In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3.

Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an α2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks α2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.

Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner.

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

Ex vivo study of human visceral nociceptors.

OBJECTIVE: The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. DESIGN: Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. RESULTS: Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (-20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (-34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity. CONCLUSIONS: Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways.

KEY POINTS: Voltage-gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage-gated sodium channel subtype NaV 1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV 1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality-specific manner and help to direct drug discovery efforts towards novel visceral analgesics. ABSTRACT: Voltage-gated sodium channel NaV 1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific NaV 1.7 knockout mouse (NaV 1.7Nav1.8 ) and selective small-molecule NaV 1.7 antagonist PF-5198007. NaV 1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV 1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV 1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of NaV 1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed NaV 1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV 1.7 (in NaV 1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV 1.7 antagonist PF-5198007. Our data demonstrate that NaV 1.7 (in NaV 1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV 1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.