Characterization of kappa opioid receptor mediated, dynorphin-stimulated [35S]GTPγS binding in mouse striatum for the evaluation of selective KOR ligands in an endogenous setting

Abstract

Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5'GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5'GNTI, is presented using this approach.

Keywords: Drug discovery; Dynorphin; KOR antagonist; Kappa opioid receptor; Mouse striatum; Selectivity; [(35)S]GTPγS binding.

Figure 1. Dynorphin in C57 WT brain

Dynorphin peptides stimulate [³⁵S]GTPγS binding in C57Bl/6 WT mouse striatal membranes in a concentration dependent manner. (A)Dynorphin A peptides [DynA(1-17), DynA(1-13)] and (B) and Dynorphin B [DynB(1-13), DynB(1-9)] are shown in comparison to the stimulation achieved with the selective KOR agonist, U69,593. Data are presented as fold of [³⁵S]GTPγS binding over vehicle-treated conditions. Pharmacological parameters can be found in Table 1 (n = 4–9 individual assays).

Figure 2. Dynorphin in KOR-KO brain

Dynorphin peptides robustly stimulate [³⁵S]GTPγS binding in the absence of KOR. (A). KOR-KO mouse striatal membranes reveal a decrease in basal [³⁵S]GTPγS binding (Mean ± S.E.M = 72.6 ± 2.3% in KOR-KO versus 100% in WT, p < 0.0001, t-test). (B). U69,593 no longer stimulates [³⁵S]GTPγS binding in KOR-KO striatal membranes. (C–F) Dynorphin peptides (each tested is indicated in the abscissa) retain significant potency and efficacy in the absence of KOR. The data plotted in 2B is the same as plotted in figure 1 for U69,593 stimulation but on a different scale. Pharmacological parameters can be found in Table 1 (n = 4–9 individual assays); the data are plotted at the mean ± SEM.

Figure 3. KOR KO

KOR-KO striatal tissue lacks binding sites for U69,593 and MOR activity is maintained. (A) [³H]U69,593 binding assay of both wide-type and KOR knockout mice striatal tissues. Total binding was determined by the binding in the presence of vehicle and non-specific binding was determined in the presence of 10 μM cold U69,593. In the present of 2.2 – 2.5 nM [³H]U69,593, the wild-type striatal tissue show 2.7 ± 0.04 fold binding over the non-specific binding determined by addition of 10 μM U69,593 (* p <0.001, unpaired t test). The calculated receptor density bound to [³H]U69,593 is 19.1 ± 1.4 fmol/mg. In KOR-KO, striatal homogenate lacks [³H]U69,593 binding supporting that the KO mice lack KOR. (B) The MOR-selective enkephalin analog (DAMGO) retains activity KOR-KO as in WT striatal membranes implicating another potential site of action for dynorphin actions in striatum. (n = 3, data are presented as the mean ± SEM).

Figure 4. Dynorphins in MOR-KO brain

Dynorphin peptides remain potent and efficacious in MOR-KO mouse striatum. (A) Basal [³⁵S]GTPγS binding is similar between MOR-KO (Mean ± S.E.M = 92 ± 7.5%) and WT C57Bl/6 mice (100%). (B) U69,593 stimulation of [³⁵S]GTPγS binding is unaffected by the deletion of MOR. (C–F) Dynorphin peptides retain significant potency and efficacy in MOR-KO. The U69,593 stiumlation curve in WT for Figure B is the same as in Figure 1. (G) DAMGO stimulation is fully diminished in absence of MOR (curve doesn’t converge). Pharmacological parameters can be found in Table 1 (n=4–9 individual assays); the data are plotted at the mean ± SEM.

Figure 5. norBNI blocks low concentration of dynorphin signals

Dynorphin or U69,593 stimulation of [³⁵S]GTPγS binding is antagonized by 10 μM NorBNI in striatum. (A). U69,593 stimulation is fully antagonized by 10 μM norBNI. (B–E) (B–C), NorBNI does not fully block dynorphin A peptides at this concentration, (D–E) yet more efficiently competes with dynorphin B peptides. Pharmacological parameters can be found in Table 2 (n = 3–9 individual assays); the data are plotted at the mean ± SEM.

Figure 6. Antagonist competition in C57WT/KOR-KO/MOR-KO

Antagonist competition assays against 1 μM DynA(1-17) or DynB(1-9). Agonist induced responses (1 μM DynA(1-17) or DynB(1-9)) were inhibited with increasing concentrations of indicated antagonists in WT (A,B), KOR-KO (C,D), or MOR-KO (E,F) striatal preparations. All the inhibition curves are reported as the percentage of the response obtained in the absence of antagonist. Antagonist potencies (IC₅₀ values) and maximal percentages of inhibition are summarized in Table 2; (n = 3–9 individual assays); the data are plotted at the mean ± SEM.

Figure 7. Novel Antagonist competition in KOR-KO/MOR-KO

Evaluation of candidate selective KOR antagonists in striatal membranes prepared from MOR- and KOR-KO mouse striata. A. Compound 1 structure and chemical name (Frankowski et al., 2014). B. Binding affinities and K_i ratios for Compound 1 in CHO-MOR, CHO-KOR and CHO-DOR expressing CHO cell lines. Competition binding assays were performed against 2–4 nM [³H]U69,593 (KOR), 2–3 nM [³H]DAMGO (MOR) or 0.1–0.3 nM [³H]Diprenorphine (DOR). Nonspecific binding was determined by 10 μM NorBNI (KOR), Naloxone (MOR) or Naltrindole (DOR) in the cell lines from this laboratory as previously described (Zhou et al., 2013). C. Compound 1 and NorBNI displace [³H]U69,593 (2–6 nM) binding in WT C57Bl/6 mouse striatal membranes (10 μM NorBNI determining nonspecific binding). Potency values are given in the legend with mean ± SEM (n=5). D. Compound 1 partially blocks DynB(1-9) stimulated [³⁵S]GTPγS in MOR-KO striatal membrane and to a lesser extent in KOR-KO striatal membranes while NorBNI abolishes all DynB(1-9) signaling regardless of genotype (NorBNI data are from Figure 6, shown in this graph for comparison to compound 1). E. Pharmacological parameters are derived from GraphPad Prism, nonlinear regression analysis, n = 9–12; data are presented as Mean ± SEM.