A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels.

ETHNOPHARMACOLOGY RELEVANCE: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. AIM OF THE STUDY: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. MATERIALS AND METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 µM and 30.19 ± 2.07 µM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 µM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 µM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. CONCLUSION: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses.

Major depressive disorder, a prevalent and severe psychiatric condition, necessitates development of new and fast-acting antidepressants. Genetic suppression of astrocytic inwardly rectifying potassium channel 4.1 (Kir4.1) in the lateral habenula ameliorates depression-like phenotypes in mice. However, Kir4.1 remains an elusive drug target for depression. Here, we discovered a series of Kir4.1 inhibitors through high-throughput screening. Lys05, the most potent one thus far, effectively suppressed native Kir4.1 channels while displaying high selectivity against established targets for rapid-onset antidepressants. Cryogenic-electron microscopy structures combined with electrophysiological characterizations revealed Lys05 directly binds in the central cavity of Kir4.1. Notably, a single dose of Lys05 reversed the Kir4.1-driven depression-like phenotype and exerted rapid-onset (as early as 1 hour) antidepressant actions in multiple canonical depression rodent models with efficacy comparable to that of (S)-ketamine. Overall, we provided a proof of concept that Kir4.1 is a promising target for rapid-onset antidepressant effects.

Ligand activation mechanisms of human KCNQ2 channel.

The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.

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.

Discovery of selective NaV1.8 inhibitors based on 5-chloro-2-(4,4-difluoroazepan-1-yl)-6-methyl nicotinamide scaffold for the treatment of pain.

The NaV1.8 channel is a genetically validated target for pain and it is mostly expressed in the peripheral nervous system. Based on the disclosed structures of NaV1.8-selective inhibitors, we designed and synthesized a series of compounds by introducing bicyclic aromatic fragments based on the nicotinamide scaffold. In this research, a systematic structure-activity relationship study was carried out. While compound 2c possessed moderate inhibitory activity (IC50 = 50.18 ± 0.04 nM) in HEK293 cells stably expressing human NaV1.8 channels, it showed potent inhibitory activity in DRG neurons and isoform selectivity (>200-fold against human NaV1.1, NaV1.5 and NaV1.7 channels). Moreover, the analgesic potency of compound 2c was identified in a post-surgical mouse model. These data demonstrate that compound 2c can be further evaluated as a non-addictive analgesic agent with reduced cardiac liabilities.

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.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 µM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

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.

(-)-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).

CCT128930 is a novel and potent antagonist of TRPM7 channel.

Transient receptor potential melastatin 7 (TRPM7) channels represent a major magnesium (Mg2+)-uptake component in mammalian cells and are negatively modulated by internal Mg2+. However, few TRPM7 modulators were identified so far, which hindered the understanding of the TRPM7 channel functions. In this study, we identified that CCT128930, an ATP-competitive protein kinase B inhibitor reported previously, was a potent TRPM7 channel antagonist. The inhibition of CCT128930 on TRPM7 was independent of intracellular Mg2+. In the absence and presence of 300 µM Mg2+ in pipette solution, the IC50 values were 0.86 ± 0.11 µM and 0.63 ± 0.09 µM, respectively. Subtype selectivity data showed that CCT128930 preferentially inhibited TRPM7 channels compared to TRPM6 and TRPM8 isoforms. In addition, CCT128930 was found to be able to reduce the endogenous TRPM7-like currents in SH-SY5Y neuroblastoma cells. At last, multiple residues in the superficial part of the TRPM7 selectivity filter were identified to be critical for the inhibitory activity of CCT128930 which are different from the determinants of Mg2+ and reported TRPM7 antagonists. Our results indicated that CCT128930 is a novel and potent TRPM7 channel antagonist.

Spatial effects of carbon emission intensity and regional development in China.

Due to the imbalance of technological level and industrial structure in regional economic development, the same carbon source can bring differentiated carbon emission levels in different regions, thus making the carbon emission show significant regional differences. In order to explore the regional differences in China's provincial carbon emission intensity and the effect of relevant influencing factors, this paper combines EKC model and STIRPAT model to conduct research. Using carbon emission intensity and other influencing factors of China's 30 provinces ranging from 2005 to 2017 to construct a panel data, the authors use exploratory spatial data analysis and Spatial Durbin Model to study the spatial effect of carbon emission intensity in China's provincial regions and the impact of different development factors on carbon emission intensity. The results show that from 2005 to 2017, China's carbon emission intensity gradually declined from east to west and from south to north. The inter-provincial carbon emission intensity of China presents an agglomeration effect in space, and the agglomeration effect gradually weakens with time. In addition, reducing energy intensity can reduce carbon emission intensity to a large extent. By optimizing industrial structure, increasing the degree of foreign trade and promoting financial development, carbon emission intensity can also be inhibited. Therefore, reducing the energy intensity of various industries and establishing inter-regional carbon emission cooperation mechanism will be effective to control the carbon emission intensity.

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.

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.

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.

Activation of peripheral KCNQ channels relieves gout pain.

Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate-induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout.

The human ether-a-go-go-related gene activator NS1643 enhances epilepsy-associated KCNQ channels.

Human ether-a-go-go-related gene (hERG) and KCNQ channels are two classes of voltage-gated potassium channels. Specific mutations have been identified that are causal for type II long QT (LQT2) syndrome, neonatal epilepsy, and benign familial neonatal convulsions. Increasing evidence from clinical studies suggests that LQT2 and epilepsy coexist in some patients. Therefore, an integral approach to investigating and treating the two diseases is likely more effective. In the current study, we found that NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea], a previously reported hERG activator, is also an activator of KCNQ channels. It potentiates the neuronal KCNQ2, KCNQ4, and KCNQ2/Q3 channels, but not the cardiac KCNQ1. The effects of NS1643 on the KCNQ2 channel include left shifting of voltage for reaching 50% of the maximum conductance and slowing of deactivation. Analysis of the dose-response curve of NS1643 revealed an EC50 value of 2.44 ± 0.25 µM. A hydrophobic phenylalanine (F137) located at the middle region of the voltage-sensing domain was identified as critical for NS1643 activity on KCNQ2. When testing NS1643 effects in rescuing LQT2 hERG mutants and the KCNQ2 BFNC mutants, we found it is particularly efficacious in some cases. Considering the substantial relationship between LQT2 and epilepsy, these findings reveal that NS1643 is a useful compound to elucidate the causal connection of LQT2 and epilepsy. More generally, this may provide a strategy in the development of therapeutics for LQT2 and epilepsy.

Hexachlorophene is a potent KCNQ1/KCNE1 potassium channel activator which rescues LQTs mutants.

The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC(50) of 4.61 ± 1.29 µM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 µM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics.

[Determination of fatty acids in vegetable oils using comprehensive two-dimensional gas chromatography coupled to quadropole mass spectrometry].

Comprehensive two-dimensional gas chromatography with quadropole mass spectrometry (GC x GC-qMS) was applied to the detection of 31 fatty acids in vegetable oils. The sets of columns, modulation period, scan range of quadropole mass spectrometer were optimized. The results demonstrated that the separation was achieved in 50 min with the column set of DB-1 (30 m x0. 25 mm x 0.25 microm) as the 1st column and DB-Wax (3.2 m x 0.1 mm x 0. 1 microm) as the 2nd column. All fatty acids were accurately and sensitively determined while the modulation period was 3.5 s and the scan range of quadropole MS was m/z 40-350. Most of the fatty acids were identified by NIST library spectra search, the other fatty acid isomers were identified by single standard injection analysis. When applying this method to the real vegetable oil samples, not only the sensitivities were 100 times higher than those obtained with GC-qMS methods, but also some minor fatty acids were identified. This work suggested a new technical approach in analyzing fatty acid components in vegetable oils, which is meaningful to prohibit adulteration and ensuring the quality safety of edible vegetable oils.