The central pore cavity binding site is formed by transmembrane segments S5 and S6 (Figure 1). Drugs are thought to access this binding site either from the intracellular side via the S6 gate or from the lipid membrane through inter-domain fenestrations (Hille, 1977), although recent evidence has suggested direct access from the extracellular side is possible (Lee et al., 2019; Nguyen et al., 2019). This location is a hub of channel interactions with drugs and neurotoxins. Notably, local anesthetics, such as flecainide (see Figure 2A) and lidocaine, bind to this region and directly block ion conduction or trap the channel in non-conducting states (Hille, 1977). Neurotoxins such as batrachotoxin and veratridine could engage in an interesting dual-action mechanism that can stabilize the open state while partially blocking ion conduction (Wang et al., 2007)^, (Craig et al., 2020). Binding at this location often exhibits state-dependent and use-dependent effects in which binding affinity is highly dependent on the membrane potentials and frequencies of stimulation protocols.

This binding site has drawn significant interest due to the clinical use of broad-spectrum small molecule sodium channel inhibitors like local anesthetics, antiarrhythmics, and antiepileptics without adverse effects. For example, FDA has approved lacosamide for partial-onset seizures (Lattanzi et al., 2015), lamotrigine for epilepsy (Nolan et al., 2016), and carbamazepine for the treatment of trigeminal neuralgia (Cruccu et al., 2008). Because these compounds are non-selective, the use cases are limited, highlighting the need for improved therapeutics. However, the high sequence homology among Na_V isoforms in the central pore cavity has created challenges for developing selective compounds targeting this site. Investigational compounds such as Vixotrigine (Convergence/Biogen) targeting Na_V1.7 (Hinckley et al., 2021), currently in phase II clinical trials for trigeminal neuralgia and small fiber neuropathy or PF-01247324 (Pfizer) (Payne et al., 2015), PF-04531083 (Pfizer) (Bagal et al., 2014) targeting Na_V1.8 are non-selective or have modest selectivity against the on-target channel. Another Na_V1.8 targeting compound, A-803467 (Abbott/Icagen) also binds to the T-type calcium channel in low micromolar affinity, within the range of therapeutic concentration (Bladen and Zamponi, 2012). Nevertheless, the central pore cavity binding site is still a tractable target even though achieving subtype selectivity could be challenging. Perhaps, this is because broad-spectrum and state-dependent compounds could still be effective and safe for local administrations or conditions with elevated neural activities such as neuropathic pain, as demonstrated by previously approved Na_V inhibitor drugs.

This binding site is located at the entrance of the selectivity filter, mainly formed by residues from the P2 helices in each Na_V channel domain (Figure 1). The binding site is well-known for different classes of marine toxins such as tetrodotoxin (TTX), saxitoxin (STX) (see Figure 2B), and μ-conotoxin (Stevens et al., 2011) that directly block sodium ions from passing through the channel. TTX has classically been used to classify Na_V channels based on their sensitivity to the toxin: Na_V1.5, Na_V1.8, Na_V1.9 are TTX resistant (TTX-R) while the others are TTX sensitive (TTX-S). Because this binding site is broadly accessible at all membrane potentials, toxin binding here exhibits minimal state-dependent effect, distinguishing this site from all other binding sites mentioned. Mutagenesis and structural studies showed TTX and STX are located deeper in the selectivity filter while μ-conotoxins interact more dominantly with residues at the entrance of the selectivity filter and the extracellular loops of the pore (Walker et al., 2012; Pan et al., 2019). Interestingly, two residues near the binding site for these toxins located on the Na_V1.7 DIII P2 helix, T1398/I1399 (hNa_V1.7 numbering), are unique among human Na_V channels making this site attractive for subtype selectivity optimization targeting Na_V1.7.

The development of selective inhibitors for this binding site has been pioneered by SiteOne Therapeutics. Using structure–activity relationship (SAR) study interrogating the T1398/I1399 positions, SiteOne Therapeutics first obtained several selective STX analogs that have >1,000-fold selectivity for Na_V1.7 over Na_V1.4 and Na_V1.6 (Mulcahy et al., 2018). Recently, a compound named ST-2262 was reported to be potent (IC₅₀ = 72 nM) with >200-fold subtype selectivity over hNa_V1.6 and >900-fold over all other hNa_V isoforms (except for hNa_V1.9, which was not tested) (Pajouhesh et al., 2020). ST-2262 was tested on non-human primate pain models and exhibited reduced sensitivity to noxious heat. More recently, the company disclosed another saxitoxin analog named ST-2530 that is potent (IC₅₀ = 25 nM) and highly selective with >500-fold over hNa_V1.1—hNa_V1.6 and hNa_V1.8 (Beckley et al., 2021). Testing in mice, the ST-2530 was analgesic in acute pain models using noxious thermal, mechanical, and chemical stimuli when administered subcutaneously. The compound also showed reversal of thermal hypersensitivity after a surgical incision on the plantar surface of the hind paw and transiently reversed mechanical allodynia in the spared nerve injury model of neuropathic pain. Another compound from SiteOne Therapeutics, ST-2427 is currently in Phase I clinical trial.

The resting VSD II binding site is formed by the extracellular S1-S2 and S3-S4 regions on VSD II (Figure 1). Notably, this site is well-known for binding of several cysteine knot spider toxins, including ProTx-II (see Figure 2C), ProTx-III, HwTx-IV, GpTx-1, and JzTx-V (Schmalhofer et al., 2008; Xiao et al., 2008; Cardoso et al., 2015; Murray et al., 2016; Moyer et al., 2018). These peptide toxins bind with a higher affinity to the resting state of VSD II and can bind with a lower affinity to the activated state of VSD II. High-affinity binding to the resting state of VSD II prevents the voltage sensor from activation upon depolarization and traps the channel in a closed state.

Several peptide toxins targeting resting VSD II binding site are at least partially Na_V1.7 selective, making them attractive for pain therapeutic developments. However, several reports have pointed out that they have limited analgesic efficacy in animal pain models, likely caused by a narrow therapeutic window (Schmalhofer et al., 2008; Liu et al., 2014; Deuis et al., 2017). The development of pain therapeutics for this binding site focuses on engineering peptides to enhance subtype selectivity targeting Na_V1.7. Using SAR and multiattribute positional scan (MAPS), Amgen used GpTx-1, a 34-residue tarantula peptide toxin that has potent activity on Na_V1.7 (IC₅₀ = 10 nM) and promising selectivity against important off-target Na_V subtypes (20-fold over Na_V1.4 (skeletal muscle) and 1,000-fold over Na_V1.5 (cardiac)) to engineer a GpTx-1 analog (named compound 71) that is nearly 10-fold more potent than the wild-type GpTx-1 (IC₅₀ = 1.6 nM) and has >1,000-fold selectivity over Na_V1.4 and Na_V1.5 (Murray et al., 2015). However, Amgen did not advance this compound for further evaluation in vivo. Amgen also used another tarantula peptide toxin, 29-residue JzTx-V, to engineer several highly selective analogs (Wu et al., 2018). The final lead peptide (named compound AM-6120) has six modifications from the wild-type toxin, including three non-canonical amino acids. AM-6120 is highly potent (IC₅₀ = 0.8 nM), has ∼ 100 folds selectivity over Na_V1.4 and >750 folds over Na_V1.5, Na_V1.6 and Na_V1.8. Testing in animal pain models showed that the compound robustly blocked histamine-induced scratching in mice after subcutaneous administration. Notably, high plasma drug concentration (100-fold over the in vitro IC₅₀) was essential to establish the pharmacological effect. Additionally, AM-6120 had no effect in a capsaicin-induced nociception pain model. The developers suggested that AM-6120 may need high exposure in plasma to achieve effective target entanglement, a problem that has been previously characterized for Na_V1.7 small molecule inhibitors. Amgen has filed related patents for GpTx-1 and JzTx-V analogs, but no further development has been disclosed.

Using a thorough positional scanning approach, Janssen produced a library of 1,500 analogs of ProTx-II, a highly potent tarantula toxin (IC₅₀ < 1 nM) and Na_V1.7 selective ( >30 folds) to identify the lead compound JNJ63955918 (Flinspach et al., 2017). The compound is less potent (IC₅₀ ∼ 10 nM) than the wild-type but has a better selectivity profile, with 100- to >1,000-fold selectivity over Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5, and Na_V1.6. In animal models, central and peripheral administration of JNJ63955918 not only cause a pharmacological insensitivity to chemical and thermal pain and induces itch, which resemble behaviors observed in Na_V1.7 knock-out in adult mice (Gingras et al., 2014). Janssen has filed a patent for ProTx-II analogs but no further development has been disclosed.

The efforts by Amgen and Janssen were mainly performed with limited structural guidance. Recently, Genentech solved the structure of Na_V1.7/Na_VAb chimera in complex with ProTx-II in resting and activated VSD II states, revealing structural determinants of toxin binding at this site (Xu et al., 2019) (Figure 2C). The structure has revealed the channel–toxin interactions in detail and confirmed the role of F813 (hNa_V1.7) in the S3 segment of VSD II as important determinant of hNa_V1.7 selectivity (Schmalhofer et al., 2008). It also revealed a minimum contribution of the S1-S2 region to the interaction with ProTx-II, leaving room for future optimization of peptides targeting this region for subtype selectivity.

The activated VSD IV binding site is formed by the extracellular S1-S2 and S3-S4 regions on VSD IV (Figure 1). A considerable development in recent years is the discovery of sulfonamide-based small molecule compounds binding to this site that have resulted in multiple selective inhibitors against Na_V1.7. These compounds bind to activated conformations of VSD IV and trap the channel in an inactivated state. Since the activation of VSD IV is required, this binding site is available at depolarized membrane potentials, thus creating state-dependent effects of binding.

Pfizer and Icagen first discovered the binding site in the effort that led to the development of selective sulfonamide compounds ICA-121431 and PF-04856264 targeting Na_V1.3 and Na_V1.7, respectively (McCormack et al., 2013). PF-04856264 is a state-dependent modulator of Na_V1.7 with a binding preference for an inactivated state. The potency of PF-04856264 measured with protocols favoring inactivated states was 28 nM for hNa_V1.7 and >10 μM for Na_V1.3 and Na_V1.5, showing promise of this binding site for Na_V channel subtype selectivity optimization (McCormack et al., 2013). Pfizer later advanced a subsequently optimized version of PF-04856264 named PF-05089771 into clinical trials. The potency of PF-05089771 was 15 nM for hNa_V1.7 with >600 fold selectivity over Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.8, and modest selectivity over Na_V1.2 ( ∼ 8 folds) and Na_V1.6 ( ∼ 11 folds) (Swain et al., 2017). PF-05089771 was studied most extensively with multiple clinical trials completed (Mulcahy et al., 2019). Notably, in a phase II randomized, double-blind study for diabetic peripheral neuropathy, PF-05089771 was less effective than the control using pregabalin, a common drug used for the disease and did not show a significant difference to the placebo (McDonnell et al., 2018). In another phase II study to assess the efficacy of increasing oral daily doses of PF-05089771 for the treatment of postoperative dental pain, the compound did not show superiority over ibuprofen which used as a control although adverse effects were similar (US National Library of Medicine, 2021). The predefined efficacy criteria were not meet and these studies did not proceed further. Several studies on similar compounds including PF-06456384 (Pfizer) and AMG8379 (Amgen) suggested that the lack of efficacy is possibly from high binding to plasma proteins, thus leading to low concentrations at the target binding site. Specifically, high unbound plasma exposure relative to in vitro potency against Na_V1.7 was necessary to achieve pharmacodynamic effects in these studies (Kornecook et al., 2017; Pero et al., 2017; Storer et al., 2017; Sun et al., 2019).

More recently, Genentech developed and optimized other sulfonamide compounds to target Na_V1.7. Notably, Genentech captured the X-ray structure of a Na_V1.7/Na_VAb chimera in complex with sulfonamide, GX-936, revealing the structural basis for sulfonamide interaction with the activated VSD IV binding site (Ahuja et al., 2015) (Figure 2D). The Na_V1.7/Na_VAb—GX-936 structure complex, together with physiological characterizations of another sibling compound, GX-674 on Na_V1.7 VSD IV, revealed critical roles of R4 gating charge on S4 and YWxxV motif on S2 for potency and selectivity, respectively. Genentech in collaboration with Xenon Pharmaceuticals then advanced two highly potent acyl sulfonamide candidates targeting Na_V1.7, GDC-0276 (IC₅₀ = 0.4 nM) and GDC-0310 (IC₅₀ = 0.6 nM) for clinical developments (Safina et al., 2021). GDC-0276has > 21 folds selectivity over hNa_V1.1, hNa_V1.2, hNa_V1.4, hNa_V1.5, hNa_V1.6 with the highest selectivity against hNa_V1.6 ( ∼ 1,200 fold) and the lowest selectivity against hNa_V1.4 ( ∼ 21 fold). GDC-0310has > 63 fold selectivity over hNa_V1.1, hNa_V1.2, hNa_V1.5, ∼ 330 fold over Na_V1.6 but only a modest ∼ 6 folds selectivity over hNa_V1.4 (Safina et al., 2021). Xenon Pharmaceuticals and Genentech have stopped the development of GDC-0276 and GDC-0310 after Phase I trials for non-disclosed reasons.

We are not aware of any related compounds currently in clinical trials, although improvements in minimizing plasma binding have been observed in a next-generation sulfonamide compound such as AMG8379 (Kornecook et al., 2017). Whether optimizing sulfonamide compounds is still a good strategy for developing pain therapeutics remains to be seen. The activated VSD IV binding site, however, has demonstrated promise for developing subtype selective Na_V channel inhibitors.

It is worth to mention that Vertex has developed a series of pyridone amide-based compounds that are selective against Na_V1.8. The compound VX-150 achieved >400-fold selectivity over other Na_V channels (Hijma et al., 2021). Another Vertex compound VX-548 is currently in phase II clinical trials for acute pain after a bunionectomy. However, binding sites for these small molecules have not been revealed yet.