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Abstract: The Aurora Biomed – organized annual Ion Channel Retreat occurred in June of 2008 and brought together researchers from both academia and industry to share advancements regarding this important field. Topics covered included: ion channels as pain targets, ion channels as disease targets, ion channel screening technologies, ion channels in safety screening, and structure and function of ion channels.

Introduction

Since it’s inception in 2003, the Ion Channel Retreat has covered numerous topics related to ion channels. Over the years, topics and discussions have included ion channels as disease targets (i.e., pain, epilepsy, Alzheimer’s Disease), screening platforms and ion channel cell lines, hERG activity and safety pharmacology, specific ion channel families (i.e., TRP channels), and new technologies which could impact future ion channel research. The 2008 Scientific Advisory Board, which included Min Li (Johns Hopkins School of Medicine), Qiang Lu (WuXi Pharmatech), Juergen Reinhardt (Novartis Institutes for Biomedical Research), Terry Snutch (Neuromed Technologies), & Jean-Pierre Valentin (AstraZeneca), put together an exemplary program. Topics included ion channels as pain targets, ion channels as disease targets, ion channel screening technologies, ion channels in safety pharmacology, and structure and function of ion channels. This year’s group of speakers were from a variety of academic & non-profit organizations, and biotechnology & pharmaceutical companies. Distinguished researchers chaired the different sessions and included Tito Gonzales (Vertex Pharmaceuticals), Umesh Patel (Millipore), Qiang Lu, and Jean-Pierre Valentin. The 6th annual retreat brought together over 90 scientists from various fields of ion channel research, which allowed for a multitude of stimulating discussions, and the sharing of cutting-edge work.

Ion Channels as Pain Targets

After an introduction from Sophia Liang (Aurora Biomed), the Ion Channel Retreat opened with a focused session of work done on ion channels involved in pain.

Dr. Torben Neelands from Abbott Laboratories (Abbott Park, IL) discussed his work on the regulation of TRPV1 channel activity. Electrophysiological recordings of human TRPV1 receptors (expressed in HEK-293 cells) assessed the voltage-dependence of TRPV1 activation by capsaicin and blockade by A-425619, a competitive TRPV1 antagonist. The results showed that A-425619 was five-fold more potent to block capsaicin activation of TRPV1 receptors at negative versus positive potentials. The potency of A-425619 at various capsaicin concentrations was consistent with competitive antagonism of the channel. Apparent voltage-dependence of competitive TRPV1 antagonists is due to voltage-dependent changes in capsaicin affinity rather than an alteration in intrinsic antagonist affinity, thus indicating membrane voltage as a regulator of TRPV1 channel activity.

Dr. Aaron Gerlach from Icagen Inc. (Durham, NC) presented “KCNQ Channels as Targets for the Treatment of Epilepsy and Pain” in which he showed how ICA-27243, a sub-type selective KCNQ2/Q3 opener found active in a variety of rodent in vivo epilepsy and pain models, can reduce seizures and pain. In his studies, ICA-27243 was used to test the effects of selective opening of KCNQ2/3 channels in nociceptive neurons. Results from whole-cell voltage-clamp and current-clamp recordings established the activation potency of the dorsal root ganglion M-current, and robust stimulation along with hyperpolarization of the resting membrane potential respectively. In addition, ICA-27243 was shown to inhibit both spontaneous and capsaicin-stimulated action potentials. The data presented suggests that selective KCNQ2/3 openers can be used as an anti-nociceptive strategy.

Millipore’s Dr. Jeff Clare introduced the importance of voltage-gated sodium channel (Nav) inhibitors in treating a variety of disorders (arrhythmia, pain, epilepsy and bipolar disorder) by characterizing various subtypes of Nav which were sensitive, (TTX-s) or resistant (TTX-r) to tetrodotoxin. His studies involved the use of two automated platforms (Ionworks and PatchXpress) to develop robust and high throughput electrophysiology assays for profiling compounds against the Nav (1.1-1.8) family. In addition, he elaborated on the specificity of molecular interactions between Nav blockers and their binding site within the channels, with data involving the use of both rat and human Nav 1.8 mutants (alanine substitutions) as probes. His findings show that these mutations define S6 region residues involved in both activation and inactivation of Nav 1.8 channels, and also suggest the importance of nearby residues causing differing effects in Nav 1.8 versus Nav 1.2 residue mutants. This panel of mutants could thus prove useful when further exploring the molecular basis of selectivity of recently emerging Nav 1.8-selective inhibitors.

Ion Channels as Disease Targets

Dr. Yi-Xin Qian from Amgen (Thousand Oaks, CA) presented data on Kv1.3 blockers for autoimmune disease in which she hypothesized that blocking of Kv1.3 offers reduced entry of Ca2+ and T-cell activation, affection immune function. An initial library screen with 12000 compounds using IonWorks revealed 258 initial hits (2.1% hit rate) with greater than 30% inhibition. Due to the cross-reactive nature of many compounds with Kv1.1, a selectivity study was also performed on five peptide compounds. From this study, it was shown that one of the compounds (PEG-ShK) retained activity at Kv1.3, and Fc conjugation of OSK1 improved selectivity for Kv1.1. Data from the study revealed that Kv1.1 IC50 values for OSK1 and Fc-L10-OSK1 were 4 and 200-fold higher than Kv1.3 respectively. This suggests the potential of IonWorks as a valuable tool for determining the PK profile of small molecules or peptide inhibitors.

Dr. Steve Goldstein (University of Chicago, Chicago, IL) continued the discussions on Kv1.3 as a disease target through his talk on designer toxins derived from animal venom. In his study, Kaliotoxin (KTX) was isolated from scorpion venom and was shown to inhibit Kv1.1-1.3 and BK channels. His creation of the KcsA-Kv1.3 chimera demonstrated a strong binding potential to the KTX protein. It was found that wild-type KcsA could bind to the phage. By using the KcsA-Kv1.3 chimera, the power of KTX-Kv1.3 phage display in solid phase and cells was tested, which identified a scaffold-based library of approximately 11,220 novel toxins. A new toxin, Mokatoxin, which showed a high percentage of inhibition of Kv1.3, was identified and isolated after panning for phage in solid phase and cells. This strategy of using phage display to develop new toxins has proven useful in isolating new ligands that bind in situ and/or in a state-dependent manner to enhance affinity or decrease cross-reactivity over natural variants.

Dr. Alexei Bagrov from NIH-NIA (Bethesda, MD) discussed his work on the immunoneutralization of heightened levels of endogenous digitalis-like cardiotonic steroids (CTS) for the treatment of preeclampsia and cerebral salt-wasting syndrome. In experiments involving this study, rostafuroxin – a digitoxigenin derivative, was found to disrupt the interaction of endogenous ouabain and adducing on Na/K-ATPase (NKA), and exerts beneficial effects in hypertension. In addition, aldosterone antagonists reduced CTS binding to the NKA and alleviated experimental hypertension. Results from experiments showed that inhibition of protein kinase C (PKC) and activation of cGMP-dependent protein kinase (PKG) (by disruption of NKA interaction), reduced inhibitory effects of CTS on cardiovascular NKA. Thus, CTS represent potential therapeutic targets in hypertension and other diseases.

“Subtype selective T-type calcium channel blockers as a novel antiepileptic therapy” was the subject of Dr. Elizabeth Tringham’s (Neuromed Pharmaceuticals, Vancouver, BC) talk. She noted that certain compounds with various subtype selectivity for different T-type calcium channel isoforms can reduce seizures and treat epilepsy. In her studies involving a novel mouse model, it was shown that both NP1 and NP2 caused a significant time-dependent decrease in spike-wave discharges (SWDs). In more traditional models, another compound NP3 elevated the electroconvulsive seizure threshold by 58% in mice when presented with an electroconvulsive stimulus to induce generalized tonic seizures. Data obtained from this study suggest that T-type blockers may provide broad spectrum antiepileptic therapy across various seizure types.

Dr. Jesus Gonzalex (Vertex Pharmaceuticals Inc., Cambridge, MA) identified small molecule modulators that restore mutant cystic fibrosis transmembrane regulator (CFTR). CFTR mutants, believed to cause cystic fibrosis by affecting channel gating and trafficking, were treated with various compounds to increase the probability of increased trafficking (potentiators) and improve trafficking (correctors). One compound – VRT-325, was shown to increase cell surface receptor density and depolarization. In addition, it was found that another compound, VX-770, increased channel gating and demonstrated a 5-fold increase in trafficking. CFTR potentiators and correctors could have therapeutic benefit in treating cystic fibrosis.

Dr. Gary Desir (Yale University, New Haven, CT) addressed issues regarding the role of Kv1.3 in the regulation of peripheral glucose metabolism. In his studies involving mice, it was demonstrated that Kv1.3 inhibition decreased body weight, and enhanced insulin sensitivity by interacting with synaptotagmin-7 (SYT7) to increase GLUT-4 translocation and glucose uptake in skeletal muscle through a calcium dependent process. Speculation that Kv1.3 and SYT7 are components that regulate calcium-dependent GUT4 traffic and glucose metabolism, suggests that Kv1.3 could perhaps be a new pharmacologic target for the treatment of obesity and type II diabetes.

Gail Robertson from the University of Wisconsin (Madison, WI) presented her research on functional hERG channels comprised of 1a and 1b subunits, and compared it to the current understanding of hERG activity, which is mainly from studies of homo-oligomers of hERG 1a isolate. She showed that hERG heteromeric hERG currents are much larger than homomeric currents and conduct 80% more charge during an action potential, which also corresponds to an increase in rates of activation and recovery from inactivation. These differences were also seen in drug sensitivity to known blockers, such as E-4031. Further studies show that these differences can be attributed to the 1b subunit, suggesting a potential mechanism underlying inherited or acquired long QT syndrome.

Day 2:

Ion Channel Screening Technologies

Xueying Cao (Novartis, Cambridge, MA) discussed the need for high throughput screening for drug-induced QT interval prolongation (LQTS) early in the drug development process. In her talk, she illustrated the optimization of the hERG QPatch profiling, which was done at Novartis, as being part of an integrated cardiosafety assessment which includes preclinical testing, assay logistics, and quality control. In addition, she demonstrated how QPatch16 significantly increased throughput of hERG K+ channel screening by 10 to 20 folds compared to manual patch clamp, and provided gigaseal quality data with rich information content.

A novel cellular and biochemical ion channel assay developed by Invitrogen was described by Stephen Hess. By combining novel cellular engineering strategies with existing and next-generation fluorescent assay technologies, assay failures due to typical technical short falls were substantially reduced. In his discussion, he described the rapid construction of a cellular assay for the TRPM8 channel and the generation of the first commercially available Fluorescence Polarization assay for the hERG cardiac channel. Further use of cryo-preservation and division-arrest approaches to improve the reliability of cell provision to meet high-throughput assay implementation was also discussed.

Dr. Sikander Gill from Aurora Biomed Inc. (Vancouver, BC) introduced/ developed a novel high throughput screening system incorporating the use of synthetic phospholipid vesicles (liposomes) with pore forming proteins that flux K+ ions. Manufactured out of the same material as a cell membrane, membranes of the liposomes are incorporated with purified glycoproteins, solubilized membrane proteins, and cell membranes. Data was presented on the high throughput development for pore forming proteins acquired with Aurora’s Ion Channel Reader (ICR8000).

Fluxion’s (San Francisco, CA) Cristian Ionescu-Zanetti spoke about his company’s new Ion Flux electrophysiology system. This system was developed to address the need for higher throughput electrophysiology with integrated liquid handling modules, decreasing bottlenecks arising from workflow scheduling difficulties. Ion Flux works by integrating microfluidic flow channels and cell trapping sites into SBS-standard 384-well plates; all liquid-handling steps are carried out on the platform, as well as final “readings”. Throughput is maximized by the use of multiple amplifier channels and by recording from groups of cells to increase overall success rate.

Dr. Steve Smith continued the Ion Channel Technology session with data his group at Chantest (Cleveland, OH) generated during their initial validation and implementation of Molecular Devices QPatch-HT platform. The platform was used from the initial steps of clone selection right through to optimization for higher throughput screening. Dr. Smith concluded that there were three main advantages to using the system: increased throughput, less effort per datapoint, and ease-of-use for staff.

Dr. Laszlo Kiss talked in a similar vein about work his group had done at Merck (Rahway, NJ) using the IonWorks Quattro for primary screening. They created “pocket” libraries, mixtures of 10 compounds that would be screening together in a single well, thereby increasing throughput of the assay. If a well met certain criteria, the compounds would the screening individually to determine activity. They found that state-dependent inhibitors could be identified at the primary screen stage, with fewer false positives.

Dr. Howard Zhang (Wyeth Research, Princeton, NJ) and his group developed and validated a 384-well population patch clamp (PPC) Quattro IonWorks assay to screen for Nav1.7 blockers that achieved a relatively high throughput. Compounds with eitherstate-dependent or use-dependent effects were screened. In addition, validation with IonWorks Quattro using directly frozen-thawed cells was performed to further increase throughput and screening efficiency. Overall, results from this study indicate that IonWorks Quattro is a powerful tool for screening Na+ channel blockers (3-4 times the throughput of the single hole IonWorks HT).

Ion Channels in Safety Screening:

Jean-Pierre Valentin (AstraZeneca) opened the Ion Channels in Safety Screening session with an overview of the importance of this subject for all ion channel researchers. There are many examples of ion channels-mediated Adverse Drug Reactions (ADRs) affecting primary vital physiological functions (i.e., cardiovascular, respiratory and CNS systems). He mentioned many examples of ion channel mediating ADRs and discussed the importance of comprehensive pharmacological profiling of NCEs against ion channel targets to identify and reduce ADRs. In addition, he talked about the importance of integrated risk assessment during preclinical and clinical development to making decisions about the future life of the drugs.

Fraser Moss from CalTech (Pasedena, CA) continued the focus of the hERG channel, describing research his group has done to determine some of the underlying mechanisms of LQTS. They compared the effects of known hERG blockers on conventional hERG mutants T623S, S624T, and Y652F, with their wild-type effects. They further probed these differences by UAA mutagenesis, identifying a bacterial tyrosine aminoacyl-tRNA synthetase pair that rescued the expression of a hERG Y652TAG mutant, and produced currents identical to that of wild-type hERG. They are continuing this line of research with other directed mutations.

Dr. Arthur Brown spoke about the new library of ion channel-expressing cell lines that Chantest (Cleveland, OH) is developing for safety and discovery profiling. Presently, they have approx. 62 different cell lines (expecting 104 by end of 2008 and 140 by mid-2009) which have all been validated and optimized for many different screening platforms, including manual patch clamp, automated patch clamp (PatchXPress, QPatch, IonWorks Quattro) and FLIPR. “Books” in the library can be arranged into panels, for different screening profiles (CNS channel panel, arrhythmia channel panel, nAChR channel panel, etc) depending on the needs of the customer. Chantest offers many different screening options, and can work with customers using this library to create specific primary and secondary screens on the panel of interest.

Ralf Kettenhofen from Axiogenesis (Cologne, Germany) talked about the role of mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) in cardiac ion channel safety screening. Some of the techniques used today to study adverse drug interactions in the human heart can be expensive, time-consuming and can provide variable data as a result of impure cardiomyocytes populations. Axiogenesis’ Cor.AT® are produced in large-scale, stored frozen and are ready-to-use upon thawing. Experiments done on Cor.AT® cardiomyocytes show expression of typical cardiac currents; application of many known ion channel blockers lead to responses that are typical of human cardiomyocytes. These results suggest that mESC-CM can be used as a standardized and predictive tool for ion channel screening in safety pharmacology.

Day 3:

After an entertaining and leisurely boat cruise around downtown Vancouver, the third and final day of the Ion Channel Retreat was dedicated to talks revolving around the structure and function of ion Channels. This research into the how’s and why’s of ion channel activity forms the basis of all the work presented earlier during the Retreat, and the speakers from today’s session provided insights not only into the workings of ion channels, but gave us a glimpse of the future of ion channel research.

Dr. Craig January from University of Wisconsin & Cellular Dynamics International (Madison, Wisconsin) opened the session discussing work his group has done looking at paired change reversal mutations and hERG trafficking. Most of these mutations resulted in failure of the channels to traffic to the plasma membrane; however, some mutations led to membrane trafficking but improper functionality, including a subset that did not fully close, leaving channels constitutively open over a wide voltage range. These studies suggested that selective disruption of closed-open gating in hERG channels is related to specific charged amino acid residues.

Dr. Jerryl Yakkel presented data from his work at NIH/NIEHS (Research Triangle Park, North Carolina) on apolipoprotein E-derived peptide interaction with the α7 nicotinic acetylcholine receptor. His data showed that APO-E non-competitively blocked a7 nAChR, but not the mutant α7-W55A. Modeling data suggested that α7-w55A affects desensitization time, and this shown by EP where α7-w55A nAChRs had an increased desensitization time compared to α7-wt. These results provide important structural information for future therapeutic targeting of various neurodegenerative disease, including Alzheimer’s disease and schizophrenia.

The next speaker, from Washington University of St. Louis (St. Louis, Missouri), Jianmin Cui, presented research on Mg+ modulation of the interaction between the voltage sensor and cytosolic domains in BK channel activation. Previous work demonstrated that specific residues in the cytosolic domain of BK channels form the Mg+ binding site. The present work showed that an Asp side chain of the membrane-spanning domain interacts with the Arg213 in trans-membrane segment S4, enhancing voltage-sensing domain activation. Future research on this interaction could provide answers to mechanistic and therapeutic questions.

Dr. Vladislav Zarayskiy from Boston College (Boston, Massachusetts) spoke on the effects of differential ADAR-mediated RNA editing of Orai1 in store-operated Ca2+ channels (SOCs). His research program included siRNA knock-down of Orai1, differential Orai expression in RBL and SMC cell lines, overexpression of ADAR1 in RBL cells, and siRNA knock-down of endogenous ADAR1 in SMC cells. Together, these studies suggest that expression and functional activity of ADAR1 may change the selectivity of SOC channels and how Orai1 may encode different SOCs with ADAR-dependent RNA editing depending on the needs of the cells.

The session continued with Dr. Jun Chen from the Neuroscience Research group at Abbott Laboratories (Abbott Park, Illinois), who spoke about molecular determinants of species-specific activation or blockade of TRPA1 channels. He reported that electrophilic, thopaminal-containing compounds covalently modify cysteine residues, but produce opposite effects in rat (activation) and human (blockade) TRPA1. Using chimeric channels and point mutations, several residues in the upper portion of the S6 transmembrane domains were identified as critical determinants of opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, while equivalent residues of hTRPA1 determine channel block.

Kenneth Byron from Layola University (Chicago, Illinois) discussed his research into the role of KNCQ channels in vascular smooth muscle cells. Little was known about this role until he identified KCNQ currents in both A7r5 (rat aortic smooth muscle cell line) and freshly isolated rat myoctyes. Using RNA interference, functional KCNQ channels in both these sources included KCNQ5 as an essential subunit. In addition, pharmacological tools confirmed KCNQ channels played a role in mesenteric vascular resistance and mean arterial pressure in isolated pressurized mesenteric arteries. Vascular smooth muscle KCNQ channels, and known modulators, could have new therapeutic potential as anti-hypertensive/anti-vasospastic targets.

The final speaker of the session and the Ion Channel Retreat was Dr. Kewei Wang from Peking University & ChemPartner, Beijing, China). He looked at the structural relationship between Cytosolic Kv channel-interacting proteins KChIPs and Kv4 channels to determine A-type K+ current, ISA. The specific binding of KChIPs to the N-terminus of Kv4 results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Dr Wang furthered this research by showing that a single KChIP1 molecule clamps two neighbouring Kv4.3 N-termini in a 4:4 manner, providing possible mechanism for co-assembly disruption and therapeutic treatment of membrane excitability-related disorders.