effects associated with long term use. (Tetrahydrofuran-2-yl)methyl 2-amino-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-pyrrolo[2,3-b]quinoxaline-3-carboxylate (25a) is the first reported EP2 antagonist with a reversible agonist-dependent allosteric mode of action (Jiang et al., 2020). Compound 25a demonstrated selectivity against other prostanoid receptors: prostaglandin E2 receptor subtype 4 (EP4), prostaglandin I2 receptor (IP) and prostaglandin D2 receptor subtype 1 (DP1) and limited structure-activity relationships (SARs) of only fourteen analogues identified the 2,3-dihydrobenzo[b][1,4]dioxine motif as essential for EP2 modulation. Herein, this thesis reports the design, synthesis and pharmacological characterisation of 33 novel analogues of 25a, with a focus on modifications at the (tetrahydrofuran-2-yl) methyl ester moiety. From this SAR dataset, numerous analogues demonstrating an improvement in EP2 binding affinity and functional potency, compared to 25a, were identified. These parameters were determined utilising NanoBRET competition binding assays for the EP2 G protein binding site, and NanoBiT complementation assays reporting EP2 recruitment of beta-arrestin2, respectively. Additionally, these ligands displayed classical allosteric insurmountable antagonism in agonist concentration response curves (CRCs) with decreasing agonist maximal effect proportional to increasing concentrations of our negative allosteric modulators (NAMs). The development of a new computational model, based on the recently reported cryo-EM structure of EP2 (protein data base (PDB) 7CX2) which was evaluated using the SAR dataset, is also reported. This model supports that these ligands bind to an intracellular binding pocket on EP2 which is predicted to sterically block the recruitment of the heterotrimeric Gαs protein and thus inhibit downstream signalling. Overall, the findings presented in this thesis highlight the synthesis and pharmacological characterisation of fourteen novel NAMs of the prostanoid EP2 receptor and provides an insight into their allosteric mechanism of action.

Item Type

ethesis

Thesis Type

PhD

Supervisors

Mistry, Shailesh