A comprehensive approach to identifying repurposed drugs to treat SCN8A epilepsy.

OBJECTIVE: Many previous studies of drug repurposing have relied on literature review followed by evaluation of a limited number of candidate compounds. Here, we demonstrate the feasibility of a more comprehensive approach using high-throughput screening to identify inhibitors of a gain-of-function mutation in the SCN8A gene associated with severe pediatric epilepsy. METHODS: We developed cellular models expressing wild-type or an R1872Q mutation in the Nav 1.6 sodium channel encoded by SCN8A. Voltage clamp experiments in HEK-293 cells expressing the SCN8A R1872Q mutation demonstrated a leftward shift in sodium channel activation as well as delayed inactivation; both changes are consistent with a gain-of-function mutation. We next developed a fluorescence-based, sodium flux assay and used it to assess an extensive library of approved drugs, including a panel of antiepileptic drugs, for inhibitory activity in the mutated cell line. Lead candidates were evaluated in follow-on studies to generate concentration-response curves for inhibiting sodium influx. Select compounds of clinical interest were evaluated by electrophysiology to further characterize drug effects on wild-type and mutant sodium channel functions. RESULTS: The screen identified 90 drugs that significantly inhibited sodium influx in the R1872Q cell line. Four drugs of potential clinical interest-amitriptyline, carvedilol, nilvadipine, and carbamazepine-were further investigated and demonstrated concentration-dependent inhibition of sodium channel currents. SIGNIFICANCE: A comprehensive drug repurposing screen identified potential new candidates for the treatment of epilepsy caused by the R1872Q mutation in the SCN8A gene.

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels.

Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.

Subtype-selective Na(v)1.8 sodium channel blockers: identification of potent, orally active nicotinamide derivatives.

A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

Sodium channels and nociception: recent concepts and therapeutic opportunities.

Recent scientific advances have enhanced our understanding of the role voltage-gated sodium channels play in pain sensation. Human data on Nav1.7 show that gain-of-function mutations lead to enhanced pain while loss-of-function mutations lead to Congenital Indifference to Pain. Pre-clinical data from knockouts, anti-sense oligonucleotides, and siRNA for Nav1.3, 1.7, 1.8, and 1.9 have also demonstrated that specific subtypes of voltage-gated sodium channels play a role in different types of pain signaling. In addition, recent reports show that CNS penetration by voltage-gated sodium channel blockers is not required for efficacy in pre-clinical pain models while others have reported that identification of subtype-selective small molecules is possible. All of these data are converging to suggest next generation sodium channel blockers may offer the potential for novel pain therapies in the future.

The effect of kappa-opioid receptor agonists on tetrodotoxin-resistant sodium channels in primary sensory neurons.

BACKGROUND: A non-opioid receptor-mediated inhibition of sodium channels in dorsal root ganglia (DRGs) by kappa-opioid receptor agonists (kappa-ORAs) has been reported to contribute to the antinociceptive actions in animals and humans. In this study, we examined structurally diverse kappa-ORAs for their abilities to inhibit tetrodotoxin-resistant (TTX-r) sodium channels in adult rat DRGs. METHODS: Whole-cell recordings of TTX-r sodium currents were performed on cultured adult rat DRGs. Structurally diverse kappa-ORAs were studied for their abilities to inhibit TTX-r sodium channels. RESULTS: The racemic kappa-ORA, (+/-)U50,488, inhibited TTX-r sodium currents in a voltage-dependent manner, yielding IC(50) values of 49 and 8 muM, at prepulse potentials of -100 and -40 mV, respectively. Furthermore, we found that both the kappa-ORA U50,488 active enantiomer 1S,2S U50,488 and the inactive enantiomer 1R,2R U50,488 were equally potent inhibitors of TTX-r sodium currents. Structurally related kappa-ORAs, such as BRL 52537 and ICI 199,441 also inhibited TTX-r sodium currents. However, sodium channel inhibition and kappa-opioid receptor agonism have a distinct structure-activity relationship because another kappa-ORA (ICI 204,488) was inactive versus TTX-r sodium channels. We further investigated the sodium channel block of this class of compounds by studying (+/-)U50,488. (+/-)U50,488 was found to preferentially interact with the slow inactivated state of TTX-r sodium channels and to retard recovery from inactivation. CONCLUSION: Our results suggest that TTX-r sodium channels can be inhibited by many kappa-ORAs via an opioid receptor-independent mechanism. Although the potency for sodium channel inhibition is typically much less than apparent affinity for opioid receptors, sodium channel block may still contribute to the antinociceptive effects of this class of compounds.

Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain.

Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons and has been implicated in the pathophysiology of inflammatory and neuropathic pain. Recent studies using an Nav1.8 antisense oligonucleotide in an animal model of chronic pain indicated that selective blockade of Nav1.8 was analgesic and could provide effective analgesia with a reduction in the adverse events associated with nonselective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 5-substituted 2-furfuramides, which are potent, voltage-dependent blockers (IC50 < 10 nM) of the human Nav1.8 channel. Selected derivatives, such as 7 and 27, also blocked TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons with comparable potency and displayed >100-fold selectivity versus human sodium (Nav1.2, Nav1.5, Nav1.7) and human ether-a-go-go (hERG) channels. Following systemic administration, compounds 7 and 27 dose-dependently reduced neuropathic and inflammatory pain in experimental rodent models.

Discovery of potent furan piperazine sodium channel blockers for treatment of neuropathic pain.

The synthesis and pharmacological characterization of a novel furan-based class of voltage-gated sodium channel blockers is reported. Compounds were evaluated for their ability to block the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3) as well as the Na(v)1.2 and Na(v)1.5 subtypes. Benchmark compounds from this series possessed enhanced potency, oral bioavailability, and robust efficacy in a rodent model of neuropathic pain, together with improved CNS and cardiovascular safety profiles compared to the clinically used sodium channel blockers mexiletine and lamotrigine.

Tenidap, a novel anti-inflammatory agent, is an opener of the inwardly rectifying K+ channel hKir2.3.

We studied the effect of a novel anti-inflammatory agent, tenidap, on a cloned inwardly rectifying K+ channel, hKir2.3. Tenidap (a) potently potentiated 86Rb+ efflux through hKir2.3 channels expressed in Chinese hamster ovary cells (EC50=402 nM), (b) reversibly and dose-dependently increased whole-cell and macro-patch hKir2.3 currents (maximum whole-cell current response to tenidap was 230+/-27% of control; EC50=1.3 microM.), and (c) caused dose-dependent and Ba2+-sensitive membrane hyperpolarizations and concurrent decreases in input resistance. Potentiation of hKir2.3 by tenidap was unaffected by inhibitors of phospholipase A2, protein kinase C, or arachidonic acid metabolic pathways. The action of tenidap was not intracellular. Tenidap also had little or no effect on currents flowing through hKir2.1, Kv1.5, and micro1 Na+ channels. Our results demonstrate that tenidap is a potent opener of hKir2.3 and suggest that it can serve as a valuable pharmacological tool for studying physiological and pathological processes involving Kir2.3.

A-887826 is a structurally novel, potent and voltage-dependent Na(v)1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats.

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

Block of Nav1.8 by small molecules.

Sodium channels are key proteins in regulating neuronal excitability and accumulating data suggest that specific subtypes of voltage-dependent sodium channels are important in signaling various types of pain. Consistent with this theme, Jarvis et al.(7) recently reported the identification of a subtype-selective Na(v)1.8 blocker that was active in several pre-clinical models of pain. During the course of these studies compounds were also identified that showed large differences in potency when tested on Na(v)1.8 channels from different species. This addendum illustrates one of these compounds along with the potency correlation between recombinant and native tetrodotoxin-resistant sodium channels for additional examples. These data show that significant differences can be observed for sodium channel blockers across species and highlight the importance of considering this possibility when searching for new compounds and research tools to probe sodium channel function.

A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na(v)1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na(v)1.8 (IC(50) = 8 nM) and was >100-fold selective vs. human Na(v)1.2, Na(v)1.3, Na(v)1.5, and Na(v)1.7 (IC(50) values >or=1 microM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Decrease of inward rectification as a mechanism for arachidonic acid-induced potentiation of hKir2.3.

Previously, we showed that arachidonic acid (AA) potentiates currents flowing through a cloned human inwardly rectifying K(+) channel, hKir2.3. The mechanism by which this potentiation occurs is not understood. Here, we report that this potentiation is mediated by multiple mechanisms and that one of them, which we studied in more detail, is consistent with AA-induced decrease of inward rectification. AA (10 micro M) potentiation of hKir2.3 whole-cell current increased with depolarization (40% greater at -47 mV than at -127 mV) and decreased with elevated extracellular [K(+)] (158+/-21%, 56+/-8% and 38+/-9% in 5.4, 70 and 135 mM K(+), respectively). Hyperpolarization elicited inward currents consisting of an instantaneous and two time-dependent components with time constants (at -97 mV) of 6.4+/-1.1 ms and 27.8+/-4.1 ms, respectively. AA (10 microM) significantly decreased the slow time constant (14.1+/-0.7 ms). Consistent with the kinetic changes, AA (10 microM) right-shifted the voltage dependence of the chord conductance (mid-point shifted by +9 mV). In inside-out patches where inward rectification was minimal, AA potentiation (38+/-3%) was smaller than in whole-cell recording and was not voltage dependent. These results are consistent with the idea that AA potentiates hKir2.3 in part by decreasing inward rectification of the channel.