ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase ß subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

The mitochondrial complex V-associated large-conductance inner membrane current is regulated by cyclosporine and dexpramipexole.

Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, reactive oxygen species-producing stimuli, or elevated mitochondrial matrix calcium opens the channel, which is inhibited by cyclosporine A and ATP/ADP. Here we show that ATP and the neuroprotective drug dexpramipexole (DEX) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX- but not ATP-mediated current inhibition, and reduced binding of [(14)C]DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. [(14)C]DEX bound specifically to purified recombinant b and oligomycin sensitivity-conferring protein subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V-associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability in Working Memory Circuits.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Discovery of (S,E)-3-(2-fluorophenyl)-N-(1-(3-(pyridin-3-yloxy)phenyl)ethyl)-acrylamide as a potent and efficacious KCNQ2 (Kv7.2) opener for the treatment of neuropathic pain.

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.

Potassium channelopathies associated with epilepsy-related syndromes and directions for therapeutic intervention.

A number of mutations to members of several CNS potassium (K) channel families have been identified which result in rare forms of neonatal onset epilepsy, or syndromes of which one prominent characteristic is a form of epilepsy. Benign Familial Neonatal Convulsions or Seizures (BFNC or BFNS), also referred to as Self-Limited Familial Neonatal Epilepsy (SeLNE), results from mutations in 2 members of the KV7 family (KCNQ) of K channels; while generally self-resolving by about 15 weeks of age, these mutations significantly increase the probability of generalized seizure disorders in the adult, in some cases they result in more severe developmental syndromes. Epilepsy of Infancy with Migrating Focal Seizures (EIMSF), or Migrating Partial Seizures of Infancy (MMPSI), is a rare severe form of epilepsy linked primarily to gain of function mutations in a member of the sodium-dependent K channel family, KCNT1 or SLACK. Finally, KCNMA1 channelopathies, including Liang-Wang syndrome (LIWAS), are rare combinations of neurological symptoms including seizure, movement abnormalities, delayed development and intellectual disabilities, with Liang-Wang syndrome an extremely serious polymalformative syndrome with a number of neurological sequelae including epilepsy. These are caused by mutations in the pore-forming subunit of the large-conductance calcium-activated K channel (BK channel) KCNMA1. The identification of these rare but significant channelopathies has resulted in a resurgence of interest in their treatment by direct pharmacological or genetic modulation. We will briefly review the genetics, biophysics and pharmacology of these K channels, their linkage with the 3 syndromes described above, and efforts to more effectively target these syndromes.

The Difficult Path to the Discovery of Novel Treatments in Psychiatric Disorders.

CNS diseases, including psychiatric disorders, represent a significant opportunity for the discovery and development of new drugs and therapeutic treatments with the potential to have a significant impact on human health. CNS diseases, however, present particular challenges to therapeutic discovery efforts, and psychiatric diseases/disorders may be among the most difficult. With specific exceptions such as psychostimulants for ADHD, a large number of psychiatric patients are resistant to existing treatments. In addition, clinicians have no way of knowing which psychiatric patients will respond to which drugs. By definition, psychiatric diagnoses are syndromal in nature; determinations of efficacy are often self-reported, and drug discovery is largely model-based. While such models of psychiatric disease are amenable to screening for new drugs, whether cellular or whole-animal based, they have only modest face validity and, more importantly, predictive validity. Multiple academic, pharmaceutical industry, and government agencies are dedicated to the translation of new findings about the neurobiology of major psychiatric disorders into the discovery and advancement of novel therapies. The collaboration of these agencies provide a pathway for developing new therapeutics. These efforts will be greatly helped by recent advances in understanding the genetic bases of psychiatric disorders, the ongoing search for diagnostic and therapy-responsive biomarkers, and the validation of new animal models.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability and Working Memory.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels coimmunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ +current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Cardiovascular effects of GLP-1 receptor agonism.

Glucagon-like peptide-1 (GLP-1) receptor agonists are extensively used in type 2 diabetic patients for the effective control of hyperglycemia. It is now clear from outcomes trials that this class of drugs offers important additional benefits to these patients due to reducing the risk of developing major adverse cardiac events (MACE). This risk reduction is, in part, due to effective glycemic control in patients; however, the various outcomes trials, further validated by subsequent meta-analysis of the outcomes trials, suggest that the risk reduction in MACE is also dependent on glycemic-independent mechanisms operant in cardiovascular tissues. These glycemic-independent mechanisms are likely mediated by GLP-1 receptors found throughout the cardiovascular system and by the complex signaling cascades triggered by the binding of agonists to the G-protein coupled receptors. This heterogeneity of signaling pathways underlying different downstream effects of GLP-1 agonists, and the discovery of biased agonists favoring specific signaling pathways, may have import in the future treatment of MACE in these patients. We review the evidence supporting the glycemic-independent evidence for risk reduction of MACE by the GLP-1 receptor agonists and highlight the putative mechanisms underlying these benefits. We also comment on the different signaling pathways which appear important for mediating these effects.

Electrochemical Carbamazepine Aptasensor for Therapeutic Drug Monitoring at the Point of Care.

Monitoring the anti-epileptic drug carbamazepine (CBZ) is crucial for proper dosing, optimizing a patient's clinical outcome, and managing their medication regimen. Due to its narrow therapeutic window and concentration-related toxicity, CBZ is prescribed and monitored in a highly personalized manner. We report an electrochemical conformation-changing aptasensor with two assay formats: a 30 min assay for routine monitoring and a 5 min assay for rapid emergency testing. To enable "sample-to-answer" testing, a de novo CBZ aptamer (K d < 12 nM) with conformational switching due to a G-quadruplex motif was labeled with methylene blue and immobilized on a gold electrode. The electrode fabrication and detection conditions were optimized using electrochemical techniques and visualized by atomic force microscopy (AFM). The aptasensor performance, including reproducibility, stability, and interference, was characterized using electrochemical impedance spectroscopy and voltammetry techniques. The aptasensor exhibited a wide dynamic range in buffer (10 nM to 100 µM) with limits of detection of 1.25 and 1.82 nM for the 5 and 30 min assays, respectively. The clinical applicability is demonstrated by detecting CBZ in finger prick blood samples (<50 µL). The proposed assays provide a promising method to enable point-of-care monitoring for timely personalized CBZ dosing.

Targeting putative components of the mitochondrial permeability transition pore for novel therapeutics.

Few discoveries have influenced drug discovery programs more than the finding that mitochondrial membranes undergo swings in permeability in response to cellular perturbations. The conductor of these permeability changes is the aptly named mitochondrial permeability transition pore which, although not yet precisely defined, is comprised of several integral proteins that differentially act to regulate the flux of ions, proteins and metabolic byproducts during the course of cellular physiological functions but also pathophysiological insults. Pursuit of the pore's exact identity remains a topic of keen interest, but decades of research have unearthed provocative functions for the integral proteins leading to their evaluation to develop novel therapeutics for a wide range of clinical indications. Chief amongst these targeted, integral proteins have been the Voltage Dependent Anion Channel (VDAC) and the F1FO ATP synthase. Research associated with the roles and ligands of VDAC has been extensive and we will expand upon 3 examples of ligand:VDAC interactions for consideration of drug discovery projects: Tubulin:VDAC1, Hexokinase I/II:VDAC1 and olesoxime:VDAC1. The discoveries that cyclosporine blocks mitochondrial permeability transition via binding to cyclophilin D, and that cyclophilin D is an important component of F1FO ATP synthase, has heightened interest in the F1FO ATP synthase as a focal point for drug discovery, and we will discuss 2 plausible campaigns associated with disease indications. To date no drug has emerged from prospective targeting these integral proteins; however, continued exploration such as the approaches suggested in this Commentary will increase the likelihood of providing important therapeutics for severely unmet medical needs.

Atropisomeric 3-(beta-hydroxyethyl)-4-arylquinolin-2-ones as Maxi-K potassium channel openers.

The synthesis of a series of 3-beta-hydroxyethyl-4-arylquinolin-2-ones is described. These compounds contain hydrophilic and hydrophobic substituents ortho to the phenolic OH in the C ring of the quinolinone. Electrophysiological evaluation of the panel of compounds revealed that 11 and 16 with an unbranched ortho substituent retain activity as maxi-K ion channel openers. Members of this series of compounds can exist as stable atropisomers. Calculated estimates of the energy barrier for rotation around the aryl-aryl single bond in 3 is 31 kcal/mol. The atropisomers of (+/-)-3, (+/-)-4, and (+/-)-11 were separated by chiral HPLC and tested for their effect on maxi-K mediated outward current in hSlo injected X. laevis oocytes. The (-) isomer in each case was found to be more active than the corresponding (+) isomer, suggesting that the ion channel exhibits stereoselective activation. X-ray crystallographic structures of (+)-3 and (+)-11 were determined. Evaluation of the stability of (-)-3 at 80 degrees C in n-butanol indicated a 19.6% conversion to (+)-3 over 72 h. In human serum at 37 degrees C (-)-3 did not racemize over the course of the 30 h study.

3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl ]-1,3,4-oxadiazol-2(3H)-one, BMS-191011: opener of large-conductance Ca(2+)-activated potassium (maxi-K) channels, identification, solubility, and SAR.

Compound 8a (BMS-191011), an opener of the cloned large-conductance, Ca2+-activated potassium (maxi-K) channel, demonstrated efficacy in in vivo stroke models, which led to its nomination as a candidate for clinical evaluation. Its maxi-K channel opening properties were consistent with its structural topology, being derived by combining elements from other known maxi-K openers. However, 8a suffered from poor aqueous solubility, which complicated elucidation of SAR during in vitro evaluation. The activity of 8a in in vivo stroke models and studies directed toward improving its solubility are reported herein. Enhanced solubility was achieved by appending heterocycles to the 8a scaffold, and a notable observation was made that inclusion of a simple amino group (anilines 8k and 8l) yielded excellent in vitro maxi-K ion channel opening activity and enhanced brain-to-plasma partitioning compared to the appended heterocycles.

Targeted disruption of the mouse Mel(1b) melatonin receptor.

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

The need for new approaches in CNS drug discovery: Why drugs have failed, and what can be done to improve outcomes.

An important goal of biomedical research is to translate basic research findings into useful medical advances. In the field of neuropharmacology this requires understanding disease mechanisms as well as the effects of drugs and other compounds on neuronal function. Our hope is that this information will result in new or improved treatment for CNS disease. Despite great progress in our understanding of the structure and functions of the CNS, the discovery of new drugs and their clinical development for many CNS disorders has been problematic. As a result, CNS drug discovery and development programs have been subjected to significant cutbacks and eliminations over the last decade. While there has been recent resurgence of interest in CNS targets, these past changes in priority of the pharmaceutical and biotech industries reflect several well-documented realities. CNS drugs in general have higher failure rates than non-CNS drugs, both preclinically and clinically, and in some areas, such as the major neurodegenerative diseases, the clinical failure rate for disease-modifying treatments has been 100%. The development times for CNS drugs are significantly longer for those drugs that are approved, and post-development regulatory review is longer. In this introduction we review some of the reasons for failure, delineating both scientific and technical realities, some unique to the CNS, that have contributed to this. We will focus on major neurodegenerative disorders, which affect millions, attract most of the headlines, and yet have witnessed the fewest successes. We will suggest some changes that, when coupled with the approaches discussed in the rest of this special volume, may improve outcomes in future CNS-targeted drug discovery and development efforts. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".

Pharmacological activation and inhibition of Slack (Slo2.2) channels.

The Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene is abundantly expressed in the mammalian brain and encodes a sodium-activated K+ (KNa) channel. Although the specific roles of Slack channel subunits in neurons remain to be identified, they may play a role in the adaptation of firing rate and in protection against ischemic injury. In the present study, we have generated a stable cell line expressing the Slack channel, and have analyzed the pharmacological properties of these channels in these cells and in Xenopus oocytes. Two known blockers of KNa channels, bepridil and quinidine, inhibited Slack currents in a concentration-dependent manner and decreased channel activity in excised membrane patches. The inhibition by bepridil was potent, with an IC50 of 1.0 microM for inhibition of Slack currents in HEK cells. In contrast, bithionol was found to be a robust activator of Slack currents. When applied to the extracellular face of excised patches, bithionol rapidly induced a reversible increase in channel opening, suggesting that it acts on Slack channels relatively directly. These data establish an important early characterization of agents that modulate Slack channels, a process essential for the experimental manipulation of Slack currents in neurons.

Donepezil primarily attenuates scopolamine-induced deficits in psychomotor function, with moderate effects on simple conditioning and attention, and small effects on working memory and spatial mapping.

RATIONALE: Alzheimer's dementia (AD) patients have profound deficits in cognitive and social functions, mediated in part by a decline in cholinergic function. Acetylcholinesterase inhibitors (AChEI) are the most commonly prescribed treatment for the cognitive deficits in AD patients, but their therapeutic effects are small, and it is still not clear if they primarily affect attention, memory, or some other cognitive/behavioral functions. OBJECTIVES: The objective of the present experiments was to explore the effects of donepezil (Aricepttrade mark), an AChEI, on behavioral deficits related exclusively to cholinergic dysfunction. MATERIALS AND METHODS: The effects of donepezil were assessed in Sprague-Dawley rats with scopolamine-induced deficits in a battery of cognitive/behavioral tests. RESULTS: Scopolamine produced deficits in contextual and cued fear conditioning, the 5-choice serial reaction time test, delayed nonmatching to position, the radial arm maze, and the Morris water maze. Analyses of the pattern and size of the effects revealed that donepezil produced very large effects on scopolamine-induced deficits in psychomotor function (approximately 20-50% of the variance), moderate-sized effects on scopolamine-induced deficits in simple conditioning and attention (approximately 3-10% of the variance), but only small effects on scopolamine-induced deficits in higher cognitive functions of working memory and spatial mapping (approximately 1% of the variance). CONCLUSIONS: These results are consistent with the limited efficacy of donepezil on higher cognitive function in AD patients, and suggest that preclinical behavioral models could be used not only to determine if novel treatments have some therapeutic potential, but also to predict more precisely what the pattern and size of the effects might be.

Soluble Abeta and cognitive function in aged F-344 rats and Tg2576 mice.

Recent findings suggest that Alzheimer's dementia may be mediated by soluble beta amyloid (Abeta) more than the deposits of aggregated, insoluble Abeta, and vulnerability to cognitive deficits after scopolamine challenge may help identify AD even in patients that are still pre-symptomatic. The objectives of the present experiments were to determine if vulnerability to cognitive deficits after scopolamine challenge is related to levels of soluble Abeta, and if levels of soluble Abeta are more closely related to cognitive deficits than levels of insoluble Abeta, even in aged, transgenic mice, after they have developed very high levels of insoluble Abeta. Aged F-344 rats and young mice over-expressing the Swedish mutation in the human amyloid precursor protein (APPsw; Tg2576+) had elevated levels of soluble Abeta, and were more vulnerable to scopolamine challenge in the Morris water maze (MWM), relative to young rats and Tg2576- mice; but, among individual animals, higher levels of soluble Abeta were not correlated with vulnerability to scopolamine. On the other hand, in aged Tg2576+ mice, cognitive deficits were related to levels of soluble Abeta, not insoluble Abeta, despite the fact that the levels of insoluble Abeta were thousands of times higher than the levels of soluble Abeta. The results of the present experiments suggest that vulnerability to cognitive deficits after scopolamine challenge is not related to elevated levels of soluble Abeta, but that high levels of soluble Abeta are more closely correlated with cognitive deficits than the amount insoluble Abeta, even after large amounts of aggregated, insoluble Abeta have been deposited.

Long-lasting functional disabilities in middle-aged rats with small cerebral infarcts.

Recommendations from experts and recently established guidelines on how to improve the face and predictive validity of animal models of stroke have stressed the importance of using older animals and long-term behavioral-functional endpoints rather than relying almost exclusively on acute measures of infarct volume in young animals. The objective of the present study was to determine whether we could produce occlusions in older rats with an acceptable mortality rate and then detect reliable, long-lasting functional deficits. A reversible intraluminar suture middle cerebral artery occlusion (MCAO) procedure was used to produce small infarcts in middle-aged rats. This resulted in an acceptable mortality rate, and robust disabilities were detected in functional assays, although the degree of total tissue loss measured 90 d after MCAO was quite modest. Infarcted animals were functionally impaired relative to sham control animals even 90 d after the occlusions, and when animals were subgrouped based on amount of tissue loss, MCAO animals with only 4% tissue loss exhibited enduring neurological-behavioral impairments relative to sham-operated controls, and the functional impairments in the group with the largest infarcts (20% tissue loss) were more severe than the functional impairments in the rats with 4% tissue loss. These results suggest that this model, using reversible MCAO to produce small infarcts and long-lasting functional-behavioral deficits in older rats, may represent an advance in the relatively higher-throughput modeling of stroke and its recovery in rodents and may be useful in the development and characterization of future stroke therapies.

Use-dependent blockade of Cav2.2 voltage-gated calcium channels for neuropathic pain.

The translocation of extracellular calcium (Ca(2+)) via voltage-gated Ca(2+) channels (VGCCs) in neurons is involved in triggering multiple physiological cell functions but also the abnormal, pathophysiological responses that develop as a consequence of injury. In conditions of neuropathic pain, VGCCs are involved in supplying the signal Ca(2+) important for the sustained neuronal firing and neurotransmitter release characteristic of these syndromes. Preclinical data have identified N-type VGCCs (Ca(v)2.2) as key participants in contributing to these Ca(2+) signaling events and clinical data with the peptide blocker Prialt have now validated Ca(v)2.2 as a bona fide target for future drug discovery efforts to identify new and novel therapeutics for neuropathic pain. Imperative for the success of such an endeavor will be the ability to identify compounds selective for Ca(v)2.2, versus other VGCCs, but also compounds which demonstrate effective blockade during the pathophysiological states of neuropathic pain without compromising channel activity associated with sustaining normal housekeeping cellular functions. An approach to obtain this research target profile is to identify compounds, which are more potent in blocking Ca(v)2.2 during higher frequencies of firing as compared to the slower more physiologically-relevant frequencies. This may be achieved by identifying compounds with enhanced potency for the inactivated state of Ca(v)2.2. This commentary explores the rationale and options for engineering a use-dependent blocker of Ca(v)2.2. It is anticipated that this use-dependent profile of channel blockade will result in new chemical entities with an improved therapeutic ratio for neuropathic pain.

Synthesis and structure-activity relationship of acrylamides as KCNQ2 potassium channel openers.

A new class of acrylamides was synthesized, and the effects of these analogues on outward potassium current were evaluated by using two electrode voltage clamp recordings from Xenopus laevis oocytes expressing cloned mKCNQ2 channels. SAR studies indicated that the pharmacophore of the acrylamide series includes the (S) absolute configuration at the (1-phenyl)ethyl moiety and the alpha,beta-unsaturated acrylamide functionality with a free NH. This study identified (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide ((S)-1) and (S)-N-[1-(4-fluoro-3-morpholin-4-yl-phenyl)-ethyl]-3-(4-fluoro-phenyl)-acrylamide ((S)-2) as KCNQ2 openers for further electrophysiological evaluations. These two acrylamides demonstrated significant activity in the cortical spreading depression model of migraine as we reported previously.