| Summary: | G-protein coupled receptors (GPCRs) are one of the main targets for drug design. GPCR pharmaceutical research has bottlenecked on drug research, and the current assay systems are not creating therapeutic drugs from drug hits. The principal aim of this project was to establish a relationship between drug affinity (PKi) and drug intrinsic efficacy (Emax) using receptor conformational change parameters (measurements defined by fluorescence resonance energy transfer (FRET) experiments) to provide a new way of screening for the intrinsic efficacy of a drug. This project aimed to develop a drug screen based on the hypothesis that ligand intrinsic efficacy was linked to ligand induced receptor conformational change. Using FRET technology, the extent and rate of ligand induced receptor conformational changes were investigated. In contrast to the clear cut nature of ligand intrinsic efficacy and the extent of ligand induced receptor conformational change observed in the literature no correlation was found in this project on the FRET reporter construct A2A.4C.CFP. FRET studies revealed some key findings into the receptor activation mechanisms of the A2aR. Firstly, that different agoinsts induce different conformational states of the receptor and at different rates, thus these finding support activation models proposed by Koshland and are inline with work carried out by Kobilka. Secondly, that different agonists imposed different conformational restrictions/conditions on the resultant conformation of the second agonist applied. This finding opens up the possibility for drug design, as it is possible that the resultant conformation of a drug applied after the receptor has been activated by a previously applied agonist may result in a different pharmacological response, and hence may open up new avenues for investigation. cAMP functional studies were carried out to classify and rank the intrinsic efficacy of the test ligands and to assess effects of the mutations within the A2A.4C.CFP receptor on downstream signalling. All the ligands tested with the exception of compound 15 were reported to be full A2aR agonists in the literature. However compounds CV 1808 and 2Chloroadenosine both generated only partial responses in this assay. This finding may be reflective of differences in receptor activation mechanism of these two compounds compared to the other full agonists.
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