Molecular Models of Voltage-Gated Potassium and Sodium Channels with Ligands
Crystallographic studies of bacterial K + channels show that the pore-lining inner helices obstruct the cytoplasmic entrance to the closed channel KcsA, but depart in the open channels KvAP and MthK, suggesting a gating-hinge role for a conserved Gly in the inner helix. A different location of the gating swivel and a narrower open pore were proposed for the voltage-gated Shaker K + channels that have the Pro473-Val-Pro motif. Using molecular modeling, we have shown that the open Shaker channel must be as wide as KvAP to accommodate an open-channel blocker correolide. We further built KcsA-, MthK-, and KvAP-based homology models of the Shaker mutant Val476Cys in the locked-open state by Monte Carlo-minimizing energy with distance constraints Cys476--- Cd 2+---His486 known from experiments. The constraints caused only small deformations of the KvAP-based starting structure. The KcsA-based structure resisted the constraints, while the MthK-based structure adopted a KvAP-like conformation. The locked-open Shaker double mutant Val474Cys/Val476Cys readily binds two Cd 2+ ions at Cys474, explaining the size discrepancy between organic and inorganic blockers. The homology model of the open Na + channel was used to search the energetically preferable binding sites of batrachotoxin and veratridine inside the pore. In the energetically optimal complexes, the agonists extend from the selectivity filter to the gate region, but do not occlude the pore, leaving some space for ion permeation. The models explain mutational and electrophysiological experiments and intriguing paradoxes in the structure-activity relationships of ligands. Our results suggest that the pore region is structurally conserved between prokaryotic and eukaryotic voltage-gated ion channels. Supported by CIHR and NSERC, Canada.
Boris S. Zhorov, Ph.D.
Professor of Biophysics
McMaster University
Voltage-gated Sodium Channels: Structures Implicated in Channel Gating and Pharmacology
Todd Scheuer, Ph.D.
Research Associate Professor - Dept. of Pharmacology University of Washington
Strategies for Discovering Blockers of Voltage-gated Sodium Channels
Peripheral nervous system (PNS) voltage-gated sodium (Nav1) channels initiate and propagate the electrical impulses which signal pain responses from damaged nerves and, therefore, Nav1 channels are important targets for developing drugs to treat conditions such as neuropathic pain. However, the identification of acceptable leads for drug development has been hindered in the past by lack of functional high throughput screening (HTS) capabilities for identifying such agents in large libraries of compounds. HTS assays can be established with the PNS Nav1 channels, Nav1.7 and Nav1.8, stably expressed in cell lines using fluorescence resonance energy transfer (FRET) technology with membrane potential sensitive FRET dye pairs and an appropriate plate reader platform, such as the ABS voltage ion probe reader (VIPR), to measure the FRET interactions of these dyes in a high density multi-well format. Counter-screens on other Nav1 channels, such as the Nav1.5 cardiac channel, can be formulated using the same approach. All Nav1 VIPR assays are configured using an appropriate chemical agonist to trigger opening of the target channel following pre-incubation with test compound; subsequently, membrane depolarization occurs and channel blocker activity is quantitated by interference with the depolarization process. Analysis of literature standards validates that this assay configuration renders the HTS most sensitive for identifying a wide range of structurally diverse Nav1 blockers. Additionally, this assay format can be used to support Medicinal Chemistry lead optimization. As an example of these capabilities, a HTS campaign identified a novel Nav1 channel blocker, N-{[2’-(aminosulfonyl)biphenyl-4-yl]methyl}-N’-(2,2’-bithien-5-ylmethyl)succinamide (BPBTS), which is two orders of magnitude more potent (IC 50 for M) than anticonvulsant m Nav1.7 of 0.14 and antiarrhythmic agents currently used to treat neuropathic pain. Block of Nav1 channels by BPBTS is voltage- and use-dependent, and this agent dose-dependently attenuates nociceptive behavior in the formalin test, a rat model of tonic pain. The pharmacokinetic profile of BPBTS was improved upon by synthesis of a related analog, trans-N-{[2’-(aminosulfonyl)biphenyl-4-yl]methyl}-N-methyl-N’-[4 (trifluoromethoxy)benzyl]
cyclopentane-1,2-dicarboxamide (CDA54; IC 50 M against Nav1.7). In two rat models of m of 0.29 neuropathic pain, CDA54 (10 mg kg -1 p.o.) significantly reduces nerve injury-induced behavioral hypersensitivity by 44-67% at doses that do not affect acute nociception, motor coordination or cardiac conduction, suggesting that CDA54 possesses properties that may translate to a greater therapeutic index than that of currently used Nav1 channel blockers. Together, these data support the idea that the outlined HTS strategy can identify structurally novel and potent Nav1 channel blockers that may be used as a template for development of new analgesic agents.
Greg Kaczorowski, Ph.D. Senior Director of Ion Channels Merck
Contribution of the tetrodoxin-resistant sodium channel Na v1.9 to sensory transmission and nociceptive behavior
The transmission of pain signals following injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels that may influence primary afferent excitability. Amongst the voltage-gated sodium (Na V) channels preferentially expressed in nociceptive neurons, Na V1.9 displays unique biophysical properties. Here we examined the contribution of Na V1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene which encodes Na V1.9. Our results confirm that Na V1.9 underlies the persistent tetrodotoxin-resistant current in small diameter dorsal root ganglion neurons, but suggest that this current contributes little to mechanical or thermal responsiveness in the absence of injury or to mechanical hypersensitivity following nerve injury or inflammation. However, the expression of Na V1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior following peripheral inflammation. These results suggest that inflammatory mediators modify the function of Na V1.9 to maintain inflammation-induced hyperalgesia.
Birgit T. Priest, Ph.D. Primary Research Fellow Merck
Voltage gated sodium channels contribute to injury-induced and inherited neuropathic pain syndromes
Sodium channels play a key role in acquired ch annelopathies following nerve and tissue injury, leading to neuropathic pain. Recently, their role in inherited pain syndromes began to emerge. While our group and others have documented a role for Nav1.8 and Nav1.9 channels in nociception, I will focus on our latest findings regarding two channels: Nav1.3 and Nav1.7. Recent evidence from our group has shed light on the contribution of Nav1.3 dysregulation in dorsal horn neurons to neuropathic pain in rodents with spinal cord injury (SCI) or chronic constriction injury (CCI) of the sciatic nerve. Nav1.3 is up-regulated in DRG neurons following axotomy or CCI, and in DH neurons following SCI and CCI. Following axotomy, Nav1.3 is the only sodium channel which has been shown to accumulate at the neuroma where it may participate in initiating ectopic firing. The presence of Nav1.3 in DH neurons following injury leads to exaggerated response of these neurons to stimulation of the receptor fields which is associated with pain behavior in the rats. Anti-sense knock-down of Nav1.3 in DH neurons or neurotrophic-mediated suppression of its expression in DRG neurons reduce the expression of this channel as measured by a combination of molecular, cell biology and electrophysiological assays, and ameliorate neuropathic pain behavior following injury. Beginning in 2004, mutations in SCN9A, the gene encoding Nav1.7 have been described families with erythromelalgia. Nav1.7 is abundantly expressed in small, nociceptive neurons and is present along unmyelinated fibers extending from these neurons. This channel is present within nerve endings, close to trigger zones where receptor potentials are generated. Nav1.7 is characterized by a slow closed-state inactivation leading to the production of a current in response to small, slow depolarizations thus amplifying the signal. Erythromelalgia is an autosomal dominant disorder characterized by burning pain in response to warm stimuli or moderate exercise. We have characterized the effect of the erythromelalgia mutations on the biophysical properties of Nav1.7 and have shown that they cause a hyperpolarizing shift in voltage-dependence of activation and a slowing–down of deactivation which causes tripling of the ramp current. These findings are predicted to lower the current threshold to produce an all-or-none action potential and to increase repetitive firing to a suprathreshold stimulus, both hallmarks of hyperexcitable neurons and neuropathic pain states. Also, we have shown that this channel is up-regulated in DRG neurons in the carrageenan model of inflammatory pain. The totality of this evidence points to Nav1.3 and Nav1.7 as appealing targets for pain treatment.
Sulayman Dib-Hajj, Ph.D. Research Scientist, Dept. of Neurology – Center for Neuroscience & Regeneration Medicine
Yale School of Medicine
New Approaches to Voltage-gated Sodium Channels and Pain
Voltage-gated sodium channel (Na V) blockade contributes to the drug action of class I antiarrhythmic agents, local anesthetics and some anticonvulsants. These drugs, which include mexiletine, lidocaine, and carbamazepine, are also used to treat various chronic pain syndromes, including difficult to treat neuropathic pain. Unfortunately, their utility is limited due to a small therapeutic window. With the identification of different Na V subtypes with distinct distributions and physiological roles, the opportunity exists to generate more targeted Na V blockers that may lead to improved pain therapeutics. Here we illustrate how electrical stimulation coupled with fast FRET voltage-sensitive dyes can enable the identification and optimization of novel Na V blockers that are more potent, and functionally selective compared to existing drugs.
Jesus "Tito" Gonzalez, Ph.D.
Sr. Director, Ion Transport
Vertex Pharmaceuticals
Ziconotide: A New Non-opioid N-type Calcium Channel Blocker for Treating Severe Chronic Pain
Ziconotide (PRIALT®) is a selective blocker of N-type voltage-sensitive calcium channels. Ziconotide was approved by the FDA in late 2004 as a non-opioid treatment for severe chronic pain in patients warranting intrathecal (intraspinal) therapy and who are intolerant or refractory to other treatment, such as adjunctive therapy, systemic analgesics or intrathecal opiates. Approval was based in part on: 1) safety data from 1254 pain patients and 2) analgesic efficacy demonstrated in a placebo-controlled, double-blind, randomized study of 220 patients suffering from pain of numerous underlying etiologies. This study showed ziconotide is analgesic in patients who fail to obtain adequate pain relief from previous oral and/or IT opiate therapy as well as from concomitant oral opiates. Ziconotide does not elicit analgesic tolerance, withdrawal syndrome upon discontinuation, or respiratory depression, nor does it pose a risk of drug dependency. Adverse effects attributed to ziconotide (incidence >10% of ziconotide-treated patients minus placebo-treated patients) include dizziness, ataxia, abnormal gait, confusion, memory impairment, nausea, and asthenia. Moreover, severe psychiatric symptoms may occur with ziconotide treatment. Of 1254 patients treated, 173 have continued ziconotide treatment for at least one year. Ziconotide’s clinical profile is consistent with its unique mechanism of action as determined by extensive non-clinical studies. These studies show ziconotide achieves anti-nociceptive activity by blocking presynaptic N-channels on primary nociceptors, thereby inhibiting entry of noxious signals into the CNS. Ziconotide’s non-clinical pharmacology and therapeutic profile demonstrate that N-channels play a key role in pain perception, validate N-channels as a target for analgesic therapy, and show that ziconotide is a non-opioid alternative for the treatment of severe chronic pain.
George Miljanich, Ph.D.
Head of Analgesia Research, Research Fellow
Elan Pharmaceuticals
T-type calcium channels as targets for the treatment of pain
Voltage dependent calcium channels play an important role in many physiological functions in humans. One variety of voltage gated calcium channel called T-type (or Ca V3.x), which is characterized by relatively rapid activation and inactivation kinetics, a low threshold voltage for activation, has recently become of interest as a possible target for the medical treatment of pain. These channels are expressed in the CNS and PNS, and inhibition of their function by antisense oligonucleotide or pharmacological intervention has been reported to be efficacious in a number of in vivo animal models of pain. While pharmacological blockers of T-type calcium channels are known, they typically lack potency and/or selectivity versus other families of calcium channels. This presentation will describe the discovery of novel potent T-type calcium channel inhibitors which exhibit selectivity over L and N type calcium channels as well as other ion channels and receptors. The ability of these inhibitors to exhibit efficacy in in vivo models of neuropathic pain will also be described.
Neil Castle, Ph.D. Director of Biology
Icagen, Inc
Ion Channels & Drug Safety
How to improve on the safety of new medicines
Adverse drug reactions are, in the USA , the leading cause of death after heart disease, cancer and stroke and are a major concern to the drug discovery industry. Safety issues, non-clinical and clinical, are one of the primary reasons for the failure of new chemical entities during clinical development and withdrawal of marketed drugs following regulatory approval. Inhibition of ion channels, either as a primary mechanism or as secondary effect, can result in serious adverse events. The presentation will review examples of ion channels-mediated functional side effects affecting vital functions (i.e., Cardiovascular, Respiratory and Central Nervous Systems; ICHS7A based definition) as well as the gastrointestinal system. For example block of either the hERG (human ether a-go-go related gene) potassium channel or the cardiac sodium channel can cause arrhythmias. The hERG channel is also involved in regulating the resting membrane potential and excitability of a number of other tissues. It is therefore possible that hERG block may cause drug-induced side effects in organ systems other than the heart. In the central nervous system convulsions are serious side effects of some quinolone antibiotics. Evidence suggests that these side effects may result from blockade of GABAA ligand gated ion channels. Other examples of adverse drug reaction in which ion channels are implicated include diarrhoea, constipation, nausea, hypoglycaemia and pulmonary oedema. A better understanding of the mechanisms underpinning ion channel-mediated drug-induced adverse drug reactions will allow identifying and addressing these issues before candidate drugs reach the clinical stages of development.
Jean-Pierre Valentin, Ph.D.
Director, Safety Pharmacology (Safety Assessment UK)
AstraZeneca
The Role of Cardiac Ion Channels in Safety Pharmacology
Safety Pharmacology encompasses non-clinical studies that investigate the undesirable pharmacodynamic effects of a drug at, and above, therapeutic drug concentrations. Drug action is demarcated using a battery of pre-clinical molecular, cellular, organ/tissue and system level studies to ensure drug safety in the clinic. Studies involve a hierarchy of vital physiological systems (CNS, cardiovascular and respiratory) necessary for survival deemed the “Safety Pharmacology Core Battery ”. The ICH S7A guidelines provide general study paradigms to assess the effects of drugs on vital and other major organ systems. According to the S7A guidelines, drug-induced QT prolongation and Torsades de Pointes (TdeP) arrhythmia risk is an issue that must be addressed. In 2001 the ICH Topic S7B guideline entitled “Guideline on Safety Pharmacology Studies for Assessing the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals” was developed to assess cardiac risk. However, because no single pre-clinical animal model can accurately predict clinical cardiac findings, the integrated risk assessment has been adopted which utilizes the findings from many study models to predict the potential for drug risk (i.e., TdeP) in humans. Therefore, to determine drug safety and assess human risk it is important to understand the role of novel drugs on cardiac ion channels since these channels determine genesis of either the normal cardiac action potential or precipitate development of TdeP (or other arrhythmias). An overview of the effects of drugs on ion channels responsible for cardiac repolarization using in vitro and in vivo pharmacological methods recommended by the ICH will be presented.
Michael K. Pugsley, M.Sc., Ph.D.
Principal Scientist – Pharmacology
Forest Research Institute
Non-clinical proarrhythmia models: predicting Torsades de Pointes
Prolongation of the QT interval and the cardiac action potential have been linked to a potentially fatal but rare tachyarrhythmia known as Torsades de Pointes (TdP). Nonclinical assays, such as those investigating the effect on I Kr (the hERG channel current), prolongation of the action potential duration (APD) and the QT interval, in vivo, have been developed to predict the risk of QT interval prolongation and TdP in man. However there seems to be dissociation between the risk of QT interval prolongation and the torsadogenic risk. There is an increasing mass of evidence showing that an increase in the QT interval does not necessarily lead to TdP. Thus it appears that while standard assays are very good, but not infallible, at predicting the risk of QT interval prolongation in man they do not predict the proarrhythmic risk. Recently there has been a plethora of publications suggesting that there are electrophysiological markers associated with drug-induced TdP other than hERG channel activity, APD and the QT interval, and these markers may be better predictors of TdP.
Here, three in vitro proarrhythmia models are discussed. These proarrhythmia models use electrophysiological markers such as transmural dispersion of repolarization; action potential triangulation, instability, reverse use-dependence; and the incidence of early after-depolarizations to predict the risk of TdP. The models presented have been published widely and show a good correlation to clinical outcome. Each is discussed along with its particular merits and shortcomings; none having shown a predictive value that makes it clearly superior to the others.
Proarrhythmia models, in particular in vitro models, challenge current perceptions of appropriate surrogates for TdP in man and question existing nonclinical strategies for assessing proarrhythmic risk. The rapid emergence of such models, compounded by the lack of a clear understanding of the key proarrhythmic mechanisms has resulted in a regulatory reluctance to embrace such models. The wider acceptance of proarrhythmia models is likely to occur when there is a clear understanding and agreement about the key proarrhythmia mechanisms. Regardless of regulatory acceptance, with further validation these models may still enhance pharmaceutical company decision-making to provide a rational basis for drug progression, particularly in areas of unmet medical need.
Chris Lawrence, Ph.D.
Senior Scientist (Safety Assessment UK)
AstraZeneca
Evaluation of hERG binding and functional assays for use in cardiac safety screening
The hERG (human ether-a-go-go related gene) potassium channel is required for cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and arrhythmias. Due to these potential adverse effects, it is critical to identify hERG blockers early in drug discovery. To this end, competitive binding assays were developed using 3H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG). hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points. Binding assays using HEK293-hERG membranes and 3H-dofetilide were robust (Z'=0.69±0.015, mean±SEM), highly reproducible (test-retest slope=1.04, r 2=0.98) and correlated well with IC 50 values obtained by patch clamp (slope=0.98, r 2=0.89). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC 50 <0.1 µM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate=0.5). Finally, the rubidium efflux assay gave highly reproducible results (Z'= 0.80±0.02, mean ± SEM) that correlated with patch clamp IC 50 values (slope=0.87, r 2=0.73). In conclusion, the hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.
Steve Murphy, Ph.D.
Senior Scientist
Athersys, Inc.
Ion Channel Technologies
A nonradioactive Li influx assay using Atomic Absorption Spectroscopy for identifying sodium channel modulators.
Ion channels are challenging targets in the early phases of the drug discovery process, especially due to the lack of technologies available to screen large numbers of compounds in functionally relevant assays. However, for random screening of compounds against potassium channel targets, a non-radioactive rubidium efflux assays using Atomic Absorption Spectroscopy (AAS) have been utilized successfully. Similarly Li could be used as a surrogate ion for sodium to measure sodium channel activity. Activity of sodium channel blockers in Li influx assay, and in a membrane potential assay will be discussed and compared to electrophysiological data.
Shephali Trivedi, Ph.D.
Sr. Research Bioscientist
Lead Discovery Department
AstraZeneca
NIH Chemical Genomics Center and Future High Throughput Screening
High throughput screening (HTS) in academic laboratories has been significantly increased in last five years. A new era of non-industrial HTS emerged as the recent establishment of NIH Chemical Genomics Center and other NIH sponsored academic screening centers as the part of NIH Roadmap. This effort have focused on the generation of molecular probes for the proteins of research interest and all the results from the screening including the structures and activities of compounds, and assay methods will be available to the academic investigators and public via a web site (PubChem). In this presentation, the application of new screening technologies and screening paradigms in NIH Chemical Genomics Center and the new trend of HTS will be discussed in details.
Wei Zheng, Ph.D. NIH, NHGRI, NIH
Chemical Genomics Center
Cell Free Electrophysiology for High Throughput Screening of the Human Cardiac Sodium-Calcium-Exchanger (NCX1).
We report a cell-free electrophysiology assay for the identification of human sodium-calcium exchanger (NCX1) inhibitors/activators. Being involved in the calcium homeostasis, calcium transporters are related to a number of human diseases. However, present drug discovery technologies often turn out to be inappropriate for investigating these and other membrane transporters due to their low turnover rates. Utilizing IonGates’ SURFE 2R technology, we were able to determine IC50 values of known human sodium-calcium transporter inhibitors with an automated, chip based and cell-free electrophysiology assay. The observed IC50 values were comparable to those from patch clamp and fluorescence experiments.
Sven Geibel, Ph.D.
Lead Identification Technologies
Aventis Pharma Deutschland GmbH
Improving the power of patch clamp – a novel drug application system improves data quality and increases throughput
The whole-cell patch clamp technique remains the gold standard in electrophysiology for characterization of ligand pharmacology at voltage- and ligand-gated ion channels. Such studies require the rapid exchange of test solutions around the recorded cell. Traditional experimental techniques are labour intensive and low throughput. As such there is a need for a system which increases throughput without compromising on the quality of the data. The Dynaflow TM system is a computer-controlled micro-fluidic chip-based platform which combines the traditional whole-cell patch clamp technique, with an ultra-fast multi-channel chip-based perfusion system. We have used the new Dynaflow TM system in a validation study on human a 7 nicotinic acetylcholine receptors (nAChRs) stably expressed in a rat GH4C1 cell line. Due to the rapid desensitization of a 7 nAChRs, it is vital that solution exchange is fast and complete. We have examined the effects of a number of standard agonists and antagonists and also characterised the properties of the reported positive allosteric modulators, galantamine, 5-hydroxyindole and ivermectin. We used both the Dynaflow TM system and the Biologic TM rapid solution changer RSC-160. The Dynaflow TM system increased throughput in terms of number of cells / day with rapid solution exchange resulting in higher data quality.
Gareth A. Jones , Ph.D.
Senior Scientist, Department of Biology, Psychiatry Centre of Excellence for Drug Discovery
GlaxoSmithKline
Identification of novel modulators of ion channel accessory proteins using LEPTICS ® : Kv Channel Case Study
K channel openers have been widely investigated as a potential treatment for a number of disorders including those of the urinary bladder. Voltage-gated (Kv) channels exist as tetrameric pore-forming complexes that are associated with cytosolic accessory proteins. Specific accessory proteins, such as the Kv channel beta subunits (Kvbeta subunits), are able to modulate the channel kinetics of certain pore-forming Kv channel (alpha) subunits. Lectus utilises its discovery engine, based on a proprietary proteomics platform: Leveraged Enabling Technology for Ion Channel Screening (LEPTICS ®), to carry out functional analysis of ion channel protein-protein interactions, including those between Kvbeta subunits and their interacting counterparts on the Kv channel alpha subunit (T1 domain). LEPTICS ® utilises the binding between Kv1.x T1 domains and correctly folded and functional Kvbeta subunit accessory proteins immobilised in a high-throughput format to generate a displacement assay. Supporting data describing the utility of the LEPTICS ® platform will be provided: this will include functional analyses such as binding kinetics. Furthermore, the identification of novel modulators of ion channel protein-protein interactions will also be demonstrated from one of Lectus’s in-house screening programmes identifying novel small molecule modulators of Kv channel T1 domain-Kvbeta subunit interactions. In support of these observations secondary electrophysiological assays, in native detrusor smooth muscle cells, confirm that these novel compounds are able to modulate Kv current kinetics. In conclusion, these data demonstrate a novel approach to modulating ion channel function through the identification of a new class of ion channel modulator with therapeutic potential identical to those of K channel openers.
Cardiac Safety Screening: Flux Assays and in vivo Data
Sikander Gill, Ph.D.
Director of Assay Development
Aurora Biomed Inc.
Novel Antagonists of Human Kv4.3: Why Glowing Worms Make a Good Ion Channel Screen
A novel high content ion channel assay is presented, which permits screening of 200.000 compounds per month in a 96-well format against human voltage gated and fast deactivating potassium channel Kv4.3 in transgenic C. elegans nematodes. Human Kv4.3 is an important target for the treatment of heart arrhythmia. Three novel lead series have been identified from Devgen’s focused chemical library. Hit compounds have been verified on human Kv4.3 / KChip2 in mammalian cell patch clamp experiments with a rate of >80%, eight times better than the industry standard of 10%. Furthermore this physiological assay enables the identification of compounds affecting any conformation of the ion channel under investigation during thousands of activity cycles, while deselecting general ion channel blockers, non-bioavailable compounds and unstable compounds. This enables savings of cost and time for the generation of tractable novel scaffolds. Devgen’s current lead molecules show 0.16-2.5 µM antagonistic activity on human Kv4.3, with a selectivity of 30 to >50 fold against unwanted hERG activity and no apparent activity on sodium channels (SCN5a). These compounds are expected to show beneficial activity in atrial fibrillation, a huge unmet medical need due to hERG related side effects of current pharmacological options.
Andreas Kopke , Ph.D.
Head of Pharma Business Development Devgen
A knowledge-Based Strategy to Improve Ion Channel Drug Discovery
A vast amount of data has been published in the literature (journals and patents) describing biological activities for a large number of ligands acting on ion channels. These data remain poorly accessible due to the heterogeneity of the data sources and in addition, the solutions available to access published structure-activity data are not convenient. To address these issues, we have built a unique and comprehensive knowledge-base resource regrouping all the data already published on ion channel ligand pharmacology. Chemical structures of ligands as well as precise description of targets and of all type of in vitro and in vivo pharmacological responses can be retrieved via an easy-to-use web-based query system. Our system contains 30,000 ligands with 150,000 biological activities obtained from 3,500 literature references. Such a structured database containing the knowledge accumulated within years on ion channel pharmacology can be used to choose, orient and accelerate drug discovery programs. A typical example of such a strategy can be illustrated by the use of our database to improve a virtual screening experiment. Querying the database to retrieve a training set of molecules sharing a very specific activity and selectivity profile is a quick one step process. As the chemical space explored will be very large, this approach allows the researcher to improve the hit rate obtained by screening internal or external compound databases.
François Petitet
Aureus Pharma
Studies on K+ channels using the Non-radioactive Rb+ Efflux Assay
Ion channels are very attractive drug targets in a wide variety of disease areas such as cardiovascular, CNS, and metabolic diseases. The gold standard for the study of ion channel targets is electrophysiology. However, the low throughput of this method makes it impossible to screen large numbers of compounds. Recently, a variety of high throughput technologies have become available for ion channel target screening. Functional non-radioactive ion flux assays using atomic absorption spectroscopy is one of theses technologies. This presentation will review our experience of using Rb-efflux assays for K+ channel screening. Screening was performed in a 384-well plate format on a Beckman automation system and the measurement of Rb+ was done using an ICR8000. Application of cryopreserved cells to the Rb+-efflux assay will also be discussed.
Mei Ding, Ph.D.
Research Scientist - Lead Discovery Sciences
AstraZeneca R&D Mölndal - Sweden
Ion Channels as Drug Discovery Targets
Ion Channel Drug Discovery in Non-Excitable Cells
Michael Xie, Ph.D. Associate Director, Biology (Lead Scientist – Ion Channel Group) Synta Pharmaceutical
General Anesthetic Modulation of GABA A Receptor Function: Implications for Conformational Changes During Channel Gating
Most general anesthetics act in the micromolar to millimolar concentration range. At these concentrations these hydrophobic drugs bind in crevices and cavities in many proteins making it difficult to define the specific molecular targets responsible for inducing the amnesia, analgesia and immobility that characterize the state of general anesthesia. Considerable evidence indicates that the GABA A receptors are important molecular targets for many general anesthetics. Propofol, a widely used intravenous anesthetic, appears to bind between the GABA A receptor b subunit M2 and M3 transmembrane segments. In the resting channel state, thermal motion induces significant conformational changes in the channel’s transmembrane domain. These conformational changes and similar ones that occur as the receptor is activated cause the formation of crevices and cavities in the transmembrane region that fill with water. Anesthetics may preferentially partition into these cavities stabilizing the receptor structure closer to the open state thereby increasing the open probability and potentiating the effects of submaximal GABA concentrations. The recent publication of a 4-Å resolution structure of the homologous nicotinic Acetylcholine (ACh) receptor in the closed state provides a structural framework for understanding our results. Our disulfide crosslinking and substituted-cysteine-accessibility method (SCAM) studies that provide experimental support for the applicability of the ACh receptor structure to the GABA A receptor will be reviewed.
Myles Akabas , MD, Ph.D.
Associate Professor, Department of Physiology & Biophysics and Neuroscience
Albert Einstein College of Medicine
Genetic & Functional Studies of C. elegans K + channels: KCNQ Orthologs
Fundamental advances in the molecular and structural biology of potassium channels have been made, but relatively less is known about the in vivo roles of potassium channels in the context of whole organisms. The nematode Caenorhabditis elegans possesses 69 genes resembling potassium channel subunits, including orthologs for all major human potassium channel types. We are using C. elegans as an attractive model organism for a systematic analysis of potassium channel function, through a combination of genetic and electrophysiological techniques. Forward genetic screens have identified behavioral mutants linked to many of the major classes of C. elegans potassium channel genes, including members of both the 4-transmembrane and 6-transmembrane classes of subunits. A review of these studies will be provided, with specific examples of Kv3 (egl-36) and EAG (egl-2) orthologs. Reverse genetic studies of KCNQ orthologs (kqt-1,-2, -3) will also be presented, highlighting conserved features of channel regulation. In general, gain-of-function mutations are associated with the most dramatic mutant phenotypes, whereas null mutations result in subtle or wild-type phenotypes. This suggests a significant degree of functional redundancy in the usage of potassium channels in C. elegans, which may allow compensation for the loss of any particular potassium channel, but not its abnormal activation. Implications of these findings to the use of C. elegans in drug discovery will be discussed.
Aguan Wei, Ph.D.
Research Assistant Professor of Neurobiology, Dept. of Anatomy and Neurobiology
Washington University St. Louis
Ion Channels and Diseases
Lead Discovery for Ion Channels and Transporters using 1536-uHTS
Jorg Juser, Ph.D.
PH-R-EU Molecular Screening Technology
Bayer HealthCare AG
Dead Channels Walking: Physiological Silencing of Leak Conductance by Sumoylation
Reversible, covalent modification with small ubiquitin modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the pathway is shown to operate at the plasma membrane to control ion channel function. SUMO conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 (a.k.a., TWIK1 and hOHO) had not previously shown function despite mRNA expression in heart, brain and kidney and sequence features like other two-P-loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly-rectifying channel regulated by reversible peptide linkage.
Steve A.N. Goldstein, MA, M.D., Ph.D.
Professor & Chair, Dept. of Pediatrics
Physician & Chief, Comer Children’s Hospital
Director, Institute for Molecular Pediatric Science
Potassium channels: shared therapeutic target for multiple sclerosis, type-1 diabetes mellitus and rheumatoid arthritis
George Chandy, Ph.D.
Professor, Physiology and Biophysics
UC Irvine
Cardiac Ion Channel Diseases: New Genes and New Mechanisms for Causing Arrhythmias.
The causes of acquired and inherited cardiac ion channel-arrhythmia diseases continue to expand with new mechanisms being identified. Beginning with congenital long QT syndrome, at least 11 inherited cardiac and multi-organ diseases are now linked to gene mutations in sodium, calcium or potassium channels, or signaling molecules. A consequence is that inherited arrhythmias are increasingly complex polygenetic and multi-mechanistic syndromes, and these diseases have been key in advancing our understanding of the genetic and molecular basis for them. In contrast, acquired arrhythmias such as drug-induced long QT syndrome remain mainly a monogenic problem arising from suppression of hERG potassium channels. However, new findings are adding complexity to this with the recognition of novel mechanisms for modulating or reducing hERG current. Reduced hERG current occurs directly through drug block of the channel pore, however, it also can occur indirectly through drug-induced abnormalities in ion channel protein trafficking through the secretory pathway and under some conditions the ion channel’s protein stability can be altered. This presentation will review the current understanding of the mechanisms that cause congenital and acquired cardiac arrhythmia syndromes.
Craig January, MD, Ph.D.
Professor of Medicine and Physiology
University of Wisconsin-Madison
Opportunities for drug discovery in trafficking systems and expression signals that control ion channel surface expression.
The regulation of the plasma membrane expression of ion channels provides a powerful means by which channel activity may be altered, and trafficking system elements may form potentially important access points at which pharmaceutical agents can be designed to regulate channel activity. Here I will describe the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increases outward potassium currents in cells stably expressing Kv1.5, as does treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, doubles Kv1.5 currents in cells and sustained potassium currents in isolated rat atrial myocytes. These increased currents are blocked by 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, show that this increase of potassium current density is caused by a redistribution of channels toward the plasma membrane. Co-immunoprecipitation experiments demonstrate a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which requires an intact SH3-binding domain in the Kv1.5 N-terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for our understanding of the way Kv channel surface expression is regulated.
David Fedida, Ph.D. Professor, Dept. of Physiology
Univ. of B.C. & Cardiome Pharma
The First Phase I Human Gene Transfer Trial of Ion Channel Therapy for the Treatment of Erectile Dysfunction: Preliminary Results
Objective: To test the safety of a single intracavernous injection of a plasmid vector (hMaxi-K) that expresses the hSlo -subunit of the Maxi-K channel, for the treatment of a gene, that encodes the erectile dysfunction (ED). We have endeavored to develop an improved treatment for ED based on more than a decade of mechanistic insight into the physiology, pharmacology and electrical excitability of corporal smooth muscle cell tone. The goal of this report is to briefly outline the background, rationale and extensive preclinical testing that led to the development of ion channel gene transfer with hMaxi-K, as well as to review the regulatory pathway and preliminary clinical trial results. Gene transfer using ion channel therapy specific to the smooth muscle organ of interest offers a potential new treatment with a duration of action that may last for months without advanced planning. Moreover, the pre-clinical study data have shown no contraindications to its use in man. Furthermore, published results of those studies suggest that the therapy may be effective in ameliorating the ED associated with diabetes and aging. The mechanism of action of this novel therapy uses a final common pathway, alteration of smooth muscle cell excitability, for its effect. The preclinical evidence showing safety, effectiveness and long duration of action has been highly significant , and thus, we proposed that the hMaxi-K vector also be injected intracavernously as naked DNA in the clinical trial. Naked DNA is acknowledged as an extremely safe vector without the significant potential side effects of viral vectors. Nonetheless, because ED is a nonfatal disease, stringent requirements needed to be met before the Food and Drug Administration (FDA) granted permission for the use of naked DNA in a human trial. The reasons that naked DNA is not chosen routinely as a vector in gene transfer protocols to treat cancer and genetic illness are the reported lack of efficiency in transfer into the cells of interest, as well as the reportedly short duration of effect for those indications.However, our preclinical studies in a rodent model of aging suggested that the therapeutic effect produced by this plasmid may remain for up to 6 months after a single injection Similarly in a rat model of diabetes, the same effect was found to last for at least 4 months. This activity of hMaxi-K may represent a distinct advantage over current drug therapy for ED, where there is a need for on-demand treatment that provides only a temporary effect.
Methods : Six men who fulfilled the entry criteria received hMaxi-K. Three received the lowest dose and three received an intermediate dose of the gene product, injected intracavernously as naked DNA.
Results : No drug-related adverse events occurred after the gene transfer.
Conclusion : Preliminary results indicate that, in a single dose escalation study, ion channel gene transfer with hMaxi-K can be administered to men with ED without adverse events.
Arnold Melman , MD
Professor & Chairman, Dept. of Urology Albert Einstein College of Medicine
PAP-1, a selective Small Molecule Kv1.3 Blocker
The lymphocyte K + channel Kv1.3 constitutes an attractive new pharmacological target for the selective suppression of terminally differentiated effector memory T (T EM) cells in T-cell mediated autoimmune diseases such as multiple sclerosis and type-1 diabetes. Despite Kv1.3’s obvious therapeutic importance selective small molecule blockers of this channel have so far not been identified. Most of the published compounds such as correolide, PAC and our own compound Psora-4 are potent Kv1.3 blockers but lack selectivity over the cardiac K + channel Kv1.5 raising concerns about potential side effects in vivo. By further exploring the structure-activity relationship of new Psora-4 derivatives through a combination of classical medicinal chemistry and whole-cell patch-clamp we recently identified a series of phenoxyalkoxypsoralens, which exhibit 2-50-fold selectivity for Kv1.3 over Kv1.5 depending on their exact substitution pattern. While compounds bearing methoxy-, alkyl-, halogen or several nitro-groups on their side-chain phenyl ring exhibit poor selectivity over Kv1.5, compounds with one nitro-group, no substituents or a phenoxy moiety display increasing selectivities towards Kv1.3. The most potent and “drug-like” compound of this series, 5-(4-phenoxybutoxy)psoralen (PAP-1), blocks Kv1.3 in a use-dependent manner with a Hill coefficient of 2 and Kd- value of 2 nM by binding to the C-type inactivated state of the channel. PAP-1 is 22-fold selective over Kv1.5, 35-fold selective over Kv1.1 and Kv1.4 and 125-fold selective over Kv1.2 and more than 1000-fold selective over HERG, Kv3.1, Kv3.2, calcium-activated K + channels, Na +, Ca 2+ and chloride channels. PAP-1 and several of its derivatives therefore constitute excellent new tools to further explore Kv1.3 as a target for immunosuppression in various animal models and could potentially be developed into orally available immunomodulators.
Heike Wulff, Ph.D.
Medical Pharmacology and Toxicology
University of California
Poster Sessions
Interaction of MinK and MiRP2 with KCNQ1 potassium channel S6 domain
KCNQ1 a subunits form functionally distinct voltage-gated potassium channels in the heart and colon by co-assembling with transmembrane ancillary subunits MinK and MiRP2, respectively. Mutations in any of the three subunits are sufficient to impair excitable cell function, and are associated with inherited human disease. A series of three contiguous transmembrane residues represented in both MinK (residues 57-59) and MiRP2 (residues 71-73) mediate control of gating (Melman, Y.F., Domenech, A., de la Luna, S., and McDonald, T.V. (2001) J. Biol. Chem. 276:6439-6444) via unknown residues within KCNQ1. Here, we performed tryptophan-scanning mutagenesis within the KCNQ1 S6 pore-lining domain to probe its interaction with MinK and MiRP2. The tryptophan-tolerance of three neighboring KCNQ1 positions (338-340) suggested a membrane-embedded pocket that might accommodate interaction with MinK and MiRP2. Mutations 338-340W all increased inactivation of homomeric KCNQ1 channels; F340W also altered activation by rendering it largely constitutive. Coexpression and functional evaluation of channels formed with KCNQ1 tryptophan mutants, and wild-type or mutant MinK or MiRP2, suggested KCNQ1-F339 is important in mediating the gating effects of MiRP2. KCNQ1-F340 appeared instrumental in coordinating the gating effects of MinK, probably via MinK-T58. In conclusion, we propose a model in which MinK and MiRP2 activation triplets exert effects on gating via neighboring aromatic residues F339 and F340 in the KCNQ1 S6 domain.
Gianina Panaghie, Ph.D.
Department of Medicine and Pharmacology
Cornell University
Prenatal Alcohol Exposure and Glutamatergic Synaptic Dysfunction
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype glutamate receptors are known to play a crucial role in information processing (memory) in the mammalian brain. Ethanol (EtOH) has been shown to act on several targets in the brain including the hippocampal glutamatergic synapses. Prenatal ethanol exposure in humans is associated with a variety of behavioral and physiological abnormalities including learning and memory deficits. The majority of studies investigating the neurotoxicity of EtOH in the hippocampus have focused on inhibition of N-methyl-D-aspartate (NMDA) subtype glutamate receptors. However, the effects of ethanol on AMPA receptors at CA-1 region of the hippocampus are not well studied. In the present study, we have examined the AMPA-miniature excitatory postsynaptic currents (mEPSCs) of the CA-1 region of the hippocampus of prenatal alcohol exposed (binge-like) 30 day old rats, using the whole-cell-patch-clamp technique. Hippocampal slices from ethanol-exposed rats showed significant reduction in mEPSCs frequency and amplitude by 65-75% and 30-40% respectively. Eventhough neurotoxic insults caused by teratogens are stable and permanent, some nootropic drugs have been shown to be effective in alleviating such insults. Therefore, the effects of 10 day aniracetam treatment (PND 18-28) on the prenatal alcohol exposed rats were investigated. Interestingly, aniracetam partly compensated for the reduced amplitude and frequency of AMPA-mEPSCs in alcohol exposed pups. These data indicate that prenatal ethanol exposure may result in abnormal hippocampal AMPA receptor mediated glutamatergic neurotransmission. Additionally, the aniracetam treatment proves to be effective in partly correcting this abnormality and thereby can reduce the associated cognitive deficits in fetal alcohol syndrome.
Nayana Wijayawardhane, Ph.D.
Department of Pharmaceutical Sciences - School of Pharmacy
Auburn University
Application of the FLIPR platform to the generation of K+ channel assays suitable for HTS.
K + channels form a diverse group of distinct ion channel families which play critical roles in a wide variety of physiological processes, including heart rate, muscle contraction, neurotransmitter release, neuronal excitability, insulin secretion, epithelial electrolyte transporter, cell volume regulation, and cell proliferation. Over the last decade, more than 80 K+ channel-related genes have been identified. This, coupled with the progress toward understanding the distribution, the molecular composition and the contribution of K+ channels to native currents, has made K+ channels increasingly attractive as therapeutic drug targets. In this work we illustrate the application of Fluorimetric Imaging Plate Reader (FLIPR) platform to the generation of cell based assays for different K+ channel classes, suitable for high throughput screening (HTS) of compounds. In particular we have focused our attention on the: Kv2.1 channel, belonging to the voltage-gated family, EAG2, belonging to the voltage–gated ether a go-go family, and Kir6.2-SUR1 and Kir6.2-SUR2A, belonging to the K ATP sensitive family. We have stably transfected CHO Dukx or CHO K1 with the human cDNAs and we have used FLIPR technology to develop functional assays, suitable for HTS. Kv2.1 and EAG2 are voltage gated, outward delayed rectifier, non-inactivating potassium channels; to detect their functionality we have used KCl injection, able to provoke a strong and sustained channel dependent cell membrane depolarization, recorder as RFU (relative fluorescent units) increase after loading the cells with a membrane potential sensitive dye. Kir6.2 is a weak potassium inward rectifier ATP sensitive K+ (K ATP) channel which assembles as heteromultimers with the SUR (sulfonylurea) receptors. Functionality of both Kir6.2–SUR1 and Kir6.2-SUR2A could be assessed as a strong cell membrane hyperpolarization obtained upon injection of the respective specific channel openers: Diazoxide and Pinacidil; while second injection of a channel blocker, such as Glybenclamide, showed an inhibition of the previously evoked current. All the FLIPR data were validated by electrophysiological experiments. We have demonstrated that FLIPR technology may represent a very sensitive and reliable tool for the study of K + channel functionality. Furthermore FLIPR based assays result to be particularly robust and suitable for high throughput screening of molecules, for the identification of specific channel modulators.
Alberto di Silvio, Ph.D.
Cell Biology
Axxam
Electrophysiological and Pharmacological Characterization of a Cell Line Stably Expressing hKCNQ1 and hKCNE1 Potassium Channels.
KCNQ1 and KCNE1 K+ channel subunits reconstitute the kinetics and pharmacology of the slowly activating cardiac delayed rectifier potassium current, IKs. Mutations in either subunit may underlie inherited long Q-T syndrome (LQTS), a condition which can lead to fatal ventricular arrhythmias. Given the difficulties of studying IKs in native myocytes, a mammalian cell line to model IKs would be highly desirable. hKCNQ1 and hKCNE1 were stably expressed in HEK293 cells under the control of Zeocin and G418 selection antibiotics, respectively. Step depolarizations from a holding potential (Vh) of -50 mV to potentials exceeding -30 mV elicited prominent, slowly activating outward currents which, upon repolarization to Vh, scaled with slowly deactivating tail currents. Activating and deactivating IKs currents were potently blocked by the IKs inhibitor L-768,673 (2.4 nM, IC50). A small, variable background current resistant to the IKs inhibitor lacked time-dependent kinetics. The current magnitude and activation kinetics of the time-dependent IKs current were highly sensitive to temperature (20-36°C). Following a 1-sec step to +50 mV, the averaged peak tail current at -50 mV was 1.1 ± 0.4 nA at 36°C and decreased to 0.17 ± 0.01 nA at room temperature (20-23°C). Fitting of the Boltzmann equation to tail current amplitudes following increasingly depolarized 1 sec voltage steps yielded a mid-point of current activation of 2.9 mV with a slope factor of 12.2. mV at 35°C. The time-dependent kinetics of the activating IKs current at 35°C was accurately fit with a modified 2nd order Hodgkin-Huxley gating model with two time constants. A fast and a slow time constant decreased e-fold every 56 ± 5.3 mV and 57 ± 6.4 mV, had mean values of 71 ± 8 msec and 268 ± 30 msec at + 40 mV, and showed Q10 values of 9.5 ± 1.2 and 3.2 ± 0.3, respectively. The rate of exponential decay of the IKs current upon repolarization to -50 mV was also highly temperature sensitive with a Q10 value of 4.7 ± 0.5. The deactivation time constant increased e-fold every 40 ± 5 mV over the range of -100 to -50 mV. Tail currents reversed at -72 ± 2 mV and the reversal potential was shifted -32 mV by reduction of extracellular K+ from 4 mM to 1 mM. The pharmacological, gating, and reversal potential parameters examined in this study agree well with those of published reports of native IKs current. Stable coexpression of KCNQ1/KCNE1 in HEK293 cells serves as a useful model system to study IKs gating and pharmacological inhibition.
John P. Imredy Ph.D. Preclinical Safety Evaluation
Merck & Co.
