Stott, Lisa Alice
(2017)
Determination of agonist intrinsic efficacies at the dopamine D2 receptor.
PhD thesis, University of Nottingham.
Abstract
G protein coupled receptor agonists can be described by two parameters; affinity (ability to bind a receptor) and efficacy (ability to activate a receptor once bound). While affinity is now an accessible parameter through binding studies, agonist efficacies have historically been difficult to determine. This is due to the significant system dependence of agonist responses and is particularly true of traditional maximal response and potency measurements.
The Black and Leff operational model determines agonist affinity and efficacy estimations directly from functional data. Its measure of efficacy (τ) offers improvement over maximal response determinations but remains a system dependent parameter. More recently, the Slack and Hall operational model has been described. This model uses receptor constitutive activity to estimate the system contribution to agonist responses to derive system independent measures of intrinsic agonist efficacy. This thesis explores the use of operational models in the analysis of agonist functional responses at the dopamine D2 receptor. This receptor is an important therapeutic target in the treatment of schizophrenia and Parkinson’s disease, and has well documented receptor constitutive activity.
Maximal responses and potencies in two representative functional assays, CRE-SPAP reporter gene and receptor internalisation, were determined for a panel of ligands at the D2L receptor. In the receptor internalisation assay, agonist maximal responses and potencies were decreased, consistent with an assay of reduced receptor reserve. Applying the Black and Leff operational model to the responses of four representative agonists, in the absence and presence of the irreversible antagonist phenoxybenzamine, yielded affinity and efficacy estimations consistent with the known pharmacology of the compounds. However, experiments did not provide evidence for the receptor constitutive activity necessary to apply the Slack and Hall operational model.
As such, site-directed mutagenesis was performed to generate a constitutively active D2L receptor. T6.34R mutation increased agonist potencies and relative maximal responses consistent with a receptor more likely to adopt the active state; however, potencies of the ergo-derived compounds were selectively decreased. Mutagenesis of three key binding site serine residues examined the mode of agonist binding in both wild type (WT) and T6.34R backgrounds. Each serine mutation had differential effects on the responses of structurally distinct agonists, suggesting agonists engage specific serine residues; while S5.42 and S5.46 were required for catechol agonist responses, S5.46 was essential for the responses of the ergo-derived compounds, bromocriptine and dihydroergocristine. It was unknown whether these specific modes of binding would generate functionally similar active conformations. Generally, the differences between the effects on CRE-SPAP reporter gene and receptor internalisation assay responses were sufficiently explained by differences in receptor reserve. However, there were differential effects of serine mutations on WT and T6.34R D2L receptors, particularly the opposing effect of S5.42A on quinpirole potencies in the CRE-SPAP reporter gene assay. This suggests that WT and T6.34R D2L receptors adopt different conformations.
Finally, we have described a method to determine agonist affinity and efficacy from [35S]-GTPγS binding assays using operational models, which does not rely on irreversible antagonist treatments. Buffer Na+ substitution revealed consistent constitutive activation of the D2S receptor, but not of the D2L or D2L T6.34R receptors. Agonist responses at the D2S receptor, in the presence and absence of buffer Na+, were analysed with the Black and Leff and Slack and Hall operational models respectively, generating agonist affinity and efficacy estimations. A novel operational model, written specifically for the analysis of [35S]-GTPγS binding assays, was also examined. This model provided similar affinity derivations to the Black and Leff operational model but provided a τ value that may have reduced system dependency. Although there may be compromises in the associated experimental conditions and which receptors can be investigated, the Slack and Hall operational model can be applied to constitutively active systems to provide system independent measures of agonist intrinsic efficacy.
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