Inhibition Of Potassium Channels Research Articles

Discovery of BMS-986308: A Renal Outer Medullary Potassium Channel Inhibitor for the Treatment of Heart Failure.

Recent literature reports highlight the importance of the renal outer medullary potassium (ROMK) channel in renal sodium and potassium homeostasis and emphasize the potential impact that ROMK inhibitors could have as a novel mechanism diuretic in heart failure patients. A series of piperazine-based ROMK inhibitors were designed and optimized to achieve excellent ROMK potency, hERG selectivity, and ADME properties, which led to the identification of compound 28 (BMS-986308). BMS-986308 demonstrated efficacy in the volume-loaded rat diuresis model as well as promising in vitro and in vivo profiles and was therefore advanced to clinical development.

Structure–Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels

Structure–Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels

ML365 ameliorates postoperative cognitive impairment in aged mice by inhibiting NLRP3 inflammasome activation in the hippocampus

The aim of this study was to examine the effects of ML365, a two-pore potassium channel (K2P) inhibitor, on postoperative cognitive impairment (POCD). A mouse model of POCD was constructed by subjecting aged C57BL/6 mice to exploratory laparotomy. Changes in cognitive function were assessed using the Morris water maze test. Western blotting and qPCR were used to detect hippocampal NLRP3, Caspase-1 and IL-1β expression levels on days 3 and 7 post-surgery. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression level was also assessed by western blotting. Pathological changes and nerve damage in the hippocampal CA1 and CA3 regions were detected by H&E staining, while the concentration of malondialdehyde (MDA) in the plasma was measured. We found that pretreatment with ML365 (administered intraperitoneally at a dose of 10 mg/kg) 30 min prior to exploratory laparotomy effectively ameliorated POCD in mice. ML365 pretreatment also reduced NLRP3, Caspase-1, ASC and IL-1β expression levels in the hippocampus, improved POCD-induced pathological changes in the hippocampal CA1 and CA3 areas of aged mice, and decreased levels of plasma MDA and oxidative stress. Together, our findings indicate that ML365 can alleviate POCD in mice by inhibiting NLRP3 inflammasome activation in the hippocampus.

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.

Abstract 7121: The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing cancers

Abstract Previous studies have demonstrated that WEE1 inhibitors (WEE1i) are a promising cancer therapeutic through synthetic lethality with Cyclin E overexpression. One challenge for two of the leading WEE1i, AZD1775 and ZN-c3, has been the observed off-targeting of PLK family members, namely, PLK1, PLK2 and PLK3. In addition, the therapeutic application of these inhibitors in clinical trials has been limited by adverse hematological effects, including anemia. Herein, we describe the testing of a novel WEE1i (APR-1051) that, based on preclinical studies: 1) demonstrates reduced off-targeting of PLK1, PLK2 and PLK3, and 2) has the ability to suppress the growth of CCNE1-overexpressing xenografted tumors while causing little impact on red blood cell and reticulocyte levels. APR-1051 has an IC50 of 2.2 nM for WEE1 in vitro and has shown the ability to limit the proliferation of various cancer cell lines in culture. Interestingly, the genotoxicity of APR-1051 is suppressed by increasing concentrations of PLK1 inhibitors, suggesting that off-targeting of PLK1 by other WEE1i may compromise efficacy of this targeted cancer treatment in addition to the risk of producing PLK1i-associated adverse effects. APR-1051 demonstrated effectiveness in suppressing the growth of Cyclin E-overexpressing breast and ovarian cancer cells lines, thus pinpointing Cyclin E as a potential biomarker for APR-1051 treatment. Importantly, dose and scheduling of APR-1051 that causes significant suppression of CCNE1-amplified high-grade serious ovarian cancer tumors in mice is well tolerated, with red blood cell and platelet counts remaining within non-pathogenic ranges after a 28-day treatment period. In addition, inhibition WEE1 by APR-1051 occurs at an IC50 that is 200-fold lower on average than the IC50 of hERG potassium channel inhibition, implying that APR-1051 will potentially exhibit low cardiotoxicity. APR-1051 is now progressing through IND-enabling studies. Together, these findings underscore the potential of APR-1051 as a novel WEE1i for the treatment of Cyclin E-overexpressing cancers. Citation Format: Molly M. Hansbarger, Stephen J. Rocca, Sonia Ritzmann, Joseph Vacca, Bridget Tokiwa, Jonathan Weinstein, Henry Zabierek, Justin Frye, Michael Carleton, Eric J. Brown, Oren Gilad. The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7121.

Cardiotoxicity Assessment through a Polymer-Based Cantilever Platform: An Integrated Electro-Mechanical Screening Approach.

Preclinical drug screening for cardiac toxicity has traditionally relied on observing changes in cardiomyocytes' electrical activity, primarily through invasive patch clamp techniques or non-invasive microelectrode arrays (MEA). However, relying solely on field potential duration (FPD) measurements for electrophysiological assessment can miss the full spectrum of drug-induced toxicity, as different drugs affect cardiomyocytes through various mechanisms. A more comprehensive approach, combining field potential and contractility measurements, is essential for accurate toxicity profiling, particularly for drugs targeting contractile proteins without affecting electrophysiology. However, previously proposed platform has significant limitations in terms of simultaneous measurement. The novel platform addresses these issues, offering enhanced, non-invasive evaluation of drug-induced cardiotoxicity. It features eight cantilevers with patterned strain sensors and MEA, enabling real-time monitoring of both cardiomyocyte contraction force and field potential. This system can detect minimum cardiac contraction force of ≈2 µN and field potential signals with 50µm MEA diameter, using the same cardiomyocytes in measurements of two parameters. Testing with six drugs of varied mechanisms of action, the platform successfully identifies these mechanisms and accurately assesses toxicity profiles, including drugs not inhibiting potassium channels. This innovative approach presents a comprehensive, non-invasive method for cardiac function assessment, poised to revolutionize preclinical cardiotoxicity screening.

In Silico Human Cardiomyocyte Action Potential Modeling: Exploring Ion Channel Input Combinations.

In silico modeling offers an opportunity to supplement and accelerate cardiac safety testing. With in silico modeling, computational simulation methods are used to predict electrophysiological interactions and pharmacological effects of novel drugs on critical physiological processes. The O'Hara-Rudy's model was developed to predict the response to different ion channel inhibition levels on cardiac action potential duration (APD) which is known to directly correlate with the QT interval. APD data at 30% 60% and 90% inhibition were derived from the model to delineate possible ventricular arrhythmia scenarios and the marginal contribution of each ion channel to the model. Action potential values were calculated for epicardial, myocardial, and endocardial cells, with action potential curve modeling. This study assessed cardiac ion channel inhibition data combinations to consider when undertaking in silico modeling of proarrhythmic effects as stipulated in the Comprehensive in Vitro Proarrhythmia Assay (CiPA). As expected, our data highlight the importance of the delayed rectifier potassium channel (IKr) as the most impactful channel for APD prolongation. The impact of the transient outward potassium channel (Ito) inhibition on APD was minimal while the inward rectifier (IK1) and slow component of the delayed rectifier potassium channel (IKs) also had limited APD effects. In contrast, the contribution of fast sodium channel (INa) and/or L-type calcium channel (ICa) inhibition resulted in substantial APD alterations supporting the pharmacological relevance of in silico modeling using input from a limited number of cardiac ion channels including IKr, INa, and ICa, at least at an early stage of drug development.

Unveiling the antinociceptive mechanisms of Methyl-2-(4-chloro-phenyl)-5-benzoxazoleacetate: insights from nociceptive assays in mice.

Methyl-2-(4-chloro- phenyl)-5-benzoxazoleacetate (MCBA), a synthetic benzoxazole derivative with established antipsoriatic efficacy, was investigated for potential antinociceptive effects. This study employs various nociceptive assays in mice to elucidate MCBA's antinociceptive mechanisms. MCBA's antinociceptive potential was tested against various nociception models induced by formalin, glutamate, capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor agonist, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator. It was then assessed using the hot plate test and examined within the acetic acid-induced writhing test. During the acetic acid-induced writhing test, MCBA was pre-challenged against selective receptor antagonists such as naloxone, caffeine, atropine, yohimbine, ondansetron, and haloperidol. It was also pre-challenged with ATP-sensitive potassium channel inhibitor (glibenclamide) to further elucidate its antinociceptive mechanism. The results showed that oral administration of MCBA led to a dose-dependent and significant inhibition (p < 0.05) of nociceptive effects across all evaluated models at doses of 60, 120, and 240 mg/kg. Moreover, the efficacy of MCBA's antinociceptive potential was significantly counteracted (p < 0.0001) by specific antagonists: (i) directed at adenosinergic, alpha-2 adrenergic, and cholinergic receptors using caffeine, yohimbine, and atropine, respectively; and (ii) targeting ATP-sensitive potassium channels, employing glibenclamide. Antagonists aimed at opioidergic and serotoninergic receptors (naloxone and ondansetron, respectively) had poor utility in inhibiting antinociceptive activity. Conversely, the dopaminergic receptor antagonist haloperidol potentiated locomotor abnormalities associated with MCBA treatment. MCBA-induced antinociception involves modulation of glutamatergic-, TRVP1 receptors- and PKC-signaling pathways. It impacts adenosinergic, alpha-2 adrenergic, and cholinergic receptors and opens ATP-sensitive potassium channels.

Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Inhibition of the KCa2 potassium channel in atrial fibrillation: a randomized phase 2 trial

Existing antiarrhythmic drugs to treat atrial fibrillation (AF) have incomplete efficacy, contraindications and adverse effects, including proarrhythmia. AP30663, an inhibitor of the KCa2 channel, has demonstrated AF efficacy in animals; however, its efficacy in humans with AF is unknown. Here we conducted a phase 2 trial in which patients with a current episode of AF lasting for 7 days or less were randomized to receive an intravenous infusion of 3 or 5 mg kg−1 AP30663 or placebo. The trial was prematurely discontinued because of slow enrollment during the coronavirus disease 2019 pandemic. The primary endpoint of the trial was cardioversion from AF to sinus rhythm within 90 min from the start of the infusion, analyzed with Bayesian statistics. Among 59 patients randomized and included in the efficacy analyses, the primary endpoint occurred in 42% (5 of 12), 55% (12 of 22) and 0% (0 of 25) of patients treated with 3 mg kg−1 AP30663, 5 mg kg−1 AP30663 or placebo, respectively. Both doses demonstrated more than 99.9% probability of superiority over placebo, surpassing the prespecified 95% threshold. The mean time to cardioversion, a secondary endpoint, was 47 (s.d. = 23) and 41 (s.d. = 24) minutes for 3 mg kg−1 and 5 mg kg−1 AP30663, respectively. AP30663 caused a transient increase in the QTcF interval, with a maximum mean effect of 37.7 ms for the 5 mg kg−1 dose. For both dose groups, no ventricular arrhythmias occurred and adverse event rates were comparable to the placebo group. AP30663 demonstrated AF cardioversion efficacy in patients with recent-onset AF episodes. KCa2 channel inhibition may be an attractive mechanism for rhythm control of AF that should be studied further in randomized trials. ClinicalTrials.gov registration: NCT04571385.

Artemisinin combined with low dose quinidine suppresses ventricular tachyarrhythmia in a model of Brugada syndrome

Abstract Background The mechanism of Brugada syndrome (BrS) is unclear. One of the mechanisms has been suggested that uneven dispersion of Ito channel between epicardium and endocardium induced ventricular arrhythmia. Artemisinin, which originates from a Chinese herb and is used widely as an antimalarial drug, is reported to inhibit the Ito channel in a canine experimental model previously. Objective We aimed to investigate the effect of artemisinin, alone and in combination with low dose quinidine, on the suppression of ventricular tachyarrhythmia in BrS in a canine experimental model. Methods We recorded transmural pseudo-ECG and epicardial/endocardial action potentials (APs) from coronary-perfused canine right ventricular wedge preparation. We used acetylcholine (3 μM), calcium channel blocker verapamil (1 μM), and Ito agonist NS5806 (6 – 10 μM) to mimic the BrS model. We induced ventricular arrhythmia and then perfused low dose quinidine (1 – 2 μM) alone and additionally perfused artemisinin (100 – 150 μM) to suppress the arrhythmia. We evaluated ventricular arrhythmia inducibility and measured AP duration, J wave area, notch index, and T wave dispersion. Results The provocation agents induced prominent J wave in all sample models. In 6 sample models out of 8 for artemisinin alone group, ventricular arrhythmia was induced. Artemisinin suppressed ventricular arrhythmia and recovered the AP dome in all preparations. J wave area and epicardial notch index were also decreased after artemisinin perfusion. The low dose quinidine (1 – 2 μM) did not completely suppressed the ventricular arrhythmia in 6 out of 6 sample models. However, additional artemisinin (100 – 200 μM) infusion suppressed the ventricular arrhythmia in all sample models and restored the AP dome. J wave area and epicardial notch index were also decreased. Conclusions Our findings suggest that artemisinin can alleviate ventricular arrhythmia in the BrS wedge preparation model. Combining artemisinin with low dose quinidine may further suppress ventricular arrhythmia by inhibiting potassium channels, including Ito channel. Artemisinin might reduce the therapeutic dose of quinidine by additional inhibition of potassium channels.

Unravelling the signaling pathway downstream of the neurokinin receptor in atrium: discovering novel anti-arrhythmic targets for treatment of atrial fibrillation

Abstract Background Stimulation of neurokinin receptor (NKR) 3 is anti-fibrillatory by prolonging the action potential duration (APD) of atrial cardiomyocytes via inhibition of a background potassium current (1). The NKR is a Gq/11 protein-coupled receptor, but the downstream intracellular pathway and the end-effector potassium (K+)-channel in atrium are unknown. We aimed at unravelling the components of this pathway in atrial cardiomyocytes. Methods Rabbit (New Zealand White) atrial cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Human immortalized atrial myocytes (HiAMs) were developed, and kindly provided by our collaborators (2). Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from atrial fibrillation (AF) patients undergoing minimally invasive surgical pulmonary vein isolation in the clinics. Action potentials (APs) and K+ currents were recorded at 36.5°C with the amphotericin-B-perforated patch clamp technique. RNA was isolated from rabbit atrial tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, stimulation of NKR3 prolonged APD by ∼50% and inhibited an background outward K+-current by ∼45%. Inhibition of phosphokinase C (PKC) did not prevent APD prolongation by NKR3 stimulation, but application of a diacylglycerol (DAG) analog inhibited an outward K+-current by ∼40% indicating that DAG, but not PKC, is downstream of NKR3. Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC), but not of phosphoinositide-specific PLC (PI-PLC), reduced APD increase by NKR3 stimulation by five-fold, indicating PC-PLC as the intracellular signaling molecule. In HiAMs, direct PLC activation prolonged APD by 34%. RNA sequencing analysis revealed potential end-targets of the NKR3 pathway in rabbit. Inhibition of small conductance potassium (KCNN2/KCNN3) or TASK-1 (KCNK3) channels (prolonging APD by ∼15%) did not abolish the APD prolongation effect by subsequent NKR3 stimulation (∼50%), indicating that these channels are not the end-effector K+-channels inhibited by NKR3 stimulation. Inhibition of constitutive KACh channels had little effect on APD at baseline. Nevertheless, APD shortening by agonist-induced KACh channel activity could be completely reversed by NKR3 stimulation, indicating that increased outward KACh current can be fully counterbalanced by the reduction in K+ background current. In human atrial cardiomyocytes from AF patients, stimulation of NKR3 prolonged APD by 25%, Conclusions PC-PLC and DAG, but not PI-PLC and PKC, are intracellularly downstream of NKR3 in atrial rabbit cardiomyocytes. Stimulation of the NKR pathway may have an anti-arrhythmic potential in patients with AF.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas.

Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in ∼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.

Aloperine protects pulmonary hypertension via triggering PPARγ signaling and inhibiting calcium regulatory pathway in pulmonary arterial smooth muscle cells.

Previous studies have reported the beneficial role of Aloperine (ALO), an active vasodilator purified from the seeds and leaves of the herbal plant Sophora alopecuroides L., on experimental pulmonary hypertension (PH); however, detailed mechanisms remain unclear. In this study, monocrotaline-induced PH (MCT-PH) rat model and primarily cultured rat distal pulmonary arterial smooth muscle cells (PASMCs) were used to investigate the mechanisms of ALO on experimental PH, pulmonary vascular remodeling, and excessive proliferation of PASMCs. Results showed that first, ALO significantly prevented the disease development of MCT-PH by inhibiting right ventricular systolic pressure (RVSP) and right ventricular hypertrophy indexed by the Fulton Index, normalizing the pulmonary arterials (PAs) remodeling and improving the right ventricular function indexed by transthoracic echocardiography. ALO inhibited the excessive proliferation of both PAs and PASMCs. Then, isometric tension measurements showed vasodilation of ALO on precontracted PAs isolated from both control and MCT-PH rats via activating the KCNQ channel, which was blocked by specific KCNQ potassium channel inhibitor linopirdine. Moreover, by using immunofluorescence staining and nuclear/cytosol fractionation, we further observed that ALO significantly enhanced the PPARγ nuclear translocation and activation in PASMCs. Transcriptome analyses also revealed activated PPARγ signaling and suppressed calcium regulatory pathway in lungs from MCT-PH rats treated with ALO. In summary, ALO could attenuate MCT-PH through both transient vasodilation of PAs and chronic activation of PPARγ signaling pathway, which exerted antiproliferative roles on PASMCs and remodeled PAs.NEW & NOTEWORTHY Aloperine attenuates monocrotaline-induced pulmonary hypertension (MCT-PH) in rats by inhibiting the pulmonary vascular remodeling and proliferation of pulmonary arterial smooth muscle cells (PASMCs). In mechanism, Aloperine not only exerts a transient KCNQ-dependent vasodilation in precontracted pulmonary arteries (PAs) from both control and MCT-PH rats but also activates PPARγ nuclear translocation and signaling transduction in PASMCs, which chronically inhibits the calcium regulatory pathway and proliferation of PASMCs.

Inhibition of the hERG Potassium Channel by a Methanesulphonate-Free E-4031 Analogue.

hERG (human Ether-à-go-go Related Gene)-encoded potassium channels underlie the cardiac rapid delayed rectifier (IKr) potassium current, which is a major target for antiarrhythmic agents and diverse non-cardiac drugs linked to the drug-induced form of long QT syndrome. E-4031 is a high potency hERG channel inhibitor from the methanesulphonanilide drug family. This study utilized a methanesulphonate-lacking E-4031 analogue, "E-4031-17", to evaluate the role of the methanesulphonamide group in E-4031 inhibition of hERG. Whole-cell patch-clamp measurements of the hERG current (IhERG) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT IhERG was inhibited by a half-maximal inhibitory concentration (IC50) of 15.8 nM, whilst the comparable value for E-4031-17 was 40.3 nM. Both compounds exhibited voltage- and time-dependent inhibition, but they differed in their response to successive applications of a long (10 s) depolarisation protocol, consistent with greater dissociation of E-4031-17 than the parent compound between applied commands. Voltage-dependent inactivation was left-ward voltage shifted for E-4031 but not for E-4031-17; however, inhibition by both compounds was strongly reduced by attenuated-inactivation mutations. Mutations of S6 and S5 aromatic residues (F656V, Y652A, F557L) greatly attenuated actions of both drugs. The S624A mutation also reduced IhERG inhibition by both molecules. Overall, these results demonstrate that the lack of a methanesulphonate in E-4031-17 is not an impediment to high potency inhibition of IhERG.

A Novel KCNH2 S981fs Mutation Identified by Whole-Exome Sequencing Is Associated with Type 2 Long QT Syndrome.

KCNH2 loss-of-function mutations cause long QT syndrome type 2 (LQT2), an inherited cardiac disorder associated with life-threatening ventricular arrhythmia. Through whole-exome sequencing, we discovered a novel AGCGACAC deletion (S981fs) in the hERG gene of an LQT2 patient. Using a heterologous expression system and patch clamping, we found that the mutant K channel had reduced cell surface expression and lower current amplitude compared to the wild type. However, functional expression was restored by lowering temperature and using potassium channel inhibitors or openers (E4031, cisapride, nicorandil). Co-immunoprecipitation experiments confirmed the assembly of mutant proteins with wild-type hERG. Confocal imaging showed decreased hERG distribution on the cell membrane in cells expressing S981fs. Notably, treatment with G418 significantly increased hERG current in wild-type/S981fs heterozygotes. In conclusion, our study identifies a novel hERG mutation leading to impaired Kv11.1 function due to trafficking and nonsense-mediated RNA decay defects. These findings shed light on the mechanisms underlying LQT2 and offer potential therapeutic avenues.