Thesis

Molecular characterisation and preclinical evaluation of a small molecule protease-activated receptor 2 (PAR2) antagonist in a prostate inflammation model

Downloadable Content

Download PDF
Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17553
Person Identifier (Local)
  • 201864768
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Aim: Protease-activated receptor 2 (PAR2), a G protein-coupled receptor (GPCR) activated by endogenous proteases, plays a key role in inflammation and pain. AZ8838, a potent small-molecule PAR2 antagonist, has shown strong inhibition of PAR2-triggered intracellular signalling, although its comprehensive inhibitory profile remains unclear. Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common, yet poorly understood condition characterised by persistent pelvic pain and prostate inflammation, with limited insight into the underlying molecular mechanisms. This study aimed to characterize the PAR2 inhibitory effects of AZ8838 using in vitro cellular assays and investigate the involvement of PAR2 in prostate inflammation and pain using an in vivo carrageenan-induced prostatitis (CIP) model. Methods: AZ8838 was characterised in vitro in NCTC2544-hPAR2/NF-κB-L, DU145 and HEK293 cells. Assays included NF-κB luciferase reporter activity, p65 nuclear translocation (immunofluorescence), ERK phosphorylation (Western blotting), intracellular Ca²⁺ assay and IL-8 release (ELISA). Receptor internalisation was assessed in DU145 cells transfected with PAR2-YFP. In the in vivo study, localised prostate inflammation and pelvic pain were induced in male Wistar rats by intraprostatic injection of 3% λ-carrageenan. Histological and molecular analyses of prostate and dorsal root ganglia (DRG) tissues were performed using haematoxylin and eosin (H&E) staining, immunofluorescence, qRT-PCR, and Western blotting to evaluate PAR2 expression and localisation. Pain behaviour was assessed using von Frey filaments (mechanical allodynia) and the Hargreaves test (thermal hyperalgesia). Results: In this project, AZ8838 showed significant inhibition of trypsin- and 2fLIG-induced PAR2 cellular responses, including NF-κB reporter activity (IC50 = 0.06 μM; 95% CI 0.002 – 0.136 for trypsin, and IC50 = 0.38 μM; 95% CI 0.26 – 0.52 for 2fLIG), p65 nuclear translocation, ERK phosphorylation activity (IC50 = 15.54 μM; 95% CI 9.8 – 29.74 for trypsin, and IC50 = 9.83 μM; 95% CI 6.82 – 14.6 for 2fLIG), receptor internalisation, intracellular calcium mobilisation activity (IC50 = 0.78 μM; 95% CI 0.38 – 1.32 for 2fLIG), in a concentration-dependent manner in most experiments. In NCTC2544-hPAR2/NF-κB-L, AZ8838 significantly inhibited 2fLIG-induced IL-8 release (IC50 = 0.63 μM; 95% CI 0.15 – 1.0), whereas neither trypsin nor 2fLIG stimulated IL-8 release in DU145 prostate cells. In case of TNF-α induced NF-κB reporter activity, AZ8838 did not show the same inhibitory effect. For in vivo studies, PAR2 was significantly upregulated at the protein level in prostate tissue of CIP rats, whereas its mRNA expression was paradoxically downregulated, suggesting potential post-transcriptional regulation. Interestingly, no significant changes in PAR2 expression were observed in dorsal root ganglia (DRG), despite the pronounced pain behaviours. AZ8838 pretreatment significantly attenuated mechanical allodynia and thermal hyperalgesia. Histological analyses showed a weak reduction in prostate inflammation and leukocyte infiltration. Conclusion: Overall, both the in vitro and in vivo studies results presented in this thesis provide a detailed PAR2 inhibition profile of AZ8838 and evidence for the involvement of PAR2 in prostate inflammation and pain, suggesting the therapeutic potential of PAR2 antagonism as a drug target in inflammatory pain conditions. Future studies are warranted to explore the broader mechanistic implications of PAR2 antagonism and the interaction of PAR2 with other key inflammatory and pain mediators relevant to CP/CPPS.
Advisor / supervisor
  • Plevin, Robin
  • McIntosh, Kathryn
Resource Type
DOI

Relations

Items

Thumbnail Title Date Uploaded Visibility Actions
file details PDF of thesis T17553 2025-12-17 Public Download