The mRNA Translation Inhibitor Vioprolide A Prevents Inflammatory Pain-Like Behaviour With Limited Action on Already Established Pain-Like Behaviour in Mice

Abstract

Background: Accumulating evidence indicates that pharmacological inhibition of the translational machinery is a therapeutic strategy for various diseases. However, whether inhibitors of mRNA translation might be suitable for pain therapy remains poorly understood. Here, we tested the potential analgesic effects of the natural product vioprolide A, which targets nucleolar protein 14 (NOP14) that is essential for ribosome biogenesis, in mouse models of pain.

Methods: We assessed the antinociceptive effects of vioprolide A in C57BL/6 mice using four different models: zymosan-induced peritonitis, zymosan-induced paw inflammation, complete Freund's adjuvant-induced paw inflammation and spared nerve injury. Plasma and brain levels of vioprolide A were determined in a pharmacokinetic study. Immunostaining and western blot experiments were performed to investigate the distribution and expression of NOP14 in dorsal root ganglia.

Results: Pretreatment with vioprolide A alleviated the visceral inflammatory hypersensitivity during zymosan-induced peritonitis, and it attenuated the somatic inflammatory hypersensitivity during zymosan-induced paw inflammation in a dose-dependent manner. However, treatment with vioprolide A did not affect established hypersensitivities. Pharmacokinetic measurements revealed that vioprolide A was not brain-penetrant and exhibited a short plasma half-life, which however seems to be sufficient to exert long-lasting antinociceptive effects. Tissue stainings revealed that NOP14 is expressed in a population of sensory neurons.

Conclusions: Our findings imply that vioprolide A may alleviate inflammatory nociceptive behaviours, but highlight that these effects may be limited to specific types of pain and treatment strategies.

Significance statement: The inhibitor of mRNA translation, vioprolide A, produced robust antinociception in distinct murine models of pain. This study provides evidence supporting further investigation of mRNA translation inhibitors, which attenuate pain by a novel mechanism of action that is not shared by established analgesics.

FIGURE 1

Structure of vioprolide A.

FIGURE 2

Vioprolide A reduces visceral nociceptive behaviour in a peritonitis model. (a) Illustration of the behavioural paradigm (top) and time course of the experiment (bottom). To assess the zymosan‐induced visceral nociceptive behaviour, the weight distribution changes of the front paws and hind paws were analysed using a dynamic weight bearing (DWB) device. After DWB baseline measurements, vioprolide A (VioA; 0.3 mg/kg) or vehicle (1% DMSO in 0.9% NaCl) were subcutaneously injected into the neck, and 16 h thereafter zymosan (1 mg) was intraperitoneally (i.p.) injected to induce peritonitis, followed by DWB assessment 5 h after the zymosan injection. (b) Summary graph showing that the extent of zymosan‐induced visceral nociceptive behaviour was significantly ameliorated by pretreatment with vioprolide A (t ₍₃₀₎ = 2.358, p = 0.0496, mixed‐effects analysis; n = 8–9, animals/group). Data are expressed as mean ± SEM. *p ≤ 0.05 vs. vehicle.

FIGURE 3

Vioprolide A reduces somatic inflammatory nociceptive behaviour and paw oedema. (a) Illustration of the behavioural paradigm (left) and time course of the experiment (right). To assess the zymosan‐induced somatic pain behaviour, the paw withdrawal latencies upon mechanical stimulation were determined using a dynamic plantar aesthesiometer (DPA). The paw volume was analysed using a plethysmometer. After baseline measurements, vioprolide A (VioA; 0.3, 1 or 3 mg/kg) or vehicle (1% DMSO in 0.9% NaCl) were subcutaneously injected into the neck, and 16 h thereafter zymosan (0.1 mg) was intraplantarly injected into a hindpaw. Further DPA and plethysmometer measurements were conducted during 1–74 h after the zymosan injection. (b, c) The zymosan‐induced mechanical hypersensitivity was significantly reduced after pretreatment with vioprolide A compared to vehicle in a dose‐dependent manner, as indicated in the time course of paw withdrawal latencies (b; F _3,26 = 15.44, p < 0.0001; two way repeated measures ANOVA followed by Šidák's multiple comparisons tests; *p < 0.05, VioA 3 mg/kg vs. vehicle; ***p < 0.001, VioA 3 mg/kg vs. vehicle; ^### p < 0.001, VioA 1 mg/kg vs. vehicle; n = 6–9 animals/group) and the area under the curve (c; F _3,26 = 20.55, p < 0.0001; ordinary one way ANOVA with Holm‐Šidák multiple comparison test; *p < 0.05, ****p < 0.0001 vs. vehicle; n = 6–9 animals/group). (d, e) The zymosan‐induced paw oedema was significantly reduced after pretreatment with vioprolide A compared to vehicle in a dose‐dependent manner, as indicated in the time course of paw volume (d; F _3,26 = 16.51, p < 0.0001; two‐way repeated measures ANOVA with Šidák's multiple comparisons test; *p < 0.05, VioA 3 mg/kg vs. vehicle; ****p < 0.0001, VioA 3 mg/kg vs. vehicle; ^### p < 0.001, VioA 1 mg/kg vs. vehicle; n = 6–9 animals/group) and the area under the curve (e; F _3,26 = 15.91, p < 0.0001; ordinary one way ANOVA with Holm‐Šidák multiple comparison test; **p < 0.01, ****p < 0.0001 vs. vehicle; n = 6–9 animals/group). Data are expressed as mean ± SEM.

FIGURE 4

Persisting mechanical hypersensitivity is not affected by vioprolide A. (a, b) CFA‐induced mechanical hypersensitivity. (a) Illustration of the behavioural paradigm (top) and time course (bottom). Mice were injected with 20 μL complete Freund's adjuvant (CFA) into a hindpaw. Twenty‐four hours thereafter, a mechanical hypersensitivity of the affected hindpaw (determined using a dynamic plantar aesthesiometer) was detected in all mice. Then animals were i.p. treated with vioprolide A (VioA; 0.3, 1 or 3 mg/kg; n = 6 per group), 50 mg/kg diclofenac (n = 6) or vehicle (n = 6), and the mechanical sensitivity was assessed over 24 h. (b) The time course of paw withdrawal latencies shows that none of the applied doses of vioprolide A affected the CFA‐induced mechanical hypersensitivity (F _4,25 = 1.282, p = 0.3036, two‐way repeated measures ANOVA). (c, d) Spared nerve injury (SNI)‐induced mechanical hypersensitivity. (c) Illustration of the behavioural paradigm (top) and time course (bottom). In the SNI model, neuropathic pain was induced by surgery. Fourteen days thereafter, a mechanical hypersensitivity of the affected hindpaw was detected in all mice. Then animals were i.p. treated with 0.3 mg/kg vioprolide A (n = 6) or vehicle (n = 5), and the mechanical sensitivity was assessed over 48 h. (d) The time course of paw withdrawal latencies shows that vioprolide did not affect the SNI‐induced mechanical hypersensitivity (F _1,9 = 0.0002282, p = 0.9983, two‐way repeated measures ANOVA). Data are expressed as mean ± SEM.

FIGURE 5

Distribution of the vioprolide A target NOP14 in dorsal root ganglia. (a) Immunostaining of NOP14 in lumbar dorsal root ganglia. (b) No immunoreactivity was observed by omitting the primary antibody. (c) Doublestaining of NOP14 and DAPI in DRG neurons revealed a nuclear localisation of NOP14. (d) Double immunostaining of NOP14 and NeuN demonstrates that NOP14 is expressed in neuronal nuclei. Scale bars: (a, b and d) 25 μm, (c) 5 μm.

FIGURE 6

Distribution of NOP14 in neuronal cells in cervical, thoracic and lumbar dorsal root ganglia. (a–f) Double‐immunostaining of NOP14 with peripherin (PRPH), a marker of unmyelinated C‐fibre neurons (a, c and e) and neurofilament 200 (NF200), a marker of myelinated neurons (b, d and f) were performed on different segmental locations of the DRGs. (g) Percentages of PRPH‐positive or NF200‐positive neurons that co‐express NOP14 in cervical (4858 neurons counted; n = 7 mice), thoracic (4932 neurons counted; n = 7 mice), or lumbar (5733 neurons counted; n = 7 mice) DRG neurons. Data are expressed as mean ± SEM. Scale bars: 25 μm.

FIGURE 7

Distribution of NOP14 in non‐neuronal cells in lumbar dorsal root ganglia. Double‐immunostaining of NOP14 with CD3, a marker of T cells (a), F4/80, a marker of macrophages (b) and glutamine synthetase (GS; c), a marker of satellite glial cells. Data indicate that NOP14 is localised to a fraction of CD3‐positive T cells, but not F4/80‐positive macrophages or GS‐positive satellite glial cells. Representative stainings from n = 5 mice. Scale bars: 25 μm.