GPCRs are one of the most important classes of proteins within the arena of drug discovery and therapeutics. They are involved in transmitting external stimuli across the cell membrane to elicit intracellular responses that can alter gene activation, protein synthesis and messenger signalling that can be intrinsic to health and disease. GPR119 is a GPCR shown to be involved in glucose-dependent insulin secretion with expression in the pancreatic β-cells and intestinal K- and L-cells, implicating the protein as a potential therapeutic target for type 2 diabetes. There have been many agonists published for GPR119, however none have progressed further than phase II clinical trials, largely due to lack of efficacy.
This thesis focuses on the synthesis and evaluation of chemical probes for GPR119 based on agonists developed by AstraZeneca, with differing pharmacophores exhibiting both on-target GPR119 mediated blood glucose lowering, and off-target GPR119 independent blood glucose lowering. Modifying the pharmacophores with diazirine photoreactive groups and alkyne click chemistry handles, a library of probes was synthesised. Probes exhibited a range of potencies for GPR119, with the most potent having an EC50 in the nanomolar range.
Within this thesis, the challenges of working with GPCRs are addressed: expression and identification of GPR119 protein is a difficulty encountered both within the literature and during this study. By exploring the use of epitope tags and fusion proteins, GPR119 expression was detectable using fluorescence and immunoblotting techniques. The generation of a HaloTag-GPR119 construct enabled the identification of GPR119 using proteomic methods, which has not previously been reported.
By utilising the biological tools for GPR119, evaluation of the chemical photocrosslinking probes for GPR119 was possible in cell lines over expressing HaloTag-GPR119. Dual fluorescent labelling of the protein and probe, alongside immunoblotting identified a chemical probe showing response to competition with the parent compound, presenting a potential chemical probe for GPR119.