Understanding the polypharmacology of Proteinase-activated receptor (PAR) pepducins and investigating their biophysical association using mass spectrometry imaging (MSI) technologies

• Published in: Proceedings for Annual Meeting of The Japanese Pharmacological Society Vol. WCP2018; p. OR26-5
• Main Author: Cunningham, Margaret R
• Format: Journal Article
• Language: English
• Published: Japanese Pharmacological Society 2018
• Subjects: G protein-coupled receptor (GPCR); Pepducins; Proteinase-activated receptors (PARs)
• ISSN: 2435-4953; 2435-4953
• DOI: 10.1254/jpssuppl.WCP2018.0_OR26-5

Background: Conventional antiplatelet therapies consist of drugs that target surface proteins called G protein-coupled receptors (GPCRs), Competitive antagonists, such as Vorapaxar, which inhibits proteinase-activated receptor-1 (PAR1), has been associated with severe bleeding [1]. High affinity, coupled with high potency, long half-life and slow elimination rates are thought to be contributing factors for the adverse haematological impairment [2]. This has led to efforts to design reversible ligands that inhibit PAR1 activity. Peptide-mimetic approaches, namely pepducins such as PZ-128 [3], has entered into phase II clinical trials as a promising PAR1 antiplatelet strategy [4]. In an effort to develop pepducins for the second thrombin receptor, PAR4, off-target effects of PZ-128 were identified during comparative target validation. The focus of the investigation was thereafter diverted to explore the polypharmacology of PZ-128. Methods and Results: Live cell confocal imaging of BODIPY-TMR PZ128 revealed that the ligand accumulates into endosomes inside the cell (HEK293 cells, 30μM, 30-60mins). During the same timeframe, significant cellular blebbing and ballooning is evident. Such cellular events have previously been associated with the coordination of procoagulative events in platelets [5]. Further pharmacological characterisation of PZ-128 in human coronary artery endothelial cells (HCAECs) has revealed dose-dependent calcium activity (LogEC50 -4.892, n=4) with responses sustained (up to 30mins) compared to calcium flux induced by the PAR1 endogenous ligand thrombin (approx. 2mins). Other pathways including MAP kinases were similarly activated when tested by Western blotting, albeit at later time frames (HEK293 cells, 30-60mins). Studies in mouse platelets, which do not possess PAR1, revealed similar sustained calcium flux in response to PZ-128 (LogEC50 -4.339, n=4). Conclusions: This data conflicts with the original 'membrane tethering' theory proposed for the mechanism of action of pepducins. Further work is currently under way using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to unravel the interplay between PZ-128 and its cellular targets. Investigations conducted to date strongly suggest that the activity observed is independent of PAR1, thus giving rise to the potential of PZ-128 having multiple targets.