Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

<p>Abstract</p> <p>Background</p> <p>Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.</p> <p>Results</p> <p>To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE₂injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment.</p> <p>Conclusions</p> <p>These results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.</p

Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain

Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering.

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems

Temporal and sex differences in the role of BDNF/TrkB signaling in hyperalgesic priming in mice and rats

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E2 (PGE2), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (Cx3cr1CreER × Bdnfflx/flx mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain. Keywords: BDNF, Hyperalgesic priming, TrkB, Sex differences, IL

PAR2 antagonist C391

Background and purposeProteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.Experimental approachWe modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2 , to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2 ) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice.Key resultsC391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq -dependent Ca2+ , MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation.Conclusions and implicationsC391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target

Lanthanide Labeling of a Potent Protease Activated Receptor‑2 Agonist for Time-Resolved Fluorescence Analysis

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO–dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO–dtpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO–dtpa can be utilized in an affinity binding assay to evaluate the ligand–receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands

Lanthanide Labeling of a Potent Protease Activated Receptor‑2 Agonist for Time-Resolved Fluorescence Analysis

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO–dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO–dtpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO–dtpa can be utilized in an affinity binding assay to evaluate the ligand–receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands

PAR₂ ligand structures and RTCA EC₅₀.

<p>Compound number, name, structure and the <i>in vitro</i> physiological EC₅₀ (RTCA) of each compound described in the manuscript are shown for comparison. #, compound number; Name, compound name; Structure, compound structure; RTCA, xCELLigence™ real time cell analysis; EC₅₀, half maximal effective concentration; 95% CI, 95% confidence interval.</p