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The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism., The transition between conducting and non-conducting states of K+ channels has been explained by conformational changes at the intracellular entrance to the conduction pathway. Here authors demonstrate that control over K+ currents in Kir channels is not explained by the canonical pore-gating model, as conduction is not impaired by a constricted inner helix bundle.

Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of voltage-gated potassium channel subtype 11.1 (Kv11.1) as a surrogate for risk. A shortcoming of this approach is that the measured IC50 of Kv11.1 block varies widely depending on the voltage protocol used in electrophysiological assays. In this study, we aimed to investigate the factors that contribute to these differences and to identify whether it is possible to make predictions about protocol-dependent block that might facilitate the comparison of potencies measured using different assays. Our data demonstrate that state preferential binding, together with drug-binding kinetics and trapping, is an important determinant of the protocol dependence of Kv11.1 block. We show for the first time that differences in IC50 measured between protocols occurs in a predictable way, such that machine-learning algorithms trained using a selection of simple voltage protocols can indeed predict protocol-dependent potency. Furthermore, we also show that the preference of a drug for binding to the open versus the inactivated state of Kv11.1 can also be inferred from differences in IC50 values measured between protocols. Our work therefore identifies how state preferential drug binding is a major determinant of the protocol dependence of IC50 values measured in preclinical Kv11.1 assays. It also provides a novel method for quantifying the state dependence of Kv11.1 drug binding that will facilitate the development of more complete models of drug binding to Kv11.1 and improve our understanding of proarrhythmic risk associated with compounds that block Kv11.1.

Background and Purpose: Hydroxychloroquine and chloroquine, alone or in combination with azithromycin, have been proposed as therapies for COVID-19. However, there is currently scant and inconsistent data regarding their proarrhythmic potential in these patients. Moreover, their risk profile in the setting of altered physiological states encountered in patients with COVID-19 (i.e. febrile state, electrolyte imbalances, and/or acidosis) is unknown. Experimental approach: Potency of hERG block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from three separate individuals. Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47±0.07 µM and 3.78±0.17 µM respectively, indicating proarrhythmic risk at concentrations effective against SARS-CoV-2 in vitro and proposed in COVID-19 clinical trials. Hypokalaemia and hypermagnesemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs (i.e. reduced proarrhythmic risk), whereas increased temperature decreased potency of chloroquine and hydroxychloroquine but increased potency for azithromycin. In silico simulations across genetically diverse populations predicted that 17% of individuals exhibit action potential durations >500 ms at the highest proposed therapeutic levels, equating to significant QT prolongation. Conclusion and Implications: Significant proarrhythmic risk is predicted for hydroxychloroquine and chloroquine at doses proposed to treat COVID-19. Clinicians should carefully consider the risk of such treatments, and implement long term QT interval monitoring in trials, particularly in patients with electrolyte imbalances.

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

KCNH2is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reportedKCNH2variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of anyKCNH2missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and anyKCNH2variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of anyKCNH2variant.

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

Current mandated preclinical tests for drug-induced proarrhythmia are very sensitive, but not sufficiently specific. This has led to concern that there is a high attrition rate of potentially safe drugs that could have been beneficial to patients. The comprehensive in vitro proarrhythmia initiative has proposed new metrics based around in silico risk predictions, which are informed, among other things, by measures of human ether-a-go-go-related gene channel (hERG) block kinetics. However, high-throughput patch-clamp systems set to collect these data largely operate at ambient temperature, whereas the simulations for risk prediction are carried out at physiologic temperature. The aims of this study were to: 1) determine to what degree kinetics of drug block of hERG are temperature-dependent, 2) assess the impact of any temperature dependence of drug binding kinetics on repolarization in silico, and 3) identify whether a common set of Q10 scalars can be used to extrapolate kinetic data gathered at ambient to physiologic temperatures for use in in silico proarrhythmic risk prediction. We show that, for a range of drugs, kinetics of block are temperature-dependent and, furthermore, that the degree of temperature dependence is different for each drug. As a result, no common set of Q10 scalars could describe the observed range of temperature dependencies. These results suggest that if accurate physiologic temperature models of the kinetics of drug binding are important for in silico risk prediction, the in vitro data should be acquired at physiologic temperature.

Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of voltage-gated potassium channel subtype 11.1 (K

The hERG (human ether-a-go-go-related gene) encoded potassium ion (K+) channel plays a major role in cardiac repolarization. Drug-induced blockade of hERG has been a major cause of potentially lethal ventricular tachycardia termed Torsades de Pointes (TdPs). Therefore, we presented a pharmacoinformatics strategy using combined ligand and structure based models for the prediction of hERG inhibition potential (IC50) of new chemical entities (NCEs) during early stages of drug design and development. Integrated GRid-INdependent Descriptor (GRIND) models, and lipophilic efficiency (LipE), ligand efficiency (LE) guided template selection for the structure based pharmacophore models have been used for virtual screening and subsequent hERG activity (pIC50) prediction of identified hits. Finally selected two hits were experimentally evaluated for hERG inhibition potential (pIC50) using whole cell patch clamp assay. Overall, our results demonstrate a difference of less than ±1.6 log unit between experimentally determined and predicted hERG inhibition potential (IC50) of the selected hits. This revealed predictive ability and robustness of our models and could help in correctly rank the potency order (lower μM to higher nM range) against hERG.

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide &omega, HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

Introduction The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol. Methods Data were acquired using a ‘step depolarisation’ protocol using manual patch-clamp at ambient temperature. Results Potency, kinetics and trapping characteristics of hERG block for the CiPA training panel of twelve drugs were measured. Timecourse of block and trapping characteristics could be reliably measured if the time constant for onset of block was between ~ 500 ms and ~ 15 s. Seven drugs, however had time courses of block faster than this cut-off. Discussion Here we describe the implementation of the standardized protocol for measurement of kinetics and potency of hERG block for CiPA. The results highlight the challenges in identifying a single protocol to measure hERG block over a range of kinetics. The dataset from this study is being used by the In Silico Working Group to develop models of drug binding for risk prediction and is freely available as a ‘gold standard’ ambient temperature dataset to evaluate variability across high throughput platforms.

© 2017 Elsevier Ltd κ-Hexatoxins (κ-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic κ-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BKCa or KCa1.1) channels, but not their mammalian orthologs. Despite this potent and selective action on insect KCa1.1 channels, we found that the classical KCa1.1 blockers paxilline, charybdotoxin and iberiotoxin, which all block insect KCa1.1 channels, are not lethal in crickets. We therefore used whole-cell patch-clamp analysis of cockroach dorsal unpaired median (DUM) neurons to study the effects of κ-HXTX-Hv1c on sodium-activated (KNa), delayed-rectifier (KDR) and ‘A-type’ transient (KA) K+ channels. 1 μM κ-HXTX-Hv1c failed to significantly inhibit cockroach KNa and KDR channels, but did cause a 30 ± 7% saturating inhibition of KA channel currents, possibly via a Kv4 (Shal-like) action. However, this modest action at such a high concentration of κ-HXTX-Hv1c would indicate a different lethal target. Accordingly, we assessed the actions of κ-HXTX-Hv1c on neurotransmitter-gated ion channels in cockroach DUM neurons. We found that κ-HXTX-Hv1c failed to produce any major effects on GABAA or glutamate-Cl receptors but dramatically slowed nicotine-evoked ACh receptor (nAChR) current decay and reversed nAChR desensitization. These actions occurred without any alterations to nAChR current amplitude or the nicotine concentration-response curve, and are consistent with a positive allosteric modulation of nAChRs. κ-HXTX-Hv1c therefore represents the first venom peptide that selectively modulates insect nAChRs with a mode of action similar to the excitotoxic insecticide spinosyn A. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’

Drug block of voltage-gated potassium channel subtype 11.1 human ether-a-go-go related gene (Kv11.1) (hERG) channels, encoded by the KCNH2 gene, is associated with reduced repolarization of the cardiac action potential and is the predominant cause of acquired long QT syndrome that can lead to fatal cardiac arrhythmias. Current safety guidelines require that potency of KV11.1 block is assessed in the preclinical phase of drug development. However, not all drugs that block KV11.1 are proarrhythmic, meaning that screening on the basis of equilibrium measures of block can result in high attrition of potentially low-risk drugs. The basis of the next generation of drug-screening approaches is set to be in silico risk prediction, informed by in vitro mechanistic descriptions of drug binding, including measures of the kinetics of block. A critical issue in this regard is characterizing the temperature dependence of drug binding. Specifically, it is important to address whether kinetics relevant to physiologic temperatures can be inferred or extrapolated from in vitro data gathered at room temperature in high-throughout systems. Here we present the first complete study of the temperature-dependent kinetics of block and unblock of a proarrhythmic drug, cisapride, to KV11.1. Our data highlight a complexity to binding that manifests at higher temperatures and can be explained by accumulation of an intermediate, non-blocking encounter-complex. These results suggest that for cisapride, physiologically relevant kinetic parameters cannot be simply extrapolated from those measured at lower temperatures; rather, data gathered at physiologic temperatures should be used to constrain in silico models that may be used for proarrhythmic risk prediction.

Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world’s annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors and enzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides.

The Janus- faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high- affinity blockers of invertebrate Ca(2+) -activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked KCa channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nM, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX- Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+) -activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents ( IC(50) similar to 10 mu m) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus- faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.

Vicinal disulfide bridges, in which a disulfide bond is formed between adjacent cysteine residues, constitute an unusual but expanding class of potential allosteric disulfides. Although vicinal disulfide rings (VDRs) are relatively uncommon, they have proven to be functionally critical in almost all proteins in which they have been discovered. However, it has proved difficult to test whether these sterically constrained disulfides participate in functionally important redox transformations. We demonstrate that chemical replacement of VDRs with dicarba or diselenide bridges can be used to assess whether VDRs function as allosteric disulfides. Our approach leads to the hypothesis that not all VDRs participate in functionally important redox reactions. Antioxid. Redox Signal. 19, 1976-1980. © Mary Ann Liebert, Inc.

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (κ-TRTX-Ec2a, κ-TRTX-Ec2b, and κ-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that κ-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K+ (BK Ca) channel currents with IC50 values of 3 to 25 nM. In addition, κ-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K+ currents, but only at significantly higher concentrations. κ-TRTX-Ec2a and κ-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous κ-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, κ-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, κ-TRTX-Ec2a and κ-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BKCa channels. This study therefore establishes these toxins as tools for studying the role of BKCa channels in insects and lead compounds for the development of novel insecticides. Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics.

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.