KCNJ2 inhibition mitigates mechanical injury in a human brain organoid model of traumatic brain injury.

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain and which strategies most potently mitigate these processes. We developed a high-intensity ultrasound platform to inflict mechanical injury to induced pluripotent stem cell (iPSC)-derived cortical organoids. Mechanically injured organoids elicit classic hallmarks of TBI, including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. We found that deep-layer neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuronal death after brain injury, and we present a scalable, genetically flexible cerebral organoid model that may enable the identification of additional modifiers of mechanical stress.

Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept.

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.

Novel inhibitors of the renal inward rectifier potassium channel of the mosquito vector Aedes aegypti.

The mosquito continues to be the most lethal animal to humans due to the devastating diseases that it carries and transmits. Controlling mosquito-borne diseases relies heavily on vector management using neurotoxic insecticides with limited modes of action. This has led to the emergence of resistance to pyrethroids and other neurotoxic insecticides in mosquitoes, which has reduced the efficacy of chemical control agents. Moreover, many neurotoxic insecticides are not selective for mosquitoes and negatively impact beneficial insects such as honeybees. Developing new mosquitocides with novel mechanisms of action is a clear unmet medical need; this review covers the efforts made toward this end by targeting the renal inward rectifier potassium channel (Kir) of the mosquito.

Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels.

Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin-channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K+ "knock-off" effect, suggesting the toxin functions as an out pore blocker physically occluding the K+-conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel-specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Parallel All-Optical Assay to Study Use-Dependent Functioning of Voltage-Gated Ion Channels in a Miniaturized Format.

Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.

Anti-Kir4.1 Antibodies in Multiple Sclerosis: Specificity and Pathogenicity.

The glial cells in the central nervous system express diverse inward rectifying potassium channels (Kir). They express multiple Kir channel subtypes that are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors. Dysfunction in a major astrocyte potassium channel, Kir4.1, appears as an early pathological event underlying neuronal phenotypes in several neurological diseases. The autoimmune effects on the potassium channel have not yet been fully described in the literature. However, several research groups have reported that the potassium channels are an immune target in patients with various neurological disorders. In 2012, Srivastava et al. reported about Kir4.1, a new immune target for autoantibodies in patients with multiple sclerosis (MS). Follow-up studies have been conducted by several research groups, but no clear conclusion has been reached. Most follow-up studies, including ours, have reported that the prevalence of Kir4.1-seropositive patients with MS was lower than that in the initial study. Therefore, we extensively review studies on the method of antibody testing, seroprevalence of MS, and other neurological diseases in patients with MS. Finally, based on the role of Kir4.1 in MS, we consider whether it could be an immune target in this disease.

Antidepressive effect of an inward rectifier K+ channel blocker peptide, tertiapin-RQ.

Renal outer medullary K+ channel, ROMK (Kir1.1, kcnj1) is expressed in the kidney and brain, but its role in the central nervous system remains unknown. Recent studies suggested an involvement of the ROMK channel in mental diseases. Tertiapin (TPN) is a European honey bee venom peptide and is reported to selectively block the ROMK channel. Here, we have chemically synthesized a series of mutated TPN peptides, including TPN-I8R and -M13Q (TPN-RQ), reported previously, and examined their blocking activity on the ROMK channel. Among 71 peptides tested, TPN-RQ was found to block the ROMK channel most effectively. Whole-cell patch-clamp recordings showed the essential roles of two disulfide bonds and the circular structure for the blockade activity. To examine the central role, we injected TPN-RQ intracerebroventricularly and examined the effects on depression- and anxiety-like behaviors in mice. TPN-RQ showed an antidepressive effect in tail-suspension and forced swim tests. The injection of TPN-RQ also enhanced the anxiety-like behavior in the elevated plus-maze and light/dark box tests and impaired spontaneous motor activities in balance beam and wheel running tests. Administration of TPM-RQ suppressed the anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in antidepressant-related nuclei, e.g. the dorsal raphe nucleus and locus coeruleus. TPN-RQ may exert its antidepressive effects through a different mechanism from current drugs.

Epoxyeicosatrienoic acid metabolites inhibit Kir4.1/Kir5.1 in the distal convoluted tubule.

Cytochrome P-450 (Cyp) epoxygenase-dependent metabolites of arachidonic acid (AA) have been shown to inhibit renal Na+ transport, and inhibition of Cyp-epoxygenase is associated with salt-sensitive hypertension. We used the patch-clamp technique to examine whether Cyp-epoxygenase-dependent AA metabolites inhibited the basolateral 40-pS K+ channel (Kir4.1/Kir5.1) in the distal convoluted tubule (DCT). Application of AA inhibited the basolateral 40-pS K+ channel in the DCT. The inhibitory effect of AA on the 40-pS K+ channel was specific because neither linoleic nor oleic acid was able to mimic the effect of AA on the K+ channel. Inhibition of Cyp-monooxygenase with N-methylsulfonyl-12,12-dibromododec-11-enamide or inhibition of cyclooxygenase with indomethacin failed to abolish the inhibitory effect of AA on the 40-pS K+ channel. However, the inhibition of Cyp-epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide abolished the effect of AA on the 40-pS K+ channel in the DCT. Moreover, addition of either 11,12-epoxyeicosatrienoic acid (EET) or 14,15-EET also inhibited the 40-pS K+ channel in the DCT. Whole cell recording demonstrated that application of AA decreased, whereas N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide treatment increased, Ba2+-sensitive K+ currents in the DCT. Finally, application of 14,15-EET but not AA was able to inhibit the basolateral 40-pS K+ channel in the DCT of Cyp2c44-/- mice. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral Kir4.1/Kir5.1 in the DCT and that Cyp2c44-epoxygenase plays a role in the regulation of the basolateral K+ channel in the mouse DCT.

Striatal Kir2 K+ channel inhibition mediates the antidyskinetic effects of amantadine.

Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) - not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances - not antagonizes - the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.

Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators.

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

The inhibition of Kir2.1 potassium channels depolarizes spinal microglial cells, reduces their proliferation, and attenuates neuropathic pain.

Spinal microglia change their phenotype and proliferate after nerve injury, contributing to neuropathic pain. For the first time, we have characterized the electrophysiological properties of microglia and the potential role of microglial potassium channels in the spared nerve injury (SNI) model of neuropathic pain. We observed a strong increase of inward currents restricted at 2 days after injury associated with hyperpolarization of the resting membrane potential (RMP) in microglial cells compared to later time-points and naive animals. We identified pharmacologically and genetically the current as being mediated by Kir2.1 ion channels whose expression at the cell membrane is increased 2 days after SNI. The inhibition of Kir2.1 with ML133 and siRNA reversed the RMP hyperpolarization and strongly reduced the currents of microglial cells 2 days after SNI. These electrophysiological changes occurred coincidentally to the peak of microglial proliferation following nerve injury. In vitro, ML133 drastically reduced the proliferation of BV2 microglial cell line after both 2 and 4 days in culture. In vivo, the intrathecal injection of ML133 significantly attenuated the proliferation of microglia and neuropathic pain behaviors after nerve injury. In summary, our data implicate Kir2.1-mediated microglial proliferation as an important therapeutic target in neuropathic pain.

Small-Molecule Inhibitors of Inward Rectifier Potassium (Kir) Channels Reduce Bloodmeal Feeding and Have Insecticidal Activity Against the Horn Fly (Diptera: Muscidae).

Bloodmeal feeding by the horn fly, Haematobia irritans (L.), is associated with reduced milk production and blood loss that ultimately prevents weight gain of calves and yearlings. Thus, blood feeding by H. irritans causes significant economic losses in several continents. As with other arthropods, resistance to the majority of commercialized insecticides reduces the efficacy of current control programs. Thus, innovative technologies and novel biochemical targets for horn fly control are needed. Salivary gland and Malpighian tubule function are critical for H. irritans survivorship as they drive bloodmeal acquisition and maintain ion- and fluid homeostasis during bloodmeal processing, respectively. Experiments were conducted to test the hypothesis that pharmacological modulation of H. irritans inward rectifier potassium (Kir) channels would preclude blood feeding and induce mortality by reducing the secretory activity of the salivary gland while simultaneously inducing Malpighian tubule failure. Experimental results clearly indicate structurally diverse Kir channel modulators reduce the secretory activity of the salivary gland by up to fivefold when compared to control and the reduced saliva secretion was highly correlated to a reduction in bloodmeal acquisition in adult flies. Furthermore, adult feeding on blood treated with Kir channel modulators resulted in significant mortality. In addition to validating the Kir channels of H. irritans as putative insecticide targets, the knowledge gained from this study could be applied to develop novel therapeutic technologies targeting salivary gland or Malpighian tubule function to reduce the economic burden of horn fly ectoparasitism on cattle health and production.

Structural Insights into the Inhibitory Mechanism of Insulin Secretagogues on the Pancreatic ATP-Sensitive Potassium Channel.

Sulfonylureas and glinides are commonly used oral insulin secretagogues (ISs) that act on the pancreatic ATP-sensitive potassium (KATP) channel to promote insulin secretion in order to lower the blood glucose level. Physiologically, KATP channels are inhibited by intracellular ATP and activated by Mg-ADP. Therefore, they sense the cellular energy status to regulate the permeability of potassium ions across the plasma membrane. The pancreatic KATP channel is composed of the pore-forming Kir6.2 subunits and the regulatory SUR1 subunits. Previous electrophysiological studies have established that ISs bind to the SUR1 subunit and inhibit the channel activity primarily by two mechanisms. First, ISs prevent Mg-ADP activation. Second, ISs inhibit the channel activity of Kir6.2 directly. Several cryo-EM structures of the pancreatic KATP channel determined recently have provided remarkable structural insights into these two mechanisms.

Astroglial Mechanisms of Ketamine Action Include Reduced Mobility of Kir4.1-Carrying Vesicles.

The finding that ketamine, an anaesthetic, can elicit a rapid antidepressant effect at low doses that lasts for weeks in patients with depression is arguably a major achievement in psychiatry in the last decades. However, the mechanisms of action are unclear. The glutamatergic hypothesis of ketamine action posits that ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist modulating downstream cytoplasmic events in neurons. In addition to targeting NMDARs in synaptic transmission, ketamine may modulate the function of astroglia, key homeostasis-providing cells in the central nervous system, also playing a role in many neurologic diseases including depression, which affects to 20% of the population globally. We first review studies on astroglia revealing that (sub)anaesthetic doses of ketamine attenuate stimulus-evoked calcium signalling, a process of astroglial cytoplasmic excitability, regulating the exocytotic release of gliosignalling molecules. Then we address how ketamine alters the fusion pore activity of secretory vesicles, and how ketamine affects extracellular glutamate and K+ homeostasis, both considered pivotal in depression. Finally, we also provide evidence indicating reduced cytoplasmic mobility of astroglial vesicles carrying the inward rectifying potassium channel (Kir4.1), which may regulate the density of Kir4.1 at the plasma membrane. These results indicate that the astroglial capacity to control extracellular K+ concentration may be altered by ketamine and thus indirectly affect the action potential firing of neurons, as is the case in lateral habenula in a rat disease model of depression. Hence, ketamine-altered functions of astroglia extend beyond neuronal NMDAR antagonism and provide a basis for its antidepressant action through glia.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

A family of orthologous proteins from centipede venoms inhibit the hKir6.2 channel.

Inhibitors targeting ion channels are useful tools for studying their functions. Given the selectivity of any inhibitor for a channel is relative, more than one inhibitor of different affinities may be used to help identify the channel in a biological preparation. Here, we describe a family of small proteins in centipede venoms that inhibit the pore (hKir6.2) of a human ATP-sensitive K+ channel (hKATP). While the traditional peptide-sequencing service gradually vanishes from academic institutions, we tried to identify the sequences of inhibitory proteins purified from venoms by searching the sequences of the corresponding transcriptomes, a search guided by the key features of a known hKir6.2 inhibitor (SpTx1). The candidate sequences were cross-checked against the masses of purified proteins, and validated by testing the activity of recombinant proteins against hKir6.2. The four identified proteins (SsdTx1-3 and SsTx) inhibit hKATP channels with a Kd of <300 nM, compared to 15 nM for SpTx1. SsTx has previously been discovered to block human voltage-gated KCNQ K+ channels with a 2.5 µM Kd. Given that SsTx inhibits hKir6.2 with >10-fold lower Kd than it inhibits hKCNQ, SsTx may not be suitable for probing KCNQ channels in a biological preparation that also contains more-SsTx-sensitive KATP channels.

Discovery of Small Molecule Renal Outer Medullary Potassium (ROMK) Channel Inhibitors: A Brief History of Medicinal Chemistry Approaches To Develop Novel Diuretic Therapeutics.

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir, Kir1.1) channels. It is primarily expressed in two regions of the kidney, the cortical collecting duct (CCD) and the thick ascending loop of Henle (TALH). At the CCD it tightly regulates potassium secretion while controlling potassium recycling in TALH. As loss-of-function mutations lead to salt wasting and low blood pressure, it has been surmised that inhibitors of ROMK would represent a target for new and improved diuretics for the treatment of hypertension and heart failure. In this review, we discuss and provide an overview of the medicinal chemistry approaches toward the development of small molecule ROMK inhibitors over the past decade.

Discovery and Characterization of 2-Nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)- N-(pyridin-4-ylmethyl)anilines as Novel Inhibitors of the Aedes aegypti Kir1 ( AeKir1) Channel.

Mosquito-borne arboviral diseases such as Zika, dengue fever, and chikungunya are transmitted to humans by infected adult female Aedes aegypti mosquitoes and affect a large portion of the world's population. The Kir1 channel in Ae. aegypti ( AeKir1) is an important ion channel in the functioning of mosquito Malpighian (renal) tubules and one that can be manipulated in order to disrupt excretory functions in mosquitoes. We have previously reported the discovery of various scaffolds that are active against the AeKir1 channel. Herein we report the synthesis and biological characterization of a new 2-nitro-5-(4-(phenylsulfonyl) piperazin-1-yl)- N-(pyridin-4-ylmethyl)anilines scaffold as inhibitors of AeKir1. This new scaffold is more potent in vitro compared to the previously reported scaffolds, and the molecules kill mosquito larvae.

Electrophysiological and Pharmacological Characterization of Human Inwardly Rectifying Kir2.1 Channels on an Automated Patch-Clamp Platform.

Inwardly rectifying IK1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding Kir2.1 channels, which primarily conduct ventricular IK1, are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of Kir2.1-mediated IK1 currents is a cardiosafety concern during new drug discovery and development. Kir2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of Kir2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human Kir2.1 channels. We reproduced key electrophysiological and pharmacological features known for native IK1, including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the Kir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of this assay authorizes medium throughput screening for cardiosafety purposes and could help to enrich the currently limited Kir2.1 pharmacology.