Naphthylisoquinoline alkaloids, a new structural template inhibitor of Nav1.7 sodium channel.

Voltage-gated sodium channel 1.7 (Nav1.7) remains one of the most promising drug targets for pain relief. In the current study, we conducted a high-throughput screening of natural products in our in-house compound library to discover novel Nav1.7 inhibitors, then characterized their pharmacological properties. We identified 25 naphthylisoquinoline alkaloids (NIQs) from Ancistrocladus tectorius to be a novel type of Nav1.7 channel inhibitors. Their stereostructures including the linkage modes of the naphthalene group at the isoquinoline core were revealed by a comprehensive analysis of HRESIMS, 1D, and 2D NMR spectra as well as ECD spectra and single-crystal X-ray diffraction analysis with Cu Kα radiation. All the NIQs showed inhibitory activities against the Nav1.7 channel stably expressed in HEK293 cells, and the naphthalene ring in the C-7 position displayed a more important role in the inhibitory activity than that in the C-5 site. Among the NIQs tested, compound 2 was the most potent with an IC50 of 0.73 ± 0.03 µM. We demonstrated that compound 2 (3 µM) caused dramatical shift of steady-state slow inactivation toward the hyperpolarizing direction (V1/2 values were changed from -39.54 ± 2.77 mV to -65.53 ± 4.39 mV, which might contribute to the inhibition of compound 2 against the Nav1.7 channel. In acutely isolated dorsal root ganglion (DRG) neurons, compound 2 (10 µM) dramatically suppressed native sodium currents and action potential firing. In the formalin-induced mouse inflammatory pain model, local intraplantar administration of compound 2 (2, 20, 200 nmol) dose-dependently attenuated the nociceptive behaviors. In summary, NIQs represent a new type of Nav1.7 channel inhibitors and may act as structural templates for the following analgesic drug development.

Diversity-oriented synthesis of marine polybrominated diphenyl ethers as potential KCNQ potassium channel activators.

Natural polybrominated diphenyl ethers, often isolated from marine sponges, have been reported to possess various biological activities, such as antibacterial, antioxidant and antidiabetic effects. Via a high throughput screening of our marine natural product library, the polybrominated diphenyl ether 3 was found to display a KCNQ potassium channel activation effect. To obtain more compound 3 related natural products and their derivatives for further bioactivity study, a diversity-oriented synthesis was conducted, leading to the successful synthesis of five polybrominated diphenyl ether natural products (1-4, 6) and 30 new derivatives. Compound 3 was found to preferentially potentiate KCNQ1 potassium channel, whereas 17h relatively activated KCNQ2 potassium channel. The structure-activity relationship was analyzed assisted by molecular docking and 17h was further conducted for its agonistic mechanism study on KCNQ2 channel. This research work may give an insight for the discovery of marine polybrominated diphenyl ether derived new drug leads.

SCN8A epileptic encephalopathy mutations display a gain-of-function phenotype and divergent sensitivity to antiepileptic drugs.

De novo missense mutations in SCN8A gene encoding voltage-gated sodium channel NaV1.6 are linked to a severe form of early infantile epileptic encephalopathy named early infantile epileptic encephalopathy type13 (EIEE13). The majority of the patients with EIEE13 does not respond favorably to the antiepileptic drugs (AEDs) in clinic and has a significantly increased risk of death. Although more than 60 EIEE13-associated mutations have been discovered, only few mutations have been functionally analyzed. In this study we investigated the functional influences of mutations N1466T and N1466K, two EIEE13-associated mutations located in the inactivation gate, on sodium channel properties. Sodium currents were recorded from CHO cells expressing the mutant and wide-type (WT) channels using the whole-cell patch-clamp technique. We found that, in comparison with WT channels, both the mutant channels exhibited increased window currents, persistent currents (INaP) and ramp currents, suggesting that N1466T and N1466K were gain-of-function (GoF) mutations. Sodium channel inhibition is one common mechanism of currently available AEDs, in which topiramate (TPM) was effective in controlling seizures of patients carrying either of the two mutations. We found that TPM (100 µM) preferentially inhibited INaP and ramp currents but did not affect transient currents (INaT) mediated by N1466T or N1466K. Among the other 6 sodium channel-inhibiting AEDs tested, phenytoin and carbamazepine displayed greater efficacy than TPM in suppressing both INaP and ramp currents. Functional characterization of mutants N1466T and N1466K is beneficial for understanding the pathogenesis of EIEE13. The divergent effects of sodium channel-inhibiting AEDs on INaP and ramp currents provide insight into the development of therapeutic strategies for the N1466T and N1466K-associated EIEE13.

(-)-Naringenin 4',7-dimethyl Ether Isolated from Nardostachys jatamansi Relieves Pain through Inhibition of Multiple Channels.

(-)-Naringenin 4',7-dimethyl ether ((-)-NRG-DM) was isolated for the first time by our lab from Nardostachys jatamansi DC, a traditional medicinal plant frequently used to attenuate pain in Asia. As a natural derivative of analgesic, the current study was designed to test the potential analgesic activity of (-)-NRG-DM and its implicated mechanism. The analgesic activity of (-)-NRG-DM was assessed in a formalin-induced mouse inflammatory pain model and mustard oil-induced mouse colorectal pain model, in which the mice were intraperitoneally administrated with vehicle or (-)-NRG-DM (30 or 50 mg/kg) (n = 10 for each group). Our data showed that (-)-NRG-DM can dose dependently (30~50 mg/kg) relieve the pain behaviors. Notably, (-)-NRG-DM did not affect motor coordination in mice evaluated by the rotarod test, in which the animals were intraperitoneally injected with vehicle or (-)-NRG-DM (100, 200, or 400 mg/kg) (n = 10 for each group). In acutely isolated mouse dorsal root ganglion neurons, (-)-NRG-DM (1~30 µM) potently dampened the stimulated firing, reduced the action potential threshold and amplitude. In addition, the neuronal delayed rectifier potassium currents (IK) and voltage-gated sodium currents (INa) were significantly suppressed. Consistently, (-)-NRG-DM dramatically inhibited heterologously expressed Kv2.1 and Nav1.8 channels which represent the major components of the endogenous IK and INa. A pharmacokinetic study revealed the plasma concentration of (-)-NRG-DM is around 7 µM, which was higher than the effective concentrations for the IK and INa. Taken together, our study showed that (-)-NRG-DM is a potential analgesic candidate with inhibition of multiple neuronal channels (mediating IK and INa).

Discovery of aryl sulfonamide-selective Nav1.7 inhibitors with a highly hydrophobic ethanoanthracene core.

Nav1.7 channels are mainly distributed in the peripheral nervous system. Blockade of Nav1.7 channels with small-molecule inhibitors in humans might provide pain relief without affecting the central nervous system. Based on the facts that many reported Nav1.7-selective inhibitors contain aryl sulfonamide fragments, as well as a tricyclic antidepressant, maprotiline, has been found to inhibit Nav1.7 channels, we designed and synthesized a series of compounds with ethanoanthracene and aryl sulfonamide moieties. Their inhibitory activity on sodium channels were detected with electrophysiological techniques. We found that compound 10o potently inhibited Nav1.7 channels stably expressed in HEK293 cells (IC50 = 0.64 ± 0.30 nmol/L) and displayed a high Nav1.7/Nav1.5 selectivity. In mouse small-sized dorsal root ganglion neurons, compound 10o (10, 100 nmol/L) dose-dependently decreased the sodium currents and dramatically suppressed depolarizing current-elicited neuronal discharge. Preliminary in vivo experiments showed that compound 10o possessed good analgesic activity: in a mouse visceral pain model, administration of compound 10o (30-100 mg/kg, i.p.) effectively and dose-dependently suppressed acetic acid-induced writhing.

Antiepileptic geissoschizine methyl ether is an inhibitor of multiple neuronal channels.

Geissoschizine methyl ether (GM) is an indole alkaloid isolated from Uncaria rhynchophyll (UR) that has been used for the treatment of epilepsy in traditional Chinese medicine. An early study in a glutamate-induced mouse seizure model demonstrated that GM was one of the active ingredients of UR. In this study, electrophysiological technique was used to explore the mechanism underlying the antiepileptic activity of GM. We first showed that GM (1-30 µmol/L) dose-dependently suppressed the spontaneous firing and prolonged the action potential duration in cultured mouse and rat hippocampal neurons. Given the pivotal roles of ion channels in regulating neuronal excitability, we then examined the effects of GM on both voltage-gated and ligand-gated channels in rat hippocampal neurons. We found that GM is an inhibitor of multiple neuronal channels: GM potently inhibited the voltage-gated sodium (NaV), calcium (CaV), and delayed rectifier potassium (IK) currents, and the ligand-gated nicotinic acetylcholine (nACh) currents with IC50 values in the range of 1.3-13.3 µmol/L. In contrast, GM had little effect on the voltage-gated transient outward potassium currents (IA) and four types of ligand-gated channels (γ-amino butyric acid (GABA), N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainite (AMPA/KA receptors)). The in vivo antiepileptic activity of GM was validated in two electricity-induced seizure models. In the maximal electroshock (MES)-induced mouse seizure model, oral administration of GM (50-100 mg/kg) dose-dependently suppressed generalized tonic-clonic seizures. In 6-Hz-induced mouse seizure model, oral administration of GM (100 mg/kg) reduced treatment-resistant seizures. Thus, we conclude that GM is a promising antiepileptic candidate that inhibits multiple neuronal channels.

Inhibitory effects of lappaconitine on the neuronal isoforms of voltage-gated sodium channels.

Lappaconitine (LA) has been widely used for postoperative and cancer pain control. LA exhibits excellent analgesic activity with a longer effective time than common local anesthetics such as tetracaine and bupivacaine. However, the mechanisms underlying the featured analgesic activity of LA remain largely unknown. Here, we report that LA is an inhibitor of voltage-gated sodium channel 1.7 (Nav1.7) stably expressed in human embryonic kidney (HEK293) cells. LA inhibited Nav1.7 in a voltage-dependent manner with an IC50 value (with 95% confidence limits) of 27.67 (15.68-39.66) µmol/L when the cell was clamped at -70 mV. In comparison with the quick and reversible inhibition of Nav1.7 by tetracaine and bupivacaine, the inhibitory effect of LA was rather slow and irreversible. It took more than 10 min to achieve steady-state inhibition when LA (300 µmol/L) was administered. Unlike tetracaine and bupivacaine, LA affected neither the voltage-dependent activation nor the inactivation of the channels. Five residues in domain III and domain IV have been reported to be critical for the effects of the two local anesthetics on Nav channels. But our mutant study revealed that only two residues (F1737, N1742) located in domain IV were necessary for the inhibitory activity of LA. The slow onset, irreversibility, and lack of influence on channel activation and inactivation accompanied with the different molecular determinants suggest that LA may inhibit Nav1.7 channels in a manner different from local anesthetics. These results may help to understand the featured analgesic activity of LA, thus benefiting its application in the clinic and future drug development.

Enhancing inactivation rather than reducing activation of Nav1.7 channels by a clinically effective analgesic CNV1014802.

The Nav1.7 channel represents a promising target for pain relief. In the recent decades, a number of Nav1.7 channel inhibitors have been developed. According to the effects on channel kinetics, these inhibitors could be divided into two major classes: reducing activation or enhancing inactivation. To date, however, only several inhibitors have moved forward into phase 2 clinical trials and most of them display a less than ideal analgesic efficacy, thus intensifying the controversy regarding if an ideal candidate should preferentially affect the activation or inactivation state. In the present study, we investigated the action mechanisms of a recently clinically confirmed inhibitor CNV1014802 using both electrophysiology and site-directed mutagenesis. We found that CNV1014802 inhibited Nav1.7 channels through stabilizing a nonconductive inactivated state. When the cells expressing Nav1.7 channels were hold at 70 mV or 120 mV, the half maximal inhibitory concentration (IC50) values (with 95% confidence limits) were 1.77 (1.20-2.33) and 71.66 (46.85-96.48) µmol/L, respectively. This drug caused dramatic hyperpolarizing shift of channel inactivation but did not affect activation. Moreover, CNV1014802 accelerated the onset of inactivation and delayed the recovery from inactivation. Notably, application of CNV1014802 (30 µmol/L) could rescue the Nav1.7 mutations expressed in CHO cells that cause paroxysmal extreme pain disorder (PEPD), thereby restoring the impaired inactivation to those of the wild-type channel. Our study demonstrates that CNV1014802 enhances the inactivation but does not reduce the activation of Nav1.7 channels, suggesting that identifying inhibitors that preferentially affect inactivation is a promising approach for developing drugs targeting Nav1.7.

Development of Novel Alkoxyisoxazoles as Sigma-1 Receptor Antagonists with Antinociceptive Efficacy.

A novel series of sigma (σ) receptor ligands based on an alkoxyisoxazole scaffold has been designed and synthesized. Preliminary receptor binding assays identified highly potent (Ki < 1 nM) and selective σ1 ligands devoid of binding interactions with the monoamine transporters DAT, NET, and SERT. In particular, compound 53 was shown to possess significant antinociceptive activity in the mouse formalin-induced inflammation pain model when administered intraperitoneally at 40 and 80 mg/kg. Initial pharmacokinetics evaluation indicated an excellent brain exposure following oral dosing in mice, suggesting that further investigation into the use of alkoxyisoxazoles as σ1 ligands for antinociception is warranted. This study supports the notion that selective σ1 antagonism could be a useful strategy in the development of novel antipain therapy.