Human native kappa opioid receptor functions not predicted by recombinant receptors: Implications for drug design

Abstract

If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues.

Figure 1

Gi activation and internalisation of κ receptors by asimadoline and ICI204448 in HEK293 cells expressing human (panels A and C) and mouse (panels B and D) κ receptors. Gi activation (panels A and B) was measured as Gi heterotrimer complex dissociation/conformational changes using bioluminescence resonance energy transfer (BRET), calculated by the ratio of emission of Venus (535 nm) to Rluc8 (480 nm) (mBRET). Receptor internalization (panels C and D) was monitored by Time-Resolved Fluorescence Energy Transfer (TR-FRET) at 37 °C, calculated as the ratio of emission of terbium cryptate (620 nm) to fluorescein (520 nm). Concentration-response curves for asimadoline and ICI204448 were obtained by measuring at steady state (80 min). For each receptor, three independent experiments were conducted for asimadoline and ICI204448, and the effects calculated as % inhibition of mBRET (Gi activation) measured at the lowest concentrations tested (normalised as 100%) and as % increase in TR-FRET ratio (internalisation) and the results expressed as means ± SD.

Figure 2. Concentration-dependent inhibition of cholinergically-mediated, EFS-evoked contractions of mouse isolated colon muscle loops.

Panels A and B show representative trace examples of ICI 204448 (A) and asimadoline (B) in the proximal colon. Panels C and D show the effect of pre-incubation of the kappa opioid receptor antagonist norbinaltorphimine 300 nM on the concentration response relationship for the inhibition of EFS evoked after contractions of ICI 204448 (C) and asimadoline (D). Data are expressed as means ± SEM; n = 3–4 each concentration.

Figure 3. Concentration-dependent inhibition of EFS-evoked contractions of human isolated colon.

Panels A and B show representative trace examples of ICI 204448 (A) and asimadoline (B). Note the slow onset of activity of asimadoline. Panels C,D show concentration response relationships for the inhibition of contractions during EFS by ICI204448 and asimadoline in ascending (C) and descending (D) human colon muscle strips. Data are expressed as means ± SEM; n = 4–5 each concentration.

Figure 4. Inhibition by ICI204448 and asimadoline of cholinergically-mediated, EFS-evoked contractions of human isolated colon in the presence of L-NAME 300 μM.

Representative trace examples are shown for ICI 204448 (panel A) and asimadoline (panel B). The concentration-response relationships for the inhibition of contractions by ICI204448 and asimadoline are shown in panel C for the ascending and in panel D for descending colon. Panel E shows the effect of pre-incubation of the kappa opioid receptor antagonist norbinaltorphimine 3–300 nM on the concentration-response relationship for the inhibition of contractions by ICI 2044448 in the descending colon. Panel F shows a linear regression of the relationship between log₁₀[norbinaltorphimine] and the dose ratio −1 (DR-1) of the EC₅₀s of ICI 204448 in the presence of 3–300 nM norbinaltorphimine. Data are expressed as means ± SEM; n = 4–6 each concentration or 3–4 each concentration in the experiments with norbinaltorphimine.

Figure 5. Receptor occupancy studies in human descending colon.

In these experiments, conducted in the presence of L-NAME 300 μM, asimadoline or vehicle was added 30 min before application of an approximately EC₈₀ concentration of ICI204448 (60 nM). The inhibition of EFS-evoked contractions caused by asimadoline alone (▲), before application of ICI204448, is shown alongside the inhibition of contractions caused by ICI204448 in the presence of asimadoline (⦁). Data are expressed as means ± SEM; n = 3–4, each concentration tested.

Figure 6. Alignment of OPRK orthologues.

Human, rat and mouse OPRK orthologues were aligned using ClustalX. Amino acid alterations in the third intracellular loop and C-terminus of OPRK were evaluated for potential impact on G-protein coupling, which is known to occur particularly at intracellular charged residues (indicated in magenta) in the 3rd Intracellular loop and C-terminal region. In the 3rd Intracellular loop region, Ala308Val is a neutral substitution in rat from an aliphatic hydrophobic residue to a tiny hydrophobic residue. In the C-terminal region in rat and mouse, Leu348Ile and Ser358Asn are preferred and neutral substitutions respectively, however both are immediately adjacent to charged residues, therefore impact on G-protein coupling cannot be excluded. Four residues across a 6 amino acid region would appear most likely to impact G-protein coupling. The residues which include Tyr369Ser (unpreferred substitution of aromatic polar to tiny polar residue), Leu370Met (preferred), Ile373Val (preferred), Asp374Gly (neutral substitution of charged polar residue to a tiny hydrophobic residue).