Identification and characterization of novel candidate compounds targeting 6- and 7-transmembrane μ-opioid receptor isoforms

Abstract

Background and purpose: The μ-opioid receptor (μ receptor) is the primary target for opioid analgesics. The 7-transmembrane (TM) and 6TM μ receptor isoforms mediate inhibitory and excitatory cellular effects. Here, we developed compounds selective for 6TM- or 7TM-μ receptors to further our understanding of the pharmacodynamic properties of μ receptors.

Experimental approach: We performed virtual screening of the ZINC Drug Now library of compounds using in silico 7TM- and 6TM-μ receptor structural models and identified potential compounds that are selective for 6TM- and/or 7TM-μ receptors. Subsequently, we characterized the most promising candidate compounds in functional in vitro studies using Be2C neuroblastoma transfected cells, behavioural in vivo pain assays using various knockout mice and in ex vivo electrophysiology studies.

Key results: Our virtual screen identified 30 potential candidate compounds. Subsequent functional in vitro cellular assays shortlisted four compounds (#5, 10, 11 and 25) that demonstrated 6TM- or 7TM-μ receptor-dependent NO release. In in vivo pain assays these compounds also produced dose-dependent hyperalgesic responses. Studies using mice that lack specific opioid receptors further established the μ receptor-dependent nature of identified novel ligands. Ex vivo electrophysiological studies on spontaneous excitatory postsynaptic currents in isolated spinal cord slices also validated the hyperalgesic properties of the most potent 6TM- (#10) and 7TM-μ receptor (#5) ligands.

Conclusion and implications: Our novel compounds represent a new class of ligands for μ receptors and will serve as valuable research tools to facilitate the development of opioids with significant analgesic efficacy and fewer side-effects.

Keywords: 6TM-μ receptor; 7TM-μ receptor; opioid; opioid receptor isoform; pain.

FIGURE 1

Tier-based strategy for virtual drug screening. We initiated the screening campaign by exploring the ability of 350,000 representative entries of the ZINC Drug Now repository (containing ~11 million commercially available compounds) to bind to the 6TM-μ receptor (6-TM-MOR) and 7TM-μ receptor (7-TM-MOR). The initial low number (i.e. 10) of independent docking attempts allowed the identification of best classes of putative binders. The isolated entries underwent two more exhaustive tiers of independent docking calculations (i.e. 100 and 500 attempts) that were designed to filter out putative hits with favourable binding energy towards μ receptor isoforms (additional details in the Methods section). We were able to purchase a total of 22 compounds that were retrieved from the top lists of (i) 6TM-, (ii) 7TM-selective, and (iii) non-isoform-selective docking solutions

FIGURE 2

Chemical structures of compounds 5, 10, 11 and 25

FIGURE 3

Dose-dependent NO release for (a–d) compounds 5, 10, 11 and 25 in Be2C cells transfected with 7TM-μ receptors (7TM-MOR), 6TM-μ receptors (6TM-MOR), β₂-adrenoceptors (β₂-AR) or 6TM-MOR+β₂-AR. (a) Compound 5 (n = 4, 4, 3 and 4 per dose tested in 7TM-MOR, 6TM-MOR, β₂-AR and 6TM-MOR/β₂-AR transfected cell line, respectively) induced a dose-dependent release of NO in 7TM-MOR transfected cells only. Compounds (b) 10 (n = 3, 4, 3 and 4 per dose tested in 7TM-MOR, 6TM-MOR, β₂-AR and 6TM-MOR/β₂-AR transfected cell line, respectively), (c) 11 (n = 3, 4, 3 and 4 per dose tested in 7TM-MOR, 6TM-MOR, β₂-AR and 6TM-MOR/β₂-AR transfected cell line, respectively) and (d) 25 (n = 3 for log doses −8.0, −7.0 and −6.0 per transfected cell line and n = 3, 5, 3 and 5 in 7TM-MOR, 6TM-MOR, β₂-AR and 6TM-MOR/β₂-AR transfected cell lines, respectively, for log doses −5.0, −4.3 and −4.0) induced a dose-dependent release of NO in 6TM-MOR transfected cells, and this effect was potentiated by co-transfection with β₂-AR. None of the compounds released NO when the cells were transfected only with β₂-AR. Data expressed as mean ± SEM

FIGURE 4

Antagonistic effects of the non-selective opioid receptor antagonist, naloxone, and the selective β₂-adrenoceptor (β₂-AR) antagonist, ICI 118,551, on NO-release induced by compounds 5, 10, 11 and 25. (a) NO release induced by 10 μM compound 5 (n = 5, 3 and 4 for vehicle, naloxone and ICI 118,551, respectively) in 7TM-MOR transfected cells was abolished by pretreatment with 10 μM naloxone but not by ICI 118,551. (b–d) NO release induced by 10 μM compounds (b) 10, (c) 11 or (d) 25 (n = 5, 4 and 4 for vehicle, naloxone and ICI 118,551 per transfected cell line per compound) was blocked by pretreatment with 10 μM ICI 118,551 in cells transfected with 6TM-MOR alone or together with β₂-AR. Data expressed as mean ± SEM

FIGURE 5

Temporal changes in the mean ± SEM ΔTWL values assessed using the tail-withdrawal assay (47°C). Subcutaneous administration of compound (a) 5 (n = 7, 8 and 7 respectively for 10, 20 and 40 mgkg⁻¹) (b) 10 (n = 7, 8 and 7, respectively for 10, 20 and 40 mgkg⁻¹) (c) 11 (n = 7, 10 and 7, respectively for 10, 20 and 40 mgkg⁻¹) and (d) 25 (n = 7, 10 and 7, respectively for 10, 20 and 40 mgkg⁻¹) but not vehicle (n = 10), produced significant dose-dependent temporal changes in the thermal hyperalgesia of naïve adult C57BL/6 mice. In contrast, administration of compounds (e) 22 and 28 (n = 7 per compound) did not elicit any temporal changes in the ΔTWL values, relative to vehicle (n = 4). (f) Dose response curves (DRCs) for each of the compounds tested in the tail-withdrawal nociceptive assay. Data presented as mean ± SEM. *P < 0.05, significantly different from vehicle (20% PEG); two-way ANOVA, with post hoc Bonferroni test

FIGURE 6

Effect of low-dose naloxone and ICI 118,551 on thermal hyperalgesia induced by compounds (a) 5, (b) 10, (c) 11 and (d) 25 in naïve adult C57BL/6 mice. The temporal changes in the ΔTWL values induced by s.c. administration of ~ED₇₅ dose of (a) compound 5 (n = 8, 7 and 7 for vehicle, naloxone and ICI 118,551, respectively) was significantly reversed by i.p. administration of low dose naloxone (1 mgkg⁻¹), but not ICI 118,551 (5 mgkg⁻¹). In contrast, thermal hyperalgesia induced by s.c. administration of ~ED₇₅ doses of compounds (b) 10, (c) 11 and (d) 25 were significantly attenuated by i.p. administration of ICI 118,551 (5 mgkg⁻¹; n = 7 per test compound), but not the non-selective opioid antagonist, naloxone (1 mgkg⁻¹; n = 7 per test compound), relative to vehicle (n = 7 per test compound). Data presented as mean ± SEM. *P < 0.05, significantly different, ns, non-significantly different, as indicated; two-way ANOVA with post hoc Bonferroni test

FIGURE 7

Pharmacological characterization of compounds (a) 5, (b) 10, (c) 11 and (d) 25 in adult male and female naïve Oprm1^−/− (n = 8, 8, 6 and 6 for compounds 5, 10, 11 and 25, respectively), Oprd1^−/− (n = 7, 7, 6 and 7 for compounds 5, 10, 11 and 25, respectively), Oprk1^−/− (n = 9, 9, 6 and 9 for compounds 5, 10, 11 and 25, respectively), Adrb1/2^−/− (n = 8, 8, 6 and 8 for compounds 5, 10, 11 and 25, respectively) and C57BL/6J mice (n = 8, 8, 7 and 10 for compounds 5, 10, 11 and 25, respectively). Homozygous ablation of Oprd1 or Oprk1 did not significantly affect the thermal hyperalgesia induced by ~ED₇₅ doses of compounds 5, 10, 11 and 25 compared with wild-type C57BL/6 mice. In contrast, subcutaneous administration of an ~ED₇₅ dose of all compounds - 5, 10, 11 and 25 - failed to produce thermal hyperalgesia in Oprm1^−/− mice lacking both 6- and 7-TM MOR. Homozygous ablation of Adrb1 and Adrb2 only partly restored the thermal hypersensitivity induced by compounds 10, 11 and 25, but not the hyperalgesic effects of compound 5. Data presented as mean ± SEM. *P < 0.05, significantly different from WT C57BL/6 mice; two-way ANOVA with post hoc Bonferroni test

FIGURE 8

Compounds 10 and 5 enhanced spontaneous excitatory transmission in spinal lamina II neurons of adult male mice (n = 6–7 neurons per group; individual values are shown). (a) Recordings of sEPSCs in the absence and presence of compound 10. (b) Recordings of sEPSCs in the absence and presence of compound 5. (c and d) Mean ± SEM changes in the sEPSCs frequency (left) and amplitude (right) before and under the action of compound (c) 10 or (d) 5. The duration of drug superfusion is shown by a horizontal bar above the chart recording, and two consecutive traces of sEPSCs for a period indicated by a short bar below the chart recording are shown in an expanded time scale. V_H = −70 mV. *P < 0.05, significantly different as indicated; Student’s t-test