Cell-based assays and instrumentation for screening ion-channel targets

Ion channels are an important class of drug targets. They comprise the molecular basis for essential physiological functions including fluid secretion, electrolyte balance, bioenergetics and membrane excitability. High-throughput screening for ion-channel function requires sensitive, simple assays and instrumentation that will report ion channel activity in living cells. This article will review relevant assay technologies for ion channels and describe voltage-sensitive probes and instruments based on fluorescence resonance energy transfer (FRET) that enable ion-channel drug discovery.     

High-throughput screening technologies for ion channel drug discovery

Ion channels are attractive targets for drug discovery as an increasing number of new ion channel targets have been uncovered in diseases, such as pain, cardiovascular disease, and neurological disorders. Despite their relevance in diseases and the variety of physiological functions they are involved in, ion channels still remain underexploited as drug targets. This, to a large extent, is attributed to the absence of screening technologies that ensure both the quality and the throughput of data. However, an increasing number of assays and technologies have evolved rapidly in the past decades. In this review, we summarized the currently available high-throughput screening technologies in ion channel drug discovery.     

Exploiting high-throughput ion channel screening technologies in integrated drug discovery

Ion channels are increasingly being implicated in disease. Although existing drugs that modulate channel function currently represent a key class of pharmaceutical agents, future ion channel drugs could help to treat an even wider variety of diseases. Despite their disease relevance, ion channels remain largely under exploited as drug targets, chiefly resulting from the absence of screening technologies that provide the throughput and quality of data required to support medicinal chemistry. Although some technical challenges still lie ahead, this historic bottleneck in drug discovery is now being bypassed by newer technologies that can be fully integrated into the early stages of drug discovery and will allow the discovery of novel therapeutic agents. Sequencing the human genome has greatly added to the number of potential drug targets but selecting suitable ion channels for drug discovery research should be based on the potential therapeutic relevance of the channel and not just the availability of suitable screens. Currently, ion channel drug discovery is focused on the need to identify compounds that can provide tractable starting points for medicinal chemistry. Advances in laboratory automation have brought significant opportunities to increase screening throughput for ion channel assays but careful assay configuration to model drug-target interactions in a physiological manner remains an essential consideration. Ion channel screening platforms are described in this review to provide some insight into the variety of technologies available for screening, together with some of their inherent advantages and limitations.     

Automated electrophysiology in drug discovery

Ion channels play essential roles in nervous system signaling, electrolyte transport, and muscle contraction. As such, ion channels are important therapeutic targets, and the search for compounds that modulate ion channels is accelerating. In order to identify and optimize ion channel modulators, assays are needed that are reliable and provide sufficient throughput for all stages of the drug discovery process. Electrophysiological assays offer the most direct and accurate characterization of channel activity and, by controlling membrane potential, can provide information about drug interactions with different conformational states. However, these assays are technically challenging and notoriously low-throughput. The recent development of several automated electrophysiology platforms has greatly increased the throughput of whole cell electrophysiological recordings, allowing them to play a more central role in ion channel drug discovery. While challenges remain, this new technology will facilitate the pharmaceutical development of ion channel modulators.     

Dynamic monitoring of beating periodicity of stem cell-derived cardiomyocytes as a predictive tool for preclinical safety assessment

BACKGROUND AND PURPOSE

Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process.

EXPERIMENTAL APPROACH

Beating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias.

KEY RESULTS

This system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes.

CONCLUSIONS AND IMPLICATIONS

Microelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment.     

Mephedrone, a new designer drug of abuse, produces acute hemodynamic effects in the rat

Mephedrone (4-methylmethcathinone) is a new and popular drug of abuse widely available on the Internet and still legal in some parts of the world. Clinical reports are now emerging suggesting that the drug displays sympathomimetic toxicity on the cardiovascular system but no studies have yet explored its cardiovascular effects. Therefore we examined the effects of mephedrone on the cardiovascular system using a combination of in vitro electrophysiology and in vivo hemodynamic and echocardiographic measurements. Patch clamp studies revealed that mephedrone, up to 30 μM, had little effect on the major voltage-dependent ion channels of the heart or on action potentials recorded in guinea pig myocytes. Subcutaneous administration of mephedrone (3 and 15 mg/kg) to conscious telemetry-implanted rats produced dose-dependent increases in heart rate and blood pressure which persisted after pre-treatment with reserpine. Echocardiographic analysis demonstrated that intravenous injection of mephedrone (0.3 and 1 mg/kg) increased cardiac function, including cardiac output, ejection fraction, and stroke volume, similar to methamphetamine (0.3 mg/kg). We conclude that mephedrone is not directly pro-arrhythmic, but induces substantial increases in heart rate, blood pressure and cardiac contractility and this activity contributes to the cardiovascular toxicity in people who abuse the drug.     

Screening lead compounds for QT interval prolongation

The late detection of cardiotoxic side effects, such as QT prolongation, induced by compounds of pharmacological interest can dramatically impede drug discovery and development projects, and consequently increase their cost. The launch of new drugs with undetected cardiotoxic side effects could have hazardous consequences and could trigger lethal cardiac dysrhythmias in patients. It is desirable, therefore, to test for the potential cardiotoxic side effects of compounds at an early stage of drug development. Electrophysiological test systems and cellular-based fluorometric high-throughput assays are now available for cloned human cardiac ion channels. These test systems are important tools in the preclinical safety evaluation of drugs and newly developed compounds.     

Ions in the fire: recent ion-channel research and approaches to pain therapy.

Ion channels form a diverse and sophisticated collection of membrane-bound proteins. They are influenced by many endogenous compounds and physiological stimuli and modulate neuronal activity. It is thus not surprising that they provide attractive targets for the design of novel therapeutics. In this article, recent ion channel research and its relevance to modulation of sensory transmission is assessed. In pain research, specific blockade or activation of ion channels has long been considered a desired route for identification of analgesics. Historically, this has proven difficult to attain due to the incidence of side-effects associated with most ion-channel modulators. The recent discovery of several novel classes of ion channels, each of which has a specific distribution and role in sensory processing and nociception, has provided a plethora of targets for pharmaceutical intervention with the promise of an improved therapeutic index.     

Neuronal nicotinic acetylcholine receptors as drug targets

Neuronal nicotinic acetylcholine receptors (nAChRs) are an important class of ion channels that have been associated with a number of neurological conditions. A great deal of research has been focused on attempting to understand the exact physiological role of these receptors. As drug targets, the nAChRs are quite complex, both in their structure (multiple receptor subtypes) and their physiological function. Initially, the difficulty encountered in identifying small-molecule modulators led to doubts about the validity of this class of receptors as drug targets. More recently, in vitro and in vivo data, homology modelling, and the identification of small-molecule agonists, have confirmed nAChRs as valid drug discovery targets. In fact, several compounds are now in clinical development for the treatment of pain, smoking cessation and cognitive disorders.     

Neuronal nicotinic acetylcholine receptors as drug targets

Neuronal nicotinic acetylcholine receptors (nAChRs) are an important class of ion channels that have been associated with a number of neurological conditions. A great deal of research has been focused on attempting to understand the exact physiological role of these receptors. As drug targets, the nAChRs are quite complex, both in their structure (multiple receptor subtypes) and their physiological function. Initially, the difficulty encountered in identifying small-molecule modulators led to doubts about the validity of this class of receptors as drug targets. More recently, in vitro and in vivo data, homology modelling, and the identification of small-molecule agonists, have confirmed nAChRs as valid drug discovery targets. In fact, several compounds are now in clinical development for the treatment of pain, smoking cessation and cognitive disorders.     

Subtype-selective targeting of voltage-gated sodium channels

Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future.     

Nonradioactive rubidium ion efflux assay and its applications in drug discovery and development

The recent sequencing of the human genome has created comprehensive information of all potential drug targets. Based on current estimations for the total number of genes, around 400 poreforming ion channel genes can be expected corresponding to about 1.3% of the human genome. Since many ion channels are involved in diseases and the currently marketed drugs act only on a small fraction of these pore-forming membrane proteins, there is a big opportunity for innovative ion channel drug discovery. In fact, recent advances in the development of functional ion channel assays are currently enabling a more systematic exploitation of this important target class. In particular, fluorescence-based methods, automated electrophysiology, and ion flux assays are most important in this regard. This article will briefly describe these methods focusing on the nonradioactive Rb(+) efflux assay that I developed in the early 1990s since it has found widespread application in drug discovery and development and greatly displaced (86)Rb(+) assays for the analysis of K(+) and nonselective cation channels in the pharmaceutical industry.     

Mechanisms of arsenic-induced prolongation of cardiac repolarization

Arsenic trioxide (As(2)O(3)) produces dramatic remissions in patients with relapsed or refractory acute promyelocytic leukemia. Its clinical use is burdened by QT prolongation, torsade de pointes, and sudden cardiac death. In the present study, we analyzed the molecular mechanisms leading to As(2)O(3)-induced abnormalities of cardiac electrophysiology. Using biochemical and electrophysiological methods, we show that long-term exposure to As(2)O(3) increases cardiac calcium currents and reduces surface expression of the cardiac potassium channel human ether-a-go-go-related gene (HERG) at clinically relevant concentrations of 0.1 to 1.5 microM. In ventricular myocytes, As(2)O(3) increases action potential duration measured at 30 and 90% of repolarization. As(2)O(3) interferes with hERG trafficking by inhibition of hERG-chaperone complexes and increases calcium currents by a faster cellular process. We propose that an increase in cardiac calcium current and reduced trafficking of hERG channels to the cell surface cause QT prolongation and torsade de pointes in patients treated with As(2)O(3). Our results suggest that calcium-channel antagonists will be useful in normalizing QT prolongation during As(2)O(3) therapy. As(2)O(3) is the first example of a drug that produces hERG liability by inhibition of ion-channel trafficking. Other drugs that interfere with proteins in the processing pathway of cardiac ion channels may be proarrhythmic for similar reasons.     

Ion channels in drug discovery and development

An excellent meeting on ion channels in drug discovery and development was organised by the Strategic Research Institute. Recent progress in the molecular and cellular biology of ion channels, their localisation and their physio-pathological roles, was presented by a selected number of academic researchers. The status of development, by pharmaceutical companies, of drugs targeting specific ion channel subtypes (e.g., AMPA receptors, nicotinic receptors, GABA(A) receptors, K(+) channels, transient receptor potential channels) and with different modalities (agonists versus potentiators) was reviewed. More comparative data on the emerging ion channel screening technologies are now available and were shared in a number of presentations. Finally, cardiac ion channel liability, in the context of drug discovery and development, was thoroughly discussed.     

Targeting N-type and T-type calcium channels for the treatment of pain

Severe chronic pain afflicts a large number of people worldwide but satisfactory relief from such pain is difficult to achieve with drugs that are currently available, and so there is a great need for the development of new, efficacious and safe analgesics. Voltage-gated calcium-permeable ion channels are multi-subunit complexes that regulate neuronal excitability, action-potential firing patterns and neurotransmission in nociceptive pathways. Although multiple subtypes of voltage-gated calcium channels exist, pharmacological and ion-channel gene knockdown approaches in animals have revealed N-type and T-type calcium channels to be particularly attractive molecular targets for the discovery and development of new analgesic drugs. The recent approval of Prialt (Elan Pharmaceuticals) provides the ultimate target validation for N-type calcium channels, namely proof that they are key regulators of nociceptive signaling in humans.     

Ion Channels In Drug Discovery and Development

An excellent meeting on ion channels in drug discovery and development was organised by the Strategic Research Institute. Recent progress in the molecular and cellular biology of ion channels, their localisation and their physio-pathological roles, was presented by a selected number of academic researchers. The status of development, by pharmaceutical companies, of drugs targeting specific ion channel subtypes (e.g., AMPA receptors, nicotinic receptors, GABA_A receptors, K⁺ channels, transient receptor potential channels) and with different modalities (agonists versus potentiators) was reviewed. More comparative data on the emerging ion channel screening technologies are now available and were shared in a number of presentations. Finally, cardiac ion channel liability, in the context of drug discovery and development, was thoroughly discussed.     

Therapeutic scope of modulation of non-voltage-gated cation channels

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non voltage gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.     

Therapeutic scope of modulation of non-voltage-gated cation channels

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non-voltage-gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.     

An assessment of the present and future roles of non-ligand gated ion channel modulators as CNS therapeutics

Few approved drugs have, as their primary known mechanism of action, modulation of non-ligand gated ion channels. However, these proteins are important regulators of neuronal function through their control of sodium, potassium, calcium and chloride flux, and are ideal candidates as drug discovery targets. Recent progress in the molecular biology and pharmacology of ion channels suggests that many will be associated with specific pharmacological profiles that will include both activators and inhibitors. Ion channels, through their regulation by G-proteins, are a major component of the final common pathway of many drugs acting at classical neuronal receptors. Thus, targeting of the ion channels themselves may confer different profiles of efficacy and specificity to drug action in the brain and spinal cord. Three areas for drug discovery are profiled that the authors consider prime targets for ion channel based therapies, anticonvulsant drugs, cognition enhancing drugs and drugs for improving neurone survival following ischaemia.     

An assessment of the present and future roles of non-ligand gated ion channel modulators as CNS therapeutics

Few approved drugs have, as their primary known mechanism of action, modulation of non-ligand gated ion channels. However, these proteins are important regulators of neuronal function through their control of sodium, potassium, calcium and chloride flux, and are ideal candidates as drug discovery targets. Recent progress in the molecular biology and pharmacology of ion channels suggests that many will be associated with specific pharmacological profiles that will include both activators and inhibitors. Ion channels, through their regulation by G-proteins, are a major component of the final common pathway of many drugs acting at classical neuronal receptors. Thus, targeting of the ion channels themselves may confer different profiles of efficacy and specificity to drug action in the brain and spinal cord. Three areas for drug discovery are profiled that the authors consider prime targets for ion channel based therapies, anticonvulsant drugs, cognition enhancing drugs and drugs for improving neurone survival following ischaemia.