Protease-activated receptor-2 (PAR2) is a G-protein coupled receptor that is activated through proteolytic cleavage of the N-terminus leading to the coupling to a number of defined second messenger systems though G-protein engagement. Early signalling events such as the mobilisation of intracellular Ca2+ and downstream cascades including ERK MAP kinase mediate an array of cellular effects stimulated through PAR2. Many studies have demonstrated the involvement of PAR2 in a number of disease pathologies including arthritis, GI disorders and inflammatory pain. However, treatments for these and other conditions have been limited by the lack of potent and selective small-molecule antagonists. Recently, a number of new putative antagonists including GB88 and AZ8838 have been proposed to be effective in cells and in vivo. However, given the relative lack of information about these compounds this thesis examined the characteristics of these compounds in a number of PAR2 mediated cellular assays.In Chapter 3 it was found that 2f-LIGRLO-NH2 and trypsin activated NFÆB-transcriptional activity in a PAR2 overexpressing cell line with potencies as expected from other studies.Somewhat surprisingly, GB88 and a number of derivatives generated in-house, behaved as partial agonists compared to synthetic peptide 2f-LIGRLO-NH2 in stimulating reporter activity with reduced efficacy but moderate potency. They were largely ineffective as antagonists. In HEK293 where PAR2 expression was moderate, GB88 derivatives also stimulated the phosphorylation of ERK again with reduced efficacy and lower what compared with synthetic peptide. Moreover, intracellular calcium mobilisation mediated by PAR2 coupling to Gq/11 as determined by treatment with YM254890 was also activated by GB88 and related compounds with similar characteristics. In neither assay did GB88 act as a antagonist compound.Studies in chapter four examined the effect of the novel PAR2 antagonist, AZ8838 both as a racaemic mixture and as a pure compound. It was found to inhibit PAR2 induced NFÆB-transcriptional activity in NFÆB-Reporter cells in a concentration dependent manner confirming its identification as an allosteric modulator.AZ8838 also decreased ERK and p38 MAP kinase stimulated by either 2f-LIGRLO-NH2 or trypsin in NFÆB-Reporter cells as well as inhibiting phosphorylation of ERK in HEK293 cells. In addition, AZ8838 had the ability to reduce PAR2-mediated calcium mobilisation in a time dependent fashion with maximum inhibition observed following preincubation for 30 minutes or more. These effects were consistent for the S-AZ8838 isomer whilst R-AZ8838 is not.Taking together these studies suggest that GB88 may have different pharamcological prpoerties in different systems but that AZ8838 has the potential to be a truly breakthrough compound. If the ADMET properties of AZ8838 are good this compound could be used in clincal studies for the treatment of inflammatroy disorders.
|Date of Award||28 Jan 2019|
- University Of Strathclyde
|Supervisor||Robin Plevin (Supervisor) & Andrew Paul (Supervisor)|