The discovery of benzenesulfonamide-based potent and selective inhibitors of voltage-gated sodium channel Nav1.7

The voltage gated sodium channel Na_v1.7 represents an interesting target for the treatment of pain. Human genetic studies have identified the crucial role of Na_v1.7 in pain signaling. Herein, we report the design and synthesis of a novel series of benzenesulfonamide-based Na_v1.7 inhibitors. Structural–activity relationship (SAR) studies were undertaken towards improving Na_v1.7 activity and minimizing CYP inhibition. These efforts resulted in the identification of compound 12k, a highly potent Na_v1.7 inhibitor with a thousand-fold selectivity over Na_v1.5 and negligible CYP inhibition.     

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 (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.     

Discovery of selective,orally bioavailable,N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice

The voltage-gated sodium channel Na_v1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately, drugs thought to garner efficacy via Na_v1 inhibition have undesirable side effect profiles due to their lack of selectivity over channel isoforms. Herein we report the discovery of a novel series of orally bioavailable arylsulfonamide Na_v1.7 inhibitors with high levels of selectivity over Na_v1.5, the Na_v isoform responsible for cardiovascular side effects, through judicious use of parallel medicinal chemistry and physicochemical property optimization. This effort produced inhibitors such as compound 5 with excellent potency, selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch model suggestive of target modulation.     

Benzoxazolinone aryl sulfonamides as potent,selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model

Studies on human genetics have suggested that inhibitors of the Na_v1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Na_v1.7 inhibitors with excellent selectivity against the Na_v1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Na_v1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.     

Localization of Nav1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction,pheromone sensing and immune function

Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Na_v1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Na_v1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Na_v1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Na_v1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Na_v1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Na_v1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.     

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Na_v1.5 and Na_v1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Na_v1.5 or Na_v1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Na_v1.5 and Na_v1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Na_v1.5 or Na_v1.7. The recovery from inactivation of Na_v1.5 or Na_v1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Na_v1.5 than lidocaine, but not Na_v1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Na_v1.5, as compared to Na_v1.7; and mexiletine exhibits stronger use-dependent block of Na_v1.5. The differential gating properties of Na_v1.5 and Na_v1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.     

Discovery of morpholine-based aryl sulfonamides as Nav1.7 inhibitors

Replacement of the piperidine ring in the lead benzenesulfonamide Na_v1.7 inhibitor 1 with a weakly basic morpholine core resulted in a significant reduction in Na_v1.7 inhibitory activity, but the activity was restored by shortening the linkage from methyleneoxy to oxygen. These efforts led to a series of morpholine-based aryl sulfonamides as isoform-selective Na_v1.7 inhibitors. This report describes the synthesis and SAR of these analogs.     

Discovery of new indole-based acylsulfonamide Nav1.7 inhibitors

Screening of 100 acylsulfonamides from the Bristol-Myers Squibb compound collection identified the C3-cyclohexyl indole 6 as a potent Na_v1.7 inhibitor. Replacement of the C2 furanyl ring of 6 with a heteroaryl moiety or truncation of this group led to the identification of 4 analogs with hNa_v1.7 IC₅₀ values under 50?nM. Fluorine substitution of the truncated compound 12 led to 34 with improved potency and isoform selectivity. The inverted indole 36 also maintained good activity. Both 34 and 36 exhibited favorable CYP inhibition profiles, good membrane permeability and a low efflux ratio and, therefore, represent new leads in the search for potent and selective Na_v1.7 inhibitors to treat pain.     

The discovery of benzoxazine sulfonamide inhibitors of NaV1.7: Tools that bridge efficacy and target engagement

The voltage-gated sodium channel Na_V1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of Na_V1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat Na_V1.7 versus human Na_V1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse Na_V1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.     

Investigation of the selectivity of one type of small-molecule inhibitor for three Nav channel isoforms based on the method of computer simulation

Voltage-gated sodium (Na_v) channels play a pivotal role for the changes in membrane potential and belong to large membrane proteins that compose four voltage sensor domains (VSD1–4). In this study, we describe the binding mode and selectivity of one of the aryl sulfonamide sodium channel inhibitors, PF-04856264, for the VSD4s in Na_v1.4, Na_v1.5 and Na_v1.7, respectively, through molecular dynamics simulation and enhanced post-dynamics analyses. Our results show that there are three binding site regions (BSR1–3) in the combination of the ligand and receptors, of which BSR1 and BSR3 contribute to the selectivity and affinity of the ligand to the receptor. What’s more, the 39th residue (Y39 in VSD4^hNav1.4/ VSD4^hNav1.7 and A39 in VSD4^hNav1.5) and N42 in BSR1, the 84th residue (L84 in VSD4^hNav1.4, T84 in VSD4^hNav1.5, and M84 in VSD4^hNav1.7) in BSR2 and the conserved positive charged residues in BSR3 have major contributions to the interaction between the ligand and receptor. Further analysis reveals that if the 39th residue has a benzene ring structure, the connection of BSR1 and the ligand would be much stronger through π-stacking interaction. On the other hand, the strength and number of the hydrogen bonds formed by the ligand and the conserved arginines on S4 determine the contribution of BSR3 to the total free binding energy. We anticipate this study pave the way for the design of more effective and safe treatment for pain that selectively target Na_v1.7.     

Discovery of pyrrolo-benzo-1,4-diazines as potent Nav1.7 sodium channel blockers

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized and displayed potent Na_v1.7 inhibitory activity and moderate selectivity over Na_v1.5. The syntheses, structure–activity relationships, and selected pharmacokinetic data of these analogs are described. Compound 41 displayed anti-nociceptive efficacy in the rat CFA pain model at 100 mpk oral dosing.     

Potency optimization of Huwentoxin-IV on hNav1.7: A neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNa_v1.7 (IC₅₀ determined herein as 17 ± 2 nM). Na_v1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure–function study into Na_v1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu¹, Glu⁴, Phe⁶ and Tyr³³ were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNa_v1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNa_v1.5. Peptide 47 (Gly¹, Gly⁴, Trp³³-HwTx) demonstrated the largest potency increase on hNa_v1.7 (IC₅₀ 0.4 ± 0.1 nM).     

Substituted biaryl pyrazoles as sodium channel blockers

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Na_v1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.     

A novel benzazepinone sodium channel blocker with oral efficacy in a rat model of neuropathic pain

A series of benzazepinones were synthesized and evaluated for block of Na_v1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.     

Discovery of isoxazole voltage gated sodium channel blockers for treatment of chronic pain

A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na_v1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na_v1.7 inhibitory activity.     

Bioavailable pyrrolo-benzo-1,4-diazines as Nav1.7 sodium channel blockers for the treatment of pain

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized to improve the profile of the previous lead compound 1. The syntheses, structure–activity relationships, and selected pharmacokinetic data of these analogs are described. The optimization efforts allowed the identification of 33, a quinoline amide exhibiting potent Na_v1.7 inhibitory activity and moderate selectivity over Na_v1.5. Compound 33 displayed anti-nociceptive oral efficacy in a rat CFA inflammatory pain model at 100 mpk and in a rat spinal nerve ligation neuropathic pain model with an EC₅₀ 75 μM.     

Discovery of a biarylamide series of potent,state-dependent NaV1.7 inhibitors

The Na_V1.7 ion channel has garnered considerable attention as a target for the treatment of pain. Herein we detail the discovery and structure-activity relationships of a novel series of biaryl amides. Optimization led to the identification of several state-dependent, potent and metabolically stable inhibitors which demonstrated promising levels of selectivity over Na_V1.5 and good rat pharmacokinetics. Compound 18, which demonstrated preferential inhibition of a slow inactivated state of Na_V1.7, was advanced into a rat formalin study where upon reaching unbound drug levels several fold over the rat Na_V1.7 IC₅₀ it failed to demonstrate a robust reduction in nociceptive behavior.     

Linkage between Increased Nociception and Olfaction via a SCN9A Haplotype

Background and Aims

Mutations reducing the function of Na_v1.7 sodium channels entail diminished pain perception and olfactory acuity, suggesting a link between nociception and olfaction at ion channel level. We hypothesized that if such link exists, it should work in both directions and gain-of-function Na_v1.7 mutations known to be associated with increased pain perception should also increase olfactory acuity.

Methods

SCN9A variants were assessed known to enhance pain perception and found more frequently in the average population. Specifically, carriers of SCN9A variants rs41268673C>A (P610T; n = 14) or rs6746030C>T (R1150W; n = 21) were compared with non-carriers (n = 40). Olfactory function was quantified by assessing odor threshold, odor discrimination and odor identification using an established olfactory test. Nociception was assessed by measuring pain thresholds to experimental nociceptive stimuli (punctate and blunt mechanical pressure, heat and electrical stimuli).

Results

The number of carried alleles of the non-mutated SCN9A haplotype rs41268673C/rs6746030C was significantly associated with the comparatively highest olfactory threshold (0 alleles: threshold at phenylethylethanol dilution step 12 of 16 (n = 1), 1 allele: 10.6±2.6 (n = 34), 2 alleles: 9.5±2.1 (n = 40)). The same SCN9A haplotype determined the pain threshold to blunt pressure stimuli (0 alleles: 21.1 N/m², 1 allele: 29.8±10.4 N/m², 2 alleles: 33.5±10.2 N/m²).

Conclusions

The findings established a working link between nociception and olfaction via Na_v1.7 in the gain-of-function direction. Hence, together with the known reduced olfaction and pain in loss-of-function mutations, a bidirectional genetic functional association between nociception and olfaction exists at Na_v1.7 level.     

Aquaporin-1 Tunes Pain Perception by Interaction with Nav1.8 Na+ Channels in Dorsal Root Ganglion Neurons

Aquaporin-1 (AQP1) water channels are expressed in the plasma membrane of dorsal root ganglion (DRG) neurons. We found reduced osmotic water permeability in freshly isolated DRG neurons from AQP1^−/− versus AQP1^+/+ mice. Behavioral studies showed greatly reduced thermal inflammatory pain perception in AQP1^−/− mice evoked by bradykinin, prostaglandin E₂, and capsaicin as well as reduced cold pain perception. Patch clamp of freshly isolated DRG neurons showed reduced action potential firing in response to current injections. Single action potentials after pulse current injections showed reduced maximum inward current, suggesting impaired Na_v1.8 Na⁺ function. Whole-cell Na_v1.8 Na⁺ currents in Na_v1.8-expressing ND7-23 cells showed slowed frequency-dependent inactivation after AQP1 transfection. Immunoprecipitation studies showed AQP1- Na_v1.8 Na⁺ interaction, which was verified in live cells by single-particle tracking of quantum dot-labeled AQP1. Our results implicate the involvement of AQP1 in DRG neurons for the perception of inflammatory thermal pain and cold pain, whose molecular basis is accounted for, in part, by reduced Na_v1.8-dependent membrane Na⁺ current. AQP1 is, thus, a novel target for pain management.     

Recent progress in sodium channel modulators for pain

Voltage-gated sodium channels (Na_vs) are an important family of transmembrane ion channel proteins and Na_v drug discovery is an exciting field. Pharmaceutical investment in Na_vs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Na_v structural biology pave the way for a future of fruitful prospective Na_v drug discovery.