NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats
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Evaluation Summary:
This exciting study reports on the characterization of a novel compound that preferentially targets Nav1.6 voltage-gated sodium channels and shows substantial activity against epilepsy associated SCN8A mutations and seizure activity in a variety of animal models. This compound and approach has significant promise to be translated into a therapeutic for individuals with treatment resistant epilepsy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target Na V 1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A -related epilepsy syndromes ( SCN8A -RES), where gain-of-function (GoF) mutations lead to excess Na V 1.6 sodium current, or other indications where Na V 1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of Na V 1.6 (IC 50 0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for Na V 1.1, 134 X for Na V 1.2, 276 X for Na V 1.7, and >583 Xfor Na V 1.3, Na V 1.4, and Na V 1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting Na V 1.6 currents, including resurgent and persistent Na V 1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where Na V 1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A- developmental epileptic encephalopathy ( SCN8A -DEE) and adult focal-onset seizures.
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Author Response:
Reviewer #2 (Public Review):
In this manuscript, Johnson Jr, et al. investigated the potency and selectivity of NBI-921352, a novel Nav1.6 blocker, on different voltage-gated sodium channel (VGSC) isoforms as well as on epileptic Nav1.6 variants. NBI-921352 exhibited exquisite selectivity against Nav1.6 channels, preferentially acting on activated channels, and inhibited tested Nav1.6 variants at similar potency except for the R1617Q, a variant that is proximal to the predicted binding site of NBI-921352. Brain slice recordings revealed that NBI-921352 effectively attenuated AP firing in excitatory pyramidal neurons, but not in inhibitory interneurons. Seizure assays in three rodent models demonstrated the protective effect of NBI-921352 on electrically induced seizures in all three models.
Nav1.6-selective …
Author Response:
Reviewer #2 (Public Review):
In this manuscript, Johnson Jr, et al. investigated the potency and selectivity of NBI-921352, a novel Nav1.6 blocker, on different voltage-gated sodium channel (VGSC) isoforms as well as on epileptic Nav1.6 variants. NBI-921352 exhibited exquisite selectivity against Nav1.6 channels, preferentially acting on activated channels, and inhibited tested Nav1.6 variants at similar potency except for the R1617Q, a variant that is proximal to the predicted binding site of NBI-921352. Brain slice recordings revealed that NBI-921352 effectively attenuated AP firing in excitatory pyramidal neurons, but not in inhibitory interneurons. Seizure assays in three rodent models demonstrated the protective effect of NBI-921352 on electrically induced seizures in all three models.
Nav1.6-selective blockers have been reported before, but their relative selectivity between Nav1.6 and Nav1.2 are not great; NBI-921352 is the first blocker that shows a high Nav1.6 selectivity over Nav1.2, making it a promising candidate for the development of therapeutics of Nav1.6-related disorders including early onset encephalopathies and mental disabilities. The study on epileptic variants of Nav1.6 further supports its potential use for the treatment on SCN8a-related diseases, which was confirmed by the seizure assays. NBI-921352 will also be a valuable pharmacological tool in VGSC-related basic research.
Despite all the wonderful work the authors have completed, there are some issues should be addressed.
First, different protocols were adopted to examine the selectivity of NBI-921352 on different VGSC isoforms. NBI-921352 is a state-dependent inhibitor, holding potential may alter the potency of NBI-921352 by changing channel activation/inactivation state, and therefore, difference in voltage-clamp protocols could introduce bias in the comparison of selectivity among VGSCs.
Second, a depolarized holding potential (-45 mV) was used in the study to determine IC50 of NBI-921352 on most VGSCs, which is uncommon under physiological conditions. The selectivity of NBI-921352 on Nav1.6 vs other VGSCs under physiological conditions could be different compared to the values reported here. It is better to hold cells at physiologically-relevant membrane potentials or using action potential waveforms derived from real AP recordings in neurons. The authors should discuss these limitations, and possible impact on their assessment of selectivity against other VGSCs in their native cellular backgrounds.
There are pros and cons to any method of determining selectivity and we acknowledge that none of them are ideal for all purposes. We chose to focus on what we refer to as “molecular selectivity,” the fundamental ability for a compound to bind to the channel and stabilize the high affinity conformation. We accomplish this by choosing voltages that promote the same fraction of channels to be in the high affinity (inactivated) state. This contrasts with “functional selectivity” that may be largely driven by the distinct state-dependence of different isoforms. Our approach avoids assumptions about what the physiologically relevant voltage is since that voltage can vary depending on the tissue or cell type. For any given isoform there may be multiple physiologically relevant voltages.
Consistent with this philosophy, we bias all the channels to be in their highest affinity state (inactivated) and then use this maximal potency to compare selectivity. At more hyperpolarized, voltages, potency for all isoforms will tend to be somewhat less. We are adding more explanation of our rationale to the text, and we are adding supplemental data giving more insight into the impact of voltage on potency.
Figure 1-figure supplement 2 shows the potency of NBI-921352 after holding at a membrane potential nearer the physiologic range (-62mV). Potency at this voltage (IC50 = 53 nM) was similar to that at fully inactivated potentials evaluated in the primary potency assay described shown in Figure 1. For this reason, we anticipate that the selectivity ratios described in the manuscript will be similar to those in physiologic conditions. A note to this effect has been added to the results section.
Third, Nav1.6 is highly expressed in Purkinje neurons and motor neurons, and plays important roles in motor system. Did the authors observe any motor impairment in the behavior studies? It would be informative to examine the effect of NBI-921352 on AP firing and resurgent currents in Purkinje neurons.
Fourth, wrong statistical test was used in the current-clamp study, and there is no description of statistical methods used for seizure assays. Please add a section of statistical analysis in Materials and Methods, and list the statistical analysis method used in each experiment.
We have reanalyzed the data in figure 4 using an AUC based analysis and this is now described in the legend and p values shown in the data transparency file. We have added statistical analysis methods to the methods and the figures legends
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Evaluation Summary:
This exciting study reports on the characterization of a novel compound that preferentially targets Nav1.6 voltage-gated sodium channels and shows substantial activity against epilepsy associated SCN8A mutations and seizure activity in a variety of animal models. This compound and approach has significant promise to be translated into a therapeutic for individuals with treatment resistant epilepsy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The study presents an impressive array of biophysical experiments showing that NBI-921352 potently inhibits inactivated Nav1.6 channels from both humans and rodents. The degree of selectivity for Nav1.6 over 6 other channel subtypes (Nav1.1-Nav1.5 and Nav1.7) is excellent. However, the compound was not tested against Nav1.8 and Nav1.9 channels. It may be unlikely that NBI-921352 would inhibit either of these as they are more divergent in the likely binding region than the other isoforms test, this is not a given and is a slight weakness. While the electrophysiological data clearly show that NBI-921352 shows voltage-dependence and likely state dependence, the study lacks an assay of use-dependence using repetitive higher frequency depolarizations. In several places it is stated that NBI-921352 preferentially …
Reviewer #1 (Public Review):
The study presents an impressive array of biophysical experiments showing that NBI-921352 potently inhibits inactivated Nav1.6 channels from both humans and rodents. The degree of selectivity for Nav1.6 over 6 other channel subtypes (Nav1.1-Nav1.5 and Nav1.7) is excellent. However, the compound was not tested against Nav1.8 and Nav1.9 channels. It may be unlikely that NBI-921352 would inhibit either of these as they are more divergent in the likely binding region than the other isoforms test, this is not a given and is a slight weakness. While the electrophysiological data clearly show that NBI-921352 shows voltage-dependence and likely state dependence, the study lacks an assay of use-dependence using repetitive higher frequency depolarizations. In several places it is stated that NBI-921352 preferentially inhibits activated (inactivated or open) channels. There is no direct assay looking at inhibition of open but not-inactivated channels, so this conclusion is potentially misleading. The data on persistent and resurgent currents adds to the strength of the gating analysis, but the statement that a preference for persistent currents is a feature of all the compounds in the Nav inhibitor class is not justified by either data presented in the study or by an appropriate reference. The three seizure models employed in mice (2) and rats (1) provide compelling evidence that NBI-921352 can substantially reduce seizure activity. It is stated that NBI-921352 is rapidly cleared in mice, but it is unclear if this is different in humans, which could limit the translational potential. The manuscript reports that NBI-921352 reduces seizure activity at substantially lower plasma concentrations than the concentration that is observed to cause behavioral signs of adverse effects in rats. Unfortunately, it is not clear what behaviors were monitored and therefore these data are not easily evaluated. As Nav1.6 is the major isoform found at nodes of Ranvier in myelinated fibers, including motor neurons axons, NBI-921352 might be expected to motor activity at some concentrations.
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Reviewer #2 (Public Review):
In this manuscript, Johnson Jr, et al. investigated the potency and selectivity of NBI-921352, a novel Nav1.6 blocker, on different voltage-gated sodium channel (VGSC) isoforms as well as on epileptic Nav1.6 variants. NBI-921352 exhibited exquisite selectivity against Nav1.6 channels, preferentially acting on activated channels, and inhibited tested Nav1.6 variants at similar potency except for the R1617Q, a variant that is proximal to the predicted binding site of NBI-921352. Brain slice recordings revealed that NBI-921352 effectively attenuated AP firing in excitatory pyramidal neurons, but not in inhibitory interneurons. Seizure assays in three rodent models demonstrated the protective effect of NBI-921352 on electrically induced seizures in all three models.
Nav1.6-selective blockers have been reported …
Reviewer #2 (Public Review):
In this manuscript, Johnson Jr, et al. investigated the potency and selectivity of NBI-921352, a novel Nav1.6 blocker, on different voltage-gated sodium channel (VGSC) isoforms as well as on epileptic Nav1.6 variants. NBI-921352 exhibited exquisite selectivity against Nav1.6 channels, preferentially acting on activated channels, and inhibited tested Nav1.6 variants at similar potency except for the R1617Q, a variant that is proximal to the predicted binding site of NBI-921352. Brain slice recordings revealed that NBI-921352 effectively attenuated AP firing in excitatory pyramidal neurons, but not in inhibitory interneurons. Seizure assays in three rodent models demonstrated the protective effect of NBI-921352 on electrically induced seizures in all three models.
Nav1.6-selective blockers have been reported before, but their relative selectivity between Nav1.6 and Nav1.2 are not great; NBI-921352 is the first blocker that shows a high Nav1.6 selectivity over Nav1.2, making it a promising candidate for the development of therapeutics of Nav1.6-related disorders including early onset encephalopathies and mental disabilities. The study on epileptic variants of Nav1.6 further supports its potential use for the treatment on SCN8a-related diseases, which was confirmed by the seizure assays. NBI-921352 will also be a valuable pharmacological tool in VGSC-related basic research.
Despite all the wonderful work the authors have completed, there are some issues should be addressed.
First, different protocols were adopted to examine the selectivity of NBI-921352 on different VGSC isoforms. NBI-921352 is a state-dependent inhibitor, holding potential may alter the potency of NBI-921352 by changing channel activation/inactivation state, and therefore, difference in voltage-clamp protocols could introduce bias in the comparison of selectivity among VGSCs.
Second, a depolarized holding potential (-45 mV) was used in the study to determine IC50 of NBI-921352 on most VGSCs, which is uncommon under physiological conditions. The selectivity of NBI-921352 on Nav1.6 vs other VGSCs under physiological conditions could be different compared to the values reported here. It is better to hold cells at physiologically-relevant membrane potentials or using action potential waveforms derived from real AP recordings in neurons. The authors should discuss these limitations, and possible impact on their assessment of selectivity against other VGSCs in their native cellular backgrounds.
Third, Nav1.6 is highly expressed in Purkinje neurons and motor neurons, and plays important roles in motor system. Did the authors observe any motor impairment in the behavior studies? It would be informative to examine the effect of NBI-921352 on AP firing and resurgent currents in Purkinje neurons.
Fourth, wrong statistical test was used in the current-clamp study, and there is no description of statistical methods used for seizure assays. Please add a section of statistical analysis in Materials and Methods, and list the statistical analysis method used in each experiment.
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