Structure-function relationship of dynorphin B variants using naturally occurring amino acid substitutions

Abstract

Dynorphins (Dyn) represent the subset of endogenous opioid peptides with the highest binding affinity to kappa opioid receptors (KOPrs). Activation of the G-protein-coupled pathway of KOPrs has strong anticonvulsant effects. Dyn also bind to mu (MOPrs) and delta opioid receptors (DOPrs) with lower affinity and can activate the β-arrestin pathway. To fully exploit the therapeutic potential of dynorphins and reduce potential unwanted effects, increased selectivity for KOPrs combined with reduced activation of the mTOR complex would be favorable. Therefore, we investigated a series of dynorphin B (DynB) variants, substituted in one or two positions with naturally occurring amino acids for differential opioid receptor activation, applying competitive radio binding assays, GTPγS assays, PRESTO-Tango, and Western blotting on single-opioid receptor-expressing cells. Seven DynB derivatives displayed at least 10-fold increased selectivity for KOPrs over either MOPrs or DOPrs. The highest selectivity for KOPrs over MOPrs was obtained with DynB_G3M/Q8H, and the highest selectivity for KOPrs over DOPrs was obtained with DynB_L5S. Increased selectivity for KOPr over MOPr and DOPr was based on a loss of affinity or potency at MOPr and DOPr rather than a higher affinity or potency at KOPr. This suggests that the investigated amino acid exchanges in positions 3, 5, and 8 are of higher importance for binding and activation of MOPr or DOPr than of KOPr. In tests for signal transduction using the GTPγS assay, none of the DynB derivatives displayed increased potency. The three tested variants with substitutions of glycine to methionine in position 3 displayed reduced efficacy and are, therefore, considered partial agonists. The two most promising activating candidates were further investigated for functional selectivity between the G-protein and the β-arrestin pathway, as well as for activation of mTOR. No difference was detected in the respective read-outs, compared to wild-type DynB. Our data indicate that the assessment of affinity to KOPr alone is not sufficient to predict either potency or efficacy of peptidergic agonists on KOPr. Further assessment of downstream pathways is required to allow more reliable predictions of in vivo effects.

Keywords: G-protein-coupled receptors; epilepsy; functional selectivity; opioid receptors; selectivity.

FIGURE 1

Activation of G-protein and β-arrestin by WT and modified dynorphins: dose–response curves for DynA, DynB, DynB_L5S, or DynB_G3A/Q8A on hKOPr (A), hMOPr (B), and hDOPr (C) in the GTPγS assay are depicted. Dose–response curves for DynA, DynB, DynB _L5S, or DynB_G3A/Q8A on hKOPr in the PRESTO-Tango assay are depicted in (D). Data represent the mean ± SEM normalized to DynA.

FIGURE 2

Bias plots for DynB_WT, DynB_L5S, and DynB_G3A/Q8A at hKOPr are depicted in (A). Bias analysis was based on GTPγS and PRESTO-Tango assays. Data represent the mean ± SEM from three to six independent experiments. Western blot analysis of phosphorylation levels for hKOPr downstream targets after activation by DynA, DynB, DynB_G3A/Q8A, or DynB_L5S is shown in the other three panels. Quantitative analyses of the experiments are depicted for phospho-ERK 1/2 (B), phospho-p38 (C), and phospho-p70 (D). Representative images of Western blots are depicted for pERK and ERK (E), pP38 and P38 (F), and pP70 and P70 (G). Bars represent the mean ± SEM and are representative of five to eight independent experiments. *p < 0.05; **p < 0.01.