Synthetic ShK-like Peptide from the Jellyfish Nemopilema nomurai Has Human Voltage-Gated Potassium-Channel-Blocking Activity.

We identified a new human voltage-gated potassium channel blocker, NnK-1, in the jellyfish Nemopilema nomurai based on its genomic information. The gene sequence encoding NnK-1 contains 5408 base pairs, with five introns and six exons. The coding sequence of the NnK-1 precursor is 894 nucleotides long and encodes 297 amino acids containing five presumptive ShK-like peptides. An electrophysiological assay demonstrated that the fifth peptide, NnK-1, which was chemically synthesized, is an effective blocker of hKv1.3, hKv1.4, and hKv1.5. Multiple-sequence alignment with cnidarian Shk-like peptides, which have Kv1.3-blocking activity, revealed that three residues (3Asp, 25Lys, and 34Thr) of NnK-1, together with six cysteine residues, were conserved. Therefore, we hypothesized that these three residues are crucial for the binding of the toxin to voltage-gated potassium channels. This notion was confirmed by an electrophysiological assay with a synthetic peptide (NnK-1 mu) where these three peptides were substituted with 3Glu, 25Arg, and 34Met. In conclusion, we successfully identified and characterized a new voltage-gated potassium channel blocker in jellyfish that interacts with three different voltage-gated potassium channels. A peptide that interacts with multiple voltage-gated potassium channels has many therapeutic applications in various physiological and pathophysiological contexts.

Chemical and biophysical characterization of novel potassium channel blocker 3-fluoro-5-methylpyridin-4-amine.

4-aminopyridine (4AP) is a potassium (K+) channel blocker used clinically to improve walking in people with multiple sclerosis (MS). 4AP binds to exposed K+ channels in demyelinated axons, reducing the leakage of intracellular K+ and enhancing impulse conduction. Multiple derivatives of 4AP capable of blocking K+ channels have been reported including three radiolabeled with positron emitting isotopes for imaging demyelinated lesions using positron emission tomography (PET). However, there remains a demand for novel molecules with suitable physicochemical properties and binding affinity that can potentially be radiolabeled and used as PET radiotracers. In this study, we introduce 3-fluoro-5-methylpyridin-4-amine (5Me3F4AP) as a novel trisubstituted K+ channel blocker with potential application in PET. 5Me3F4AP has comparable potency to 4AP and the PET tracer 3-fluoro-4-aminopyridine (3F4AP). Compared to 3F4AP, 5Me3F4AP exhibits comparable basicity (pKa = 7.46 ± 0.01 vs. 7.37 ± 0.07, P-value = 0.08), greater lipophilicity (logD = 0.664 ± 0.005 vs. 0.414 ± 0.002, P-value < 0.0001) and higher permeability to an artificial brain membrane (Pe = 88.1 ± 18.3 vs. 31.1 ± 2.9 nm/s, P-value = 0.03). 5Me3F4AP is also more stable towards oxidation in vitro by the cytochrome P450 enzyme CYP2E1 (IC50 = 36.2 ± 2.5 vs. 15.4 ± 5.1, P-value = 0.0003); the enzyme responsible for the metabolism of 4AP and 3F4AP. Taken together, 5Me3F4AP has promising properties as a candidate for PET imaging warranting additional investigation.

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.

Inhibition of voltage-gated potassium channel by aripiprazole in rabbit coronary arterial smooth muscle cells.

Aripiprazole, a third-generation antipsychotic, has been widely used to treat schizophrenia. In this study, we evaluated the effect of aripiprazole on voltage-gated potassium (Kv) channels in rabbit coronary arterial smooth muscle cells using the patch clamp technique. Aripiprazole reduced the Kv current in a concentration-dependent manner with a half-maximal inhibitory concentration of 0.89 ± 0.20 µM and a Hill coefficient of 1.30 ± 0.25. The inhibitory effect of aripiprazole on Kv channels was voltage-dependent, and an additional aripiprazole-induced decrease in the Kv current was observed in the voltage range of full channel activation. The decay rate of Kv channel inactivation was accelerated by aripiprazole. Aripiprazole shifted the steady-state activation curve to the right and the inactivation curve to the left. Application of a repetitive train of pulses (1 and 2 Hz) promoted inhibition of the Kv current by aripiprazole. Furthermore, the recovery time constant from inactivation increased in the presence of aripiprazole. Pretreatment of Kv1.5 subtype inhibitor reduced the inhibitory effect of aripiprazole. However, pretreatment with Kv 7 and Kv2.1 subtype inhibitors did not change the degree of aripiprazole-induced inhibition of the Kv current. We conclude that aripiprazole inhibits Kv channels in a concentration-, voltage-, time-, and use (state)-dependent manner by affecting the gating properties of the channels.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block.

AIMS: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. METHODS AND RESULTS: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (µM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 µM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90. CONCLUSION: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Kv1.3 in the spotlight for treating immune diseases.

INTRODUCTION: Kv1.3 is the main voltage-gated potassium channel of leukocytes from both the innate and adaptive immune systems. Channel function is required for common processes such as Ca2+ signaling but also for cell-specific events. In this context, alterations in Kv1.3 are associated with multiple immune disorders. Excessive channel activity correlates with numerous autoimmune diseases, while reduced currents result in increased cancer prevalence and immunodeficiencies. AREAS COVERED: This review offers a general view of the role of Kv1.3 in every type of leukocyte. Moreover, diseases stemming from dysregulations of the channel are detailed, as well as current advances in their therapeutic research. EXPERT OPINION: Kv1.3 arises as a potential immune target in a variety of diseases. Several lines of research focused on channel modulation have yielded positive results. However, among the great variety of specific channel blockers, only one has reached clinical trials. Future investigations should focus on developing simpler administration routes for channel inhibitors to facilitate their entrance into clinical trials. Prospective Kv1.3-based treatments will ensure powerful therapies while minimizing undesired side effects.

Isolation and structural identification of a potassium ion channel Kv4.1 inhibitor SsTx-P2 from centipede venom. / 蜈蚣毒液中钾离子通道Kv4.1抑制剂SsTx-P2的分离和结构鉴定.

OBJECTIVES: To isolate a potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its sequence and structure. METHODS: Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with matrix assisted laser desorption ionization-time-of-flight mass spectrometry; its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry; its structure was established based on iterative thread assembly refinement online analysis. RESULTS: A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues NH2-ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 µmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its structure showed that SsTx-P2 shared a conserved helical structure. CONCLUSIONS: The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1 and displays structural conservation.

Benchmarking of Small Molecule Feature Representations for hERG, Nav1.5, and Cav1.2 Cardiotoxicity Prediction.

In the field of drug discovery, there is a substantial challenge in seeking out chemical structures that possess desirable pharmacological, toxicological, and pharmacokinetic properties. Complications arise when drugs interfere with the functioning of cardiac ion channels, leading to serious cardiovascular consequences. The discontinuation and removal of numerous approved drugs from the market or at late development stages in the pipeline due to such inhibitory effects further highlight the urgency of addressing this issue. Consequently, the early prediction of potential blockers targeting cardiac ion channels during the drug discovery process is of paramount importance. This study introduces a deep learning framework that computationally determines the cardiotoxicity associated with the voltage-gated potassium channel (hERG), the voltage-gated calcium channel (Cav1.2), and the voltage-gated sodium channel (Nav1.5) for drug candidates. The predictive capabilities of three feature representations─molecular fingerprints, descriptors, and graph-based numerical representations─are rigorously benchmarked. Additionally, a novel training and evaluation data set framework is presented, enabling predictive model training of drug off-target cardiotoxicity using a comprehensive and large curated data set covering these three cardiac ion channels. To facilitate these predictions, a robust and comprehensive small molecule cardiotoxicity prediction tool named CToxPred has been developed. It is made available as open source under the permissive MIT license at https://github.com/issararab/CToxPred.

Selective block of human Kv1.1 channels and an epilepsy-associated gain-of-function mutation by AETX-K peptide.

Dysfunction of the human voltage-gated K+ channel Kv1.1 has been associated with epilepsy, multiple sclerosis, episodic ataxia, myokymia, and cardiorespiratory dysregulation. We report here that AETX-K, a sea anemone type I (SAK1) peptide toxin we isolated from a phage display library, blocks Kv1.1 with high affinity (Ki ~ 1.6 pM) and notable specificity, inhibiting other Kv channels we tested a million-fold less well. Nuclear magnetic resonance (NMR) was employed both to determine the three-dimensional structure of AETX-K, showing it to employ a classic SAK1 scaffold while exhibiting a unique electrostatic potential surface, and to visualize AETX-K bound to the Kv1.1 pore domain embedded in lipoprotein nanodiscs. Study of Kv1.1 in Xenopus oocytes with AETX-K and point variants using electrophysiology demonstrated the blocking mechanism to employ a toxin-channel configuration we have described before whereby AETX-K Lys23 , two positions away on the toxin interaction surface from the classical blocking residue, enters the pore deeply enough to interact with K+ ions traversing the pathway from the opposite side of the membrane. The mutant channel Kv1.1-L296 F is associated with pharmaco-resistant multifocal epilepsy in infants because it significantly increases K+ currents by facilitating opening and slowing closure of the channels. Consistent with the therapeutic potential of AETX-K for Kv1.1 gain-of-function-associated diseases, AETX-K at 4 pM decreased Kv1.1-L296 F currents to wild-type levels; further, populations of heteromeric channels formed by co-expression Kv1.1 and Kv1.2, as found in many neurons, showed a Ki of ~10 nM even though homomeric Kv1.2 channels were insensitive to the toxin (Ki > 2000 nM).

Facilitation of hERG Activation by Its Blocker: A Mechanism to Reduce Drug-Induced Proarrhythmic Risk.

Modulation of the human Ether-à-go-go-Related Gene (hERG) channel, a crucial voltage-gated potassium channel in the repolarization of action potentials in ventricular myocytes of the heart, has significant implications on cardiac electrophysiology and can be either antiarrhythmic or proarrhythmic. For example, hERG channel blockade is a leading cause of long QT syndrome and potentially life-threatening arrhythmias, such as torsades de pointes. Conversely, hERG channel blockade is the mechanism of action of Class III antiarrhythmic agents in terminating ventricular tachycardia and fibrillation. In recent years, it has been recognized that less proarrhythmic hERG blockers with clinical potential or Class III antiarrhythmic agents exhibit, in addition to their hERG-blocking activity, a second action that facilitates the voltage-dependent activation of the hERG channel. This facilitation is believed to reduce the proarrhythmic potential by supporting the final repolarizing of action potentials. This review covers the pharmacological characteristics of hERG blockers/facilitators, the molecular mechanisms underlying facilitation, and their clinical significance, as well as unresolved issues and requirements for research in the fields of ion channel pharmacology and drug-induced arrhythmias.

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Acute Inhibition of the Human Kv1.5 Channel by H1 Receptor Antagonist Dimenhydrinate: Mode of Action.

Dimenhydrinate, an H1 receptor antagonist, is generally used for the prevention and treatment of nausea and vomiting. However, cardiac arrhythmias have been reported to be associated with the overdose of histamine H1 receptor antagonists, indicating the probable effect of antihistamines on ion channels. By using a two-microelectrode voltage clamp, we have herein studied the electrophysiological effects of dimenhydrinate on the human Kv1.5 channel in the Xenopus oocyte expression system. Dimenhydrinate acutely and reversibly suppressed the amplitudes of the peak and the steady-state current, within 6 min. The inhibitory effect of dimenhydrinate on the peak and the steady-state Kv1.5 currents increased progressively from -10 to +50 mV. At each test voltage, the drug suppressed both the peak and the steady-state currents to a similar extent. When the oocytes were stimulated at the rates of 5- and 30-s intervals, dimenhydrinate-induced a use-dependent blockade of the human Kv1.5 channel. Dimenhydrinate expedited the timecourse of the Kv1.5 channel activation more effectively than the timecourse of its inactivation. However, the activation and inactivation curves of the channel were not altered by the H1 receptor antagonist. In conclusion, we found that dimenhydrinate inhibits the human Kv1.5 channel by changing the channel's activation mode, thereby possibly increasing the possibility of triggering cardiac arrhythmias and affecting atrial fibrillation.

Cilostazol induces vasorelaxation through the activation of the eNOS/NO/cGMP pathway, prostanoids, AMPK, PKC, potassium channels, and calcium channels.

OBJECTIVE: This study aimed to investigate vasoactive effect mechanisms of cilostazol in rat thoracic aorta. MATERIALS AND METHODS: The vessel rings prepared from the thoracic aortas of the male rats were placed in the chambers of the isolated tissue bath system. The resting tone was adjusted to 1 g. Following the equilibration phase, potassium chloride or phenylephrine was used to contract the vessel rings. When achieving a steady contraction, cilostazol was applied cumulatively (10-8-10-4 M). In the presence of potassium channel blockers or signaling pathway inhibitors, the same experimental procedure was performed. RESULTS: Cilostazol exhibited a significant vasorelaxant effect in a concentration-dependent manner (pD2: 5.94 ± 0.94) (p < .001). The vasorelaxant effect level of cilostazol was significantly reduced by the endothelial nitric oxide synthase inhibitor L-NAME (10-4 M), soluble guanylate cyclase inhibitor methylene blue (10 µM), cyclooxygenase 1/2 inhibitor indomethacin (5 µM), adenosine monophosphate-activated protein kinase inhibitor compound C (10 µM), non-selective potassium channel blocker tetraethylammonium chloride (10 mM), large-conductance calcium-activated potassium channel blocker iberiotoxin (20 nM), voltage-gated potassium channel blocker 4-Aminopyridine (1 mM), and inward-rectifier potassium channel blocker BaCl2 (30 µM) (p < .001). Moreover, incubation of cilostazol (10-4 M) significantly reduced caffeine (10 mM), cyclopiazonic acid (10 µM), and phorbol 12-myristate 13-acetate-induced (100 µM) vascular contractions (p < .001). CONCLUSIONS: In the rat thoracic aorta, the vasodilator action level of cilostazol is quite noticeable. The vasorelaxant effects of cilostazol are mediated by the eNOS/NO/cGMP pathway, prostanoids, AMPK pathway, PKC, potassium channels, and calcium channels.

Inhibition of voltage-dependent K+ currents of rabbit coronary arterial smooth muscle cells by the atypical antipsychotic paliperidone.

Paliperidone, an atypical antipsychotic, is widely used to treat schizophrenia. In this study, we explored whether paliperidone inhibited the voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells. Paliperidone reduced Kv channel activity in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 16.58 ± 3.03 µM and a Hill coefficient of 0.60 ± 0.04. It did not significantly shift the steady-state activation or inactivation curves, suggesting that the drug did not affect the gating properties of Kv channels. In the presence of paliperidone, the application of 20 repetitive depolarizing pulses at 1 and 2 Hz gradually increased the inhibition of the Kv current. Further, the recovery time constant after Kv channel inactivation was increased by paliperidone, indicating that it inhibited the Kv channel in a use (state)-dependent manner. Its inhibitory effects were reduced by pretreatment with a Kv1.5 subtype inhibitor. However, pretreatment with a Kv2.1 or Kv7 inhibitor did not reduce its inhibitory effect. We conclude that paliperidone inhibits Kv channels (mainly Kv1.5 subtype channels) in a concentration- and use (state)-dependent manner without changing channel gating.

Prolonged Exposure to Remdesivir Inhibits the Human Ether-A-Go-Go-Related Gene Potassium Current.

ABSTRACT: Remdesivir, approved for the treatment of COVID-19, has been associated with heart-rate corrected QT interval (QTc) prolongation and torsade de pointes in case reports. However, data are conflicting regarding the ability of remdesivir to inhibit the human ether-a-go-go-related gene (hERG) -related current. The objective of this study was to investigate the effects remdesivir and its primary metabolite, GS-441524, on hERG-related currents. Human embryonic kidney 293 cells stably expressing hERG were treated with various concentrations of remdesivir and GS-441524. The effects of acute and prolonged exposure on hERG-related current were assessed using whole-cell configuration of voltage-clamp protocols. Acute exposure to remdesivir and GS-441524 had no effect on hERG currents and the half-activation voltage (V 1/2 ). Prolonged treatment with 100 nM and 1 µM remdesivir significantly reduced peak tail currents and hERG current density. The propensity for remdesivir to prolong QTc intervals and induce torsade de pointes in predisposed patients warrants further investigation.

The Strength of hERG Inhibition by Erythromycin at Different Temperatures Might Be Due to Its Interacting Features with the Channels.

Erythromycin is one of the few compounds that remarkably increase ether-a-go-go-related gene (hERG) inhibition from room temperature (RT) to physiological temperature (PT). Understanding how erythromycin inhibits the hERG could help us to decide which compounds are needed for further studies. The whole-cell patch clamp technique was used to investigate the effects of erythromycin on hERG channels at different temperatures. While erythromycin caused a concentration-dependent inhibition of cardiac hERG channels, it also shifted the steady-state activation and steady-state inactivation of the channel to the left and significantly accelerated the onset of inactivation at both temperatures, although temperature itself caused a profound change in the dynamics of hERG channels. Our data also suggest that the binding pattern to S6 of the channels changes at PT. In contrast, cisapride, a well-known hERG blocker whose inhibition is not affected by temperature, does not change its critical binding sites after the temperature is raised to PT. Our data suggest that erythromycin is unique and that the shift in hERG inhibition may not apply to other compounds.

hERG agonists pose challenges to web-based machine learning methods for prediction of drug-hERG channel interaction.

Pharmacological blockade of the IKr channel (hERG) by diverse drugs in clinical use is associated with the Long QT Syndrome that can lead to life threatening arrhythmia. Various computational tools including machine learning models (MLM) for the prediction of hERG inhibition have been developed to facilitate the throughput screening of drugs in development and optimise thus the prediction of hERG liabilities. The use of MLM relies on large libraries of training compounds for the quantitative structure-activity relationship (QSAR) modelling of hERG inhibition. The focus on inhibition omits potential effects of hERG channel agonist molecules and their associated QT shortening risk. It is instructive, therefore, to consider how known hERG agonists are handled by MLM. Here, two highly developed online computational tools for the prediction of hERG liability, Pred-hERG and HergSPred were probed for their ability to detect hERG activator drug molecules as hERG interactors. In total, 73 hERG blockers were tested with both computational tools giving overall good predictions for hERG blockers with reported IC50s below Pred-hERG and HergSPred cut-off threshold for hERG inhibition. However, for compounds with reported IC50s above this threshold such as disopyramide or sotalol discrepancies were observed. HergSPred identified all 20 hERG agonists selected as interacting with the hERG channel. Further studies are warranted to improve online MLM prediction of hERG related cardiotoxicity, by explicitly taking into account channel agonism as well as inhibition.

Immunosuppressive effects of new thiophene-based KV1.3 inhibitors.

Voltage-gated potassium channel KV1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca2+ homeostasis. Here, we present the structure-activity relationship, KV1.3 inhibition, and immunosuppressive effects of new thiophene-based KV1.3 inhibitors with nanomolar potency on K+ current in T-lymphocytes and KV1.3 inhibition on Ltk- cells. The new KV1.3 inhibitor trans-18 inhibited KV1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC50 value of 26.1 nM and in mammalian Ltk- cells with an IC50 value of 230 nM. The KV1.3 inhibitor trans-18 also had nanomolar potency against KV1.3 in Xenopus laevis oocytes (IC50 = 136 nM). The novel thiophene-based KV1.3 inhibitors impaired intracellular Ca2+ signaling as well as T-cell activation, proliferation, and colony formation.

Inhibitory effects of N-methyl-D-aspartate (NMDA) and α1-adrenergic receptor antagonist ifenprodil on human Kv1.5 channel.

Ifenprodil has been known to reduce cardiac contractility and cerebral vasodilation by antagonizing α1-adrenergic and N-methyl D-aspartate receptor-mediated intracellular signals. This study aimed to investigate the direct effect of ifenprodil on the human voltage-gated Kv1.5 channel (hKv1.5) by using a Xenopus oocyte expression system and a two-microelectrode voltage clamp technique. The amplitudes of hKv1.5 currents, including peak and steady state, were suppressed in a concentration-dependent manner (IC50; 43.1 and 35.5 µM, respectively) after 6 min of ifenprodil treatment. However, these effects were ~ 80% reversed by washout, suggesting that ifenprodil directly inhibited the hKv1.5 independent of membrane receptors or intracellular signals. The inhibition rate of steady state showed voltage dependence, wherein the rates increased according to test voltage depolarization. Ifenprodil reduced the time constants of hKv1.5 inactivation but has higher effects on activation. hKv1.5 inhibition by ifenprodil showed use dependency because the drug more rapidly reduced the current at the higher activation frequencies, and subsequent reduction in frequency after high activation frequency caused a partial channel block relief. Therefore, ifenprodil directly blocked the hKv1.5 in an open state and accelerated the time course of the channel inactivation, which provided a biophysical mechanism for the hKv1.5 blocking effects of ifenprodil.