11 research outputs found
Subtype-selective small molecule inhibitors reveal a fundamental role for Nav1.7 in nociceptor electrogenesis, axonal conduction and presynaptic release
Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission
Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release
<div><p>Human genetic studies show that the voltage gated sodium channel 1.7 (Na<sub>v</sub>1.7) is a key molecular determinant of pain sensation. However, defining the Na<sub>v</sub>1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na<sub>v</sub>1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na<sub>v</sub>1.7’s role in nociceptor physiology. We report that Na<sub>v</sub>1.7 is the predominant functional TTX-sensitive Na<sub>v</sub> in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na<sub>v</sub>1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na<sub>v</sub>1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.</p></div
Potency of PF-05089771 across hNa<sub>v</sub>1.7 splice variants.
<p>Potency of PF-05089771 across hNa<sub>v</sub>1.7 splice variants.</p
PF-05089771 is a potent, state-dependent and selective inhibitor of Na<sub>v</sub>1.7.
<p>A. Structure of PF-05089771 (4-(2-(3-amino-1H-pyrazol-4-yl)-4-chlorophenoxy)-5-chloro-2-fluoro-N-(thiazol-4-yl)benzenesulfonamide) B. Representative PatchXpress current recordings illustrating the near-complete block following 300 nM PF-05089771 application to half-inactivated WT hNa<sub>v</sub>1.7 channels (97% ± 3%, n = 10) which was partially reversed following a 5 min washout duration. In contrast there was minimal block following application of 300 nM PF-05089771 to resting WT hNa<sub>v</sub>1.7 channels (5% ± 3%, n = 4). Inset: PatchXpress voltage protocols for half-inactivation (upper) and resting state (lower). For a full description of the voltage protocols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#sec002" target="_blank">Methods</a>. C. Block of half-inactivated WT hNa<sub>v</sub>1.7 channels (n = 6–22 per concentration) was nearly 1000-fold more potent than resting channels (n = 4–11 per concentration) (11 nM <i>vs</i> 10 μM). D. Potency of PF-05089771 was similar across hNa<sub>v</sub>1.7 splice variants. IC<sub>50</sub> values and Hill slopes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t001" target="_blank">Table 1</a>. Data points represent n = 2–9 observations per concentration. E. PF-05089771 activity is impacted by mutation of a novel interaction site and not by local anaesthetic or toxin binding sites. Data points represent n = 3–6 observations per concentration except for hNa<sub>v</sub>1.7 where n = 6–22 observations per concentration. F. Potency of PF-05089771 was assessed on orthologous channels cloned from common preclinical species. IC<sub>50</sub> values and Hill slopes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t002" target="_blank">Table 2</a>. Data points represent n = 2–28 observations per concentration. G. PF-05089771 is a selective Na<sub>v</sub>1.7 subtype-selective inhibitor. Selectivity was assessed across a collection of heterologously expressed human Na<sub>v</sub>s on PatchXpress at the unique half inactivation voltage for each channel. Hill slopes and IC<sub>50</sub> values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t003" target="_blank">Table 3</a>. Data points represent n = 3–12 observations per concentration except for hNa<sub>v</sub>1.7 where n = 6–22 observations per concentration. Selectivity over the TTX-R Na<sub>v</sub>1.5 and Na<sub>v</sub>1.8 channels was greater than 1000-fold.</p
Evidence for functional Na<sub>v</sub>1.7 in human DRG neurons.
<p>A. Representative TTX-S current traces (recorded in the presence of 1 μM A-803467 and following graded voltage steps from -110 mV to 10 mV. B. Voltage dependence of activation (red, n = 4 for each voltage) generated from the protocol described in A and steady state fast inactivation (blue) generated by conditioning 500 msec prepulses to voltages between -110 mV and +10 mV followed by a test pulse to 0 mV from a holding potential of -110 mV (n = 4 for each voltage). Both datasets are fitted with Boltzmann functions. C. Representative timecourse relationship for peak TTX-S current following the application of 100 nM PF-05089771 and 500 nM TTX. D. Concentration-response relationship for PF-05089771 block of TTX-S current (IC<sub>50</sub>, slope: 8.4 nM, 1.1; n = 3–6 per concentration) E. Example voltage traces from a current clamp recording. Single action potentials were evoked by a 20 ms suprathreshold current step at 0.1 Hz. The scale bar refers to the voltage traces whereas the start-to-start interval is 10 s. F. Summary pie charts showing that the application of 30 and 100 nM PF-05089771 resulted in action potential block in 3/7 and 5/8 DRG neurons respectively.</p
PF-05198007 increases action potential rheobase in small diameter mDRG neurons.
<p>A. Overlayed representative voltage traces in response to graded current step injections before (blue) and after PF-05198007 application (red). Current step stimulations are shown below. B. Example timecourse of change in rheobase following PF-05198007 application and washout. C. Summary bar graph, n = 8 neurons, ** p < 0.01, ANOVA. Data are shown ±SEM.</p
Potency of PF-05089771 assessed at orthologous channels from selected species.
<p>Potency of PF-05089771 assessed at orthologous channels from selected species.</p
PF-05198007 acts peripherally and centrally to influence neurotransmitter release.
<p>A. Upper: representative evoked EPSCs during control (blue) and after 30 mins PF-05198007 application (red). Lower: representative synaptically evoked action potential trace (blue) recorded in SG neurons of the dorsal horn following dorsal root stimulation. PF-05198007 (20 mins) abolished synaptically evoked action potentials (red). B. Example time course of EPSC block following PF-05198007 application to the whole preparation. C. Action potentials induced <i>via</i> current injection steps in SG neurons were not abolished by PF-05198007 (30 nM). Representative voltage traces are shown following current injection steps of -20, 0 and 50 pA before (blue traces) and after (red traces) PF-05198007 application. Line chart shows change in firing frequency (Hz) during control and after application of PF-05198007 for all neurons tested (n = 5, p > 0.05, paired t-test). D. Example time course of EPSC block following PF-05198007 application to the dorsal root alone. E. Representative EPSC traces and summary bar graph showing that the application of PF-05198007 (30 nM) to the dorsal root alone inhibited C-fibre mediated EPSCs and resulted in a significant conduction delay (n = 7, * p < 0.05; ANOVA on Ranks). F. PF-05198007 (30 nM; n = 15: 100 nM; n = 19) reduced veratridine evoked CGRP release in spinal cord synaptosomes. Reduction was compared with mexilitine (100 μM; n = 19), Ca<sup>2+</sup> free conditions (n = 8) and TTX (500 nM; n = 6) (Data are shown ±SEM; * p < 0.05; ANOVA on Ranks).</p
Activity of PF-05089771 at human voltage-gated sodium channel isoforms.
<p>Activity of PF-05089771 at human voltage-gated sodium channel isoforms.</p
PF-05198007 reduces the capsaicin flare response in WT, but not Na<sub>v</sub>1.7<sup>Nav1.8Cre</sup> mice.
<p>A, B.Time-course plots showing the effects of PF-05198007 on skin blood flow measured before and after topical capsaicin application for WT (A) and Nav1.7<sup>Nav1.8Cre</sup> (B) mice (for each genotype, n = 8 per group). C, D. Corresponding summary bar graphs showing flare response measured as area under the curve for WT (C) and Nav1.7<sup>Nav1.8Cre</sup> (D) mice before and after PF-05198007 treatment. 1 mg/kg and 10 mg/kg PF-05198007 significantly reduced capsaicin-induced flare in WT mice (C, both 1 mg/kg and 10 mg/kg, p < 0.01, ANOVA) but had no effect in Na<sub>v</sub>1.7<sup>Nav1.8Cre</sup> mice (D, both 1 mg/kg and 10 mg/kg, p > 0.05, ANOVA).</p