Discovery of a Series of Indazole TRPA1 Antagonists

A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and <i>in vitro</i> DMPK profiles are discussed. Good <i>in vivo</i> exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds

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_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v 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_v1.7 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_v1.7 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_v1.7 splice variants.

<p>Potency of PF-05089771 across hNa_v1.7 splice variants.</p

PF-05198007 reduces the capsaicin flare response in WT, but not Na_v1.7^Nav1.8Cre 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^Nav1.8Cre (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^Nav1.8Cre (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_v1.7^Nav1.8Cre mice (D, both 1 mg/kg and 10 mg/kg, p > 0.05, ANOVA).</p

Evidence for functional Na_v1.7 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 (IC₅₀, 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

Na_v1.7 is the major TTX-sensitive Na_v channel in small diameter mDRG neurons.

<p>A. RNASeq analysis of Na_v channel mRNA from pooled small diameter mouse DRG neurons. B. Structure of PF-05198007 (4-(2-(3-amino-1H-pyrazol-4-yl)-4-(trifluoromethyl)phenoxy)-5-chloro-2-fluoro-N-(thiazol-4-yl)benzenesulfonamide C. Patch clamp data showing concentration-response relationship for PF-05198007 assessed against recombinantly expressed mouse Na_v1.7, Na_v1.6 and Na_v1.1 (IC₅₀, Slope: 5.2 nM, 1.1; 149 nM, 1.5; 174 nM, 0.7 respectively; n = 3–4 per concentration). D. Representative patch clamp current traces of peak sodium current from small diameter mouse DRG neurons in the presence of A-803467 and following concurrent application of PF-05198007 and TTX. E. Representative peak TTX-S current <i>vs</i> time plot before and after 30 nM PF-05198007 and 500 nM TTX. G. Scatter plot of cell capacitance <i>vs</i> Na_v1.7/TTX-S ratio (n = 35). Note that in every cell tested, Na_v1.7 provided the predominant TTX-S sodium conductance.</p

PF-05089771 is a potent, state-dependent and selective inhibitor of Na_v1.7.

<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_v1.7 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_v1.7 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_v1.7 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_v1.7 splice variants. IC₅₀ 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_v1.7 where n = 6–22 observations per concentration. F. Potency of PF-05089771 was assessed on orthologous channels cloned from common preclinical species. IC₅₀ 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_v1.7 subtype-selective inhibitor. Selectivity was assessed across a collection of heterologously expressed human Na_vs on PatchXpress at the unique half inactivation voltage for each channel. Hill slopes and IC₅₀ 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_v1.7 where n = 6–22 observations per concentration. Selectivity over the TTX-R Na_v1.5 and Na_v1.8 channels was greater than 1000-fold.</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), Ca²⁺ free conditions (n = 8) and TTX (500 nM; n = 6) (Data are shown ±SEM; * p < 0.05; ANOVA on Ranks).</p