Search

Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

The synthesis and preclinical characterization of novel 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are potent and selective brain penetrant P2X7 antagonists are described. Optimization efforts based on previously disclosed unsubstituted 6,7-dihydro-4H-triazolo[4,5-c]pyridines, methyl substituted 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazines, and several other series lead to the identification of a series of 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are selective P2X7 antagonists with potency at the rodent and human P2X7 ion channels. These novel P2X7 antagonists have suitable physicochemical properties, and several analogs have an excellent pharmacokinetic profile, good partitioning into the CNS and show robust in vivo target engagement after oral dosing. Improvements in metabolic stability led to the identification of JNJ-54175446 (14) as a candidate for clinical development. The drug discovery efforts and strategies that resulted in the identification of the clinical candidate are described herein.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Na(v)1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Na(v)1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Na(v)1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Na(v)1.7 and Na(v)1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Na(v)1.7 potency (E1N, E4D, Y33W, Q34S–Na(v)1.7 pIC(50) = 8.1 ± 0.08) and increased selectivity over other Na(v) isoforms (E1N, R26K, Q34S, G36I, Na(v)1.7 pIC(50) = 7.2 ± 0.1, Na(v)1.2 pIC(50) = 6.1 ± 0.18, Na(v)1.3 pIC(50) = 6.4 ± 1.0), Na(v)1.4 is inactive at 3 μm, and Na(v)1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Na(v)1.7 inhibition and propose a model for huwentoxin-IV's interaction with Na(v)1.7. These findings uncover fundamental features of huwentoxin involved in Na(v)1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

The synthesis, SAR and preclinical characterization of a series of 6-chloro- N -(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists is described herein. The lead compounds are potent inhibitors in Ca 2+ flux and whole blood IL-1β P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1β release in a mouse ex vivo model with a 50 mg/kg oral dose. Evaluation of compound 1e in the mouse SNI tactile allodynia, carrageenan-induced paw edema or CIA models resulted in no analgesic or anti-inflammatory effects.

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca2+ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ± 0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and β subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H2O2. In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology.

Maintaining the integrity of cellular membranes is critical to protecting metabolic activities and genetic information from the environment. Regulation of transport across membranes of essential chemicals, including water, nutrients, hormones and many drugs, is therefore key to cellular homeostasis and physiological processes. The two main transporter superfamilies are ATP-binding cassette (ABC) transporters that primarily function as efflux transporters, and the solute carrier (SLC) transporters. SLC transporters encompass 52 gene families with almost 400 different human transporter genes. Although long under-explored, SLC transporters are an emerging drug target class and the molecular target of several approved inhibitor drugs, such as selective serotonin reuptake inhibitors (SSRIs) for depression and sodium/glucose co-transporter (SGLT2) inhibitors for diabetes. Interestingly though, although loss-of-function mutations in numerous human SLC transporters are linked to Mendelian diseases, few reports of SLC transporter activators have appeared, and only inhibitors have been advanced to clinical studies. In this commentary, we discuss several strategies for potentiating SLC transporter function, from direct acting potentiators to modulators of transcription, translation or trafficking. We review the progress made in recent years toward the understanding of the structural and molecular basis of SLC transporter function and the pathways and mechanisms that regulate SLC expression, and describe the opportunities these new insights present for discovery of SLC transporter potentiators. Finally, we highlight the challenges associated with the various approaches and provide some thoughts on future directions that might facilitate the search for SLC potentiators with therapeutic potential.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

In the liver, citrate is a key metabolic intermediate involved in the regulation of glycolysis and lipid synthesis and reduced expression of the hepatic citrate SLC13A5 transporter has been shown to improve metabolic outcomes in various animal models. Although inhibition of hepatic extracellular citrate uptake through SLC13A5 has been suggested as a potential therapeutic approach for Type-2 diabetes and/or fatty liver disease, so far, only a few SLC13A5 inhibitors have been identified. Moreover, their mechanism of action still remains unclear, potentially limiting their utility for in vivo proof-of-concept studies. In this study, we characterized the pharmacology of the recently identified hydroxysuccinic acid SLC13A5 inhibitors, PF-06649298 and PF-06761281, using a combination of 14C-citrate uptake, a membrane potential assay and electrophysiology. In contrast to their previously proposed mechanism of action, our data suggest that both PF-06649298 and PF-06761281 are allosteric, state-dependent SLC13A5 inhibitors, with low-affinity substrate activity in the absence of citrate. As allosteric state-dependent modulators, the inhibitory potency of both compounds is highly dependent on the ambient citrate concentration and our detailed mechanism of action studies therefore, may be of value in interpreting the in vivo effects of these compounds.

Ion channels are targets of many therapeutically useful agents, and worldwide sales of ion channel-targeted drugs are estimated to be approximately US$12 billion. Nevertheless, considering that over 400 genes encoding ion channel subunits have been identified, ion channels remain significantly under-exploited as therapeutic targets. This is at least partly due to limitations in high-throughput assay technologies that support screening and lead optimization. Will the recent developments in automated electrophysiology rectify this situation? What are the other major limitations and can they be overcome? In this article, we review the status of ion channel drug discovery, discuss current challenges and propose alternative approaches that may facilitate the discovery of new drugs in the future.

As an integrator of multiple nociceptive and/or inflammatory stimuli, TRPV1 is an attractive therapeutic target for the treatment of various painful disorders. Several TRPV1 antagonists have been advanced into clinical trials and the initial observations suggest that TRPV1 antagonism may be associated with mild hyperthermia and thermal insensitivity in man. However, no clinical efficacy studies have been described to date, making an assessment of risk:benefit impossible. Furthermore, it is not clear whether these early observations are representative of all TRPV1 antagonists and whether additional clinical studies with novel TRPV1 antagonists are required in order to understand optimal compound characteristics. In the present study we describe 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729309) as a novel, TRPV1 antagonist. JNJ-39729209 displaced tritiated resiniferotoxin binding to TRPV1 and prevented TRPV1 activation by capsaicin, protons and heat. In-vivo, JNJ-39729209 blocked capsaicin-induced hypotension, induced a mild hyperthermia and inhibited capsaicin-induced hypothermia in a dose dependent manner. JNJ-39729209 showed significant efficacy against carrageenan- and CFA-evoked thermal hyperalgesia and exhibited significant anti-tussive activity in a guinea-pig model of capsaicin-induced cough. In pharmacokinetic studies, JNJ-39729209 was found to have low clearance, a moderate volume of distribution, good oral bioavailability and was brain penetrant. On the basis of these findings, JNJ-39729209 represents a structurally novel TRPV1 antagonist with potential for clinical development. The advancement of JNJ-39729209 into human clinical trials could be useful in further understanding the analgesic potential of TRPV1 antagonists.

Based upon a previously reported lead compound 1, a series of 1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines were synthesized and evaluated for improved physiochemical and pharmacokinetic properties while maintaining TRPV1 antagonist activity. Structure–activity relationship studies directed toward improving the aqueous solubility (pH 2 and fasted-state simulated intestinal fluid (SIF)) and rat pharmacokinetics led to the discovery of compound 13. Aqueous solubility of compound 13 (pH 2 = >237 μg/mL and SIF = 11 μg/mL) was significantly improved over compound 1 (pH 2 = 5 μg/mL and SIF = 0.5 μg/mL). In addition, compound 13 afforded improved rat pharmacokinetics (CL = 0.7 L/kg/h) compared to compound 1 (CL = 3.1 L/kg/h). Compound 13 was orally bioavailable and afforded a significant reversal of carrageenan-induced thermal hyperalgesia at 5 and 30 mg/kg in rats.

The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is a nonselective cation channel that is highly expressed in small-diameter sensory neurons, where it functions as a polymodal receptor, responsible for detecting potentially harmful chemicals, mechanical forces and temperatures. TRPA1 is also activated and/or sensitized by multiple endogenous inflammatory mediators. As such, TRPA1 likely mediates the pain and neurogenic inflammation caused by exposure to reactive chemicals. In addition, it is also possible that this channel may mediate some of the symptoms of chronic inflammatory conditions such as asthma. We review recent advances in the biology of TRPA1 and summarize the evidence for TRPA1 as a therapeutic drug target. In addition, we provide an update on TRPA1 medicinal chemistry and the progress in the search for novel TRPA1 antagonists.

The mammalian KCNQ (Kv7) gene family is composed of five members (KCNQ1-5). KCNQ2, Q4 and Q5 (KCNQ2-5) channels co-express with KCNQ3 to form heterotetrameric voltage-gated K(+) (KCNQ2-5/3) channels that underlie the endogenous M-current and regulate neuronal excitability in CNS and PNS neurons. Openers of one or a mixture of these channels may be an attractive therapeutic agent for epilepsy and pain. Non-selective KCNQ2-5/3 activators have shown efficacy in pre-clinical and clinical studies. However, more selective pharmacological profiles, including greater KCNQ sub-type-selective activation, could provide efficacy with fewer side effects. One such compound, ICA-27243, sub-type selectively enhances the activation of KCNQ2/3 channels and also exhibits efficacy in pre-clinical anticonvulsant models; Roeloffs et al. (2008) [15]; Wickenden et al. (2008) [27]. The binding site of non-selective KCNQ2-5/3 openers maps to the S5-S6 pore domain and is altered by mutation of a tryptophan residue (Trp236 in KCNQ2, Trp265 in KCNQ3) conserved among KCNQ2-5 channels; Schenzer et al. (2005) [19]; Wuttke et al. (2005) [30]. Here we report that the activity of the KCNQ2/3 selective opener ICA-27243 is not affected by these Trp mutations and does not map to the S5-S6 domain. Rather, the selective activity of ICA-27243 is determined by a novel site within the S1-S4 voltage-sensor domain (VSD) of KCNQ channels. The sub-type-selective activity of ICA-27243 may arise from greater sequence diversity of KCNQ family members within the ICA-27243 binding pocket, allowing for more selective small molecule-protein interactions.

Kv7.x channels are a family of six transmembrane domain, single pore-loop, voltage-gated K(+) channels. Five members of the family have been identified to date, including the cardiac channel Kv7.1 (formerly known as KvLQT1) and four neuronal Kv7.x channels, Kv7.2-5. Heteromeric channels containing Kv7.3 and either Kv7.2 or Kv7.5 are thought to underlie the neuronal M-current, a non-inactivating, slowly deactivating, sub-threshold current that has long been known to exert a powerful stabilizing influence on neuronal excitability. Modulators of these channels have the potential to influence neuronal activity in various tissues and are of much interest as therapeutic drug targets for the treatment of a variety of clinical disorders, such as epilepsy and pain. The purpose of the present article is to review the molecular, functional and behavioral evidence validating Kv7.x as drug targets for the treatment of pain. In addition, an update on pre-clinical Kv7 drug discovery efforts will be presented, along with a summary of on-going clinical trials with Kv7 channel activators.

Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of I(h) (also known as 'funny' I(f) and 'queer' I(q)), a hyperpolarization-activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.

Voltage dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders such as epilepsy and pain as well as cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic and antiarrhythmic sodium channel blockers. While patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery in which there is often a need to evaluate many thousands to hundreds of thousands of compounds. Fortunately over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms like the PatchXpressTM and Ion- WorksTM, which are now widely used in drug discovery. This review highlights the strengths and weaknesses of these two high throughput devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpressTM and IonWorksTM electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state dependent modulation of sodium channels. IonWorksTM has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpressTM has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery.

The nonselective cation channel TRPA1 (ANKTM1, p120) is a potential mediator of pain, and selective pharmacological modulation of this channel may be analgesic. Although several TRPA1 activators exist, these tend to be either reactive or of low potency and/or selectivity. The aim of the present study, therefore, was to identify novel TRPA1 agonists. Using a combination of calcium fluorescent assays and whole-cell electrophysiology, we discovered several compounds that possess potent, selective TRPA1-activating activity, including several lipid compounds (farnesyl thiosalicylic acid, farnesyl thioacetic acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), and 5,8,11,14-eicosatetraynoic acid), and two marketed drugs: disulfiram (Antabuse; a compound used in the treatment of alcohol abuse) and the antifungal agent chlordantoin. Farnesyl thiosalicylic acid activates the channel in excised patches and in the absence of calcium. Furthermore, using a quadruple TRPA1 mutant, we show that the mechanism of action of farnesyl thiosalicylic acid differs from that of the reactive electrophilic reagent allylisothiocyanate. As a TRPA1 agonist with a potentially novel mechanism of action, farnesyl thiosalicylic acid may be useful in the study of TRPA1 channels.

It is estimated that up to 30% of epilepsy patients are poorly treated with available anti-epileptic drugs (AEDs). Thus, there is a medical need for new AEDs with novel mechanisms of action to serve as alternate or adjunct therapy for the treatment of drug-resistant or refractory epilepsy. One potential anti-epileptic mechanism that has not yet been exploited is K(+) channel opening. Considerable genetic, molecular, physiological and pharmacological evidence now exists to support a role for K(+) channels such as KCNQ2/Q3, Kv1.1, KATP and GIRK2 in the control of neuronal excitability and epileptogenesis. Evidence supporting these K(+) channels as novel AED targets will be reviewed in the following article.

K(+) channels play critical roles in a wide variety of physiological processes, including the regulation of heart rate, muscle contraction, neurotransmitter release, neuronal excitability, insulin secretion, epithelial electrolyte transport, cell volume regulation, and cell proliferation. As such, K(+) channels have been recognized as potential therapeutic drug targets for many years. Unfortunately, progress toward identifying selective K(+) channel modulators has been severely hampered by the need to use native currents and primary cells in the drug-screening process. Today, however, more than 80 K(+) channel and K(+) channel-related genes have been identified, and an understanding of the molecular composition of many important native K(+) currents has begun to emerge. The identification of these molecular K(+) channel drug targets should lead to the discovery of novel drug candidates. A summary of progress is presented.

Kv1.3 is a voltage-gated potassium channel expressed on T cells that plays an important role in T cell activation. Previous studies have shown that blocking Kv1.3 channels in human T cells during activation results in reduced calcium entry, cytokine production, and proliferation. The aim of the present study was to further explore the effects of Kv1.3 blockers on the response of different human T cell subsets under various stimulation conditions. Our studies show that, unlike the immune suppressor cyclosporine A, the inhibitory effect of Kv1.3 blockers was partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulations. T cell responses to allergens including house dust mites and ragweed were partially reduced by Kv1.3 blockers. The effect of Kv1.3 inhibition was dependent on T cell subsets, with stronger effects on CCR7- effector memory compared to CCR7+ central memory CD4 T cells. Calcium entry studies also revealed a population of CD4 T cells resistant to Kv1.3 blockade. Activation of CD4 T cells was accompanied with an increase in Kv1.3 currents but Kv1.3 transcripts were found to be reduced, suggesting a posttranscriptional mechanism in the regulation of Kv1.3 activities. In summary, Kv1.3 blockers inhibit T cell activation in a manner that is highly dependent on the T cell identity and stimulation strength, These findings suggest that Kv1.3 blockers inhibit T cells in a unique, conditional manner, further refining our understanding of the therapeutic potential of Kv1.3 blockers.

Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.

An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist.We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain.JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL(-1) (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg(-1) ) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test.JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology.

This unit describes protocols to aid investigators in determining the electrophysiological and pharmacological profile of heterologously expressed voltage or calcium-activated potassium channels belonging to the Kv1.x and SK/IK gene families. Protocols for data acquisition as well as analysis are provided.

P2X7 is an ATP-gated non-selective cation channel expressed primarily on cells of hematopoietic origin, such as macrophages and microglia. Since the initial cloning of this channel, enormous progress has been made in the understanding of the physiology, pharmacology and therapeutic utility of P2X7. This article attempts to review the biology of P2X7 with a focus on the complex pharmacology of this channel. Finally, the authors discuss the role of P2X7 as an analgesic drug target and raise some of the challenges and issues that face the P2X7 research community.

TRPA1 is a nonselective cation channel belonging to the transient receptor potential (TRP) family that is expressed in peripheral sensory neurons and may play important roles in pain perception and inflammation. We found that agonist stimulation of TRPA1, along with other members of the TRP family (TRPV1–4 and TRPM8), can induce the appearance of a large pore permeable to large organic cations such as Yo-Pro (YP) and N-methyl-d-glucamine, in an agonist and divalent cation-dependent manner. YP uptake was not inhibited by a panel of putative gap junction/pannexin blockers, suggesting that gap junction proteins are not required in this process. Our data suggest that changes in the TRP channel selectivity filter itself result in a progressive but reversible pore dilation process, a process that is under strong regulation by external calcium ions. Our data suggest that calcium plays a novel role in setting the amount of time TRPA1 channels spend in a dilated state providing a mechanism that may limit sensory neuron activation by painful or irritating substances.

This article reviews evidence that hyperpolarization-activated, cation nonselective (HCN) channels, the molecular basis of the Ih current, potentially represent valid targets for novel analgesic agents. Ih is a prominent current in many peripheral sensory nerves, with highest current density typically found in large diameter neurons. Recent data suggest that Ih may represent a valid target for the treatment of spontaneous pain and allodynia associated with nerve injury. The majority of available electrophysiological and molecular evidence suggests that fast activating, weakly cyclic adenosine monophosphate (cAMP) sensitive HCN1-based channels may make a significant contribution to Ih, especially in large diameter, mechanosensitive fibers, where the Ih current appears to support abnormal spontaneous firing after nerve injury. In contrast, HCN4 channels seem to play the predominant role in cardiac pacemaker tissue. These observations raise the possibility that HCN1 selective blockers may inhibit pain associated with nerve injury without dramatic effects on heart rate. Development of novel HCN blocking analgesics presents a number of significant technical challenges. Although a number of HCN blockers are available, such as ZD-7288, ivabradine, and others, these drugs inhibit all HCN isoforms with the same potency. As a result, these compounds have powerful effects on heart rate, severely limiting their utility for non-cardiac indications such as pain. Selectivity challenges, mechanisms of compound interaction with the channel, and assay methods are described in detail.

Aryl sulfonamido tetralins based on lead compound 2a were synthesized and evaluated for Kv1.5 inhibitory activity. Several compounds having IC50 values less then 0.1 μM were identified. Kv1.5 inhibitors have the potential to be atrium-selective agents for the treatment of atrial fibrillation.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K(+) channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy (Biervert et al., 1998; Singh et al., 1998), suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. In the present study, we have identified the compound N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) as a selective activator of the neuronal M current and KCNQ2/Q3 channels. In SH-SY5Y human neuroblastoma cells, ICA-27243 produced membrane potential hyperpolarization that could be prevented by coadministration with the M-current inhibitors 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) and linopirdine. ICA-27243 enhanced both (86)Rb(+) efflux (EC(50) = 0.2 microM) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC(50) = 0.4 microM). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V((1/2)) shift of -19 mV at 10 microM). In contrast, ICA-27243 was less effective at activating KCNQ4 and KCNQ3/Q5 and was selective over a wide range of neurotransmitter receptors and ion channels such as voltage-dependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 microM) was found to reversibly suppress seizure-like activity in an ex vivo hippocampal slice model of epilepsy and demonstrated in vivo anticonvulsant activity (ED(50) = 8.4 mg/kg) in the mouse maximal electroshock epilepsy model. In conclusion, ICA-27243 represents the first member of a novel chemical class of selective KCNQ2/Q3 activators with anticonvulsant-like activity in experimental models of epilepsy.

The Elk subfamily of the Eag K+ channel gene family is represented in mammals by three genes that are highly conserved between humans and rodents. Here we report the distribution and functional properties of a member of the human Elk K+ channel gene family, KCNH8. Quantitative RT-PCR analysis of mRNA expression patterns showed that KCNH8, along with the other Elk family genes, KCNH3 and KCNH4, are primarily expressed in the human nervous system. KCNH8 was expressed at high levels, and the distribution showed substantial overlap with KCNH3. In Xenopus oocytes, KCNH8 gives rise to slowly activating, voltage-dependent K+ currents that open at hyperpolarized potentials (half-maximal activation at -62 mV). Coexpression of KCNH8 with dominant-negative KCNH8, KCNH3, and KCNH4 subunits led to suppression of the KCNH8 currents, suggesting that Elk channels can form heteromultimers. Similar experiments imply that KCNH8 subunits are not able to form heteromultimers with Eag, Erg, or Kv family K+ channels.

KCNQ2 and KCNQ3 potassium-channel subunits can form both homomeric and heteromeric channels; the latter are thought to constitute native ganglionic M channels. We have tried to deduce the stoichiometric contributions of KCNQ2 and KCNQ3 subunits to currents generated by the coexpression of KCNQ2 and KCNQ3 cDNA plasmids in Chinese hamster ovary (CHO) cells, and to native M currents in dissociated rat superior cervical ganglion (SCG) neurons, by comparing the block of these currents produced by tetraethylammonium (TEA) with the block of currents generated by a tandem KCNQ3/2 construct. TEA concentration–inhibition curves against coexpressed KCNQ2 plus KCNQ3 currents, and against native M currents in SCG neurons from 6-week-old [postnatal day 45 (P45)] rats, were indistinguishable from those for the expressed tandem construct, and fully accorded with a 1:1 stoichiometry. Inhibition curves in neurons from younger (P17) rats could be better fitted assuming an additional small proportion of current carried by KCNQ2 homomultimers. Single-cell PCR yielded signals for KCNQ2, KCNQ3, and KCNQ5 mRNAs in all SCG neurons tested from both P17 and P45 rats. Quantitative PCR of whole-ganglion mRNA revealed stable levels of KCNQ2 and KCNQ5 mRNA between P7 and P45, but excess and incrementing levels of KCNQ3 mRNA. Increasing levels of KCNQ3 protein between P17 and P45 were confirmed by immunocytochemistry. We conclude that coexpressed KCNQ2 plus KCNQ3 cDNAs generate channels with 1:1 (KCNQ2:KCNQ3) stoichiometry in CHO cells and that native M channels in SCG neurons adopt the same conformation during development, assisted by the increased expression of KCNQ3 mRNA and protein.

This unit provides an overview of the principal electrophysiological techniques commonly used for the study of ionic currents and the ion channels that mediate them. These techniques include electroencephalograms (EEGs), electrocardiograms (ECGs), single- and multiunit extracellular recording, multielectrode arrays, transepithelial recording, impedance measurements, and current-clamp, voltage-clamp, patch-clamp, and lipid bilayer recording. The unit also discusses recent advances in high-throughput, automated electrophysiological techniques for drug discovery and the use of stem cells as a tissue source.

Retigabine [N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid; D-23129] is a novel anticonvulsant, unrelated to currently available antiepileptic agents, with activity in a broad range of seizure models. In the present study, we sought to determine whether retigabine could enhance current through M-like currents in PC12 cells and KCNQ2/Q3 K(+) channels expressed in Chinese hamster ovary cells (CHO-KCNQ2/Q3). In differentiated PC12 cells, retigabine enhanced a linopirdine-sensitive current. The effect of retigabine was associated with a slowing of M-like tail current deactivation in these cells. Retigabine (0.1 to 10 microM) induced a potassium current and hyperpolarized CHO cells expressing KCNQ2/Q3 cells but not in wild-type cells. Retigabine-induced currents in CHO-KCNQ2/Q3 cells were inhibited by 60.6 +/- 11% (n = 4) by the KCNQ2/Q3 blocker, linopirdine (10 microM), and 82.7 +/- 5.4% (n = 4) by BaCl(2) (10 mM). The mechanism by which retigabine enhanced KCNQ2/Q3 currents involved large, drug-induced, leftward shifts in the voltage dependence of channel activation (-33.1 +/- 2.6 mV, n = 4, by 10 microM retigabine). Retigabine shifted the voltage dependence of channel activation with an EC(50) value of 1.6 +/- 0.3 microM (slope factor was 1.2 +/- 0.1, n = 4 to 5 cells per concentration). Retigabine (0.1 to 10 microM) also slowed the rate of channel deactivation, predominantly by increasing the contribution of a slowly deactivating tail current component. Our findings identify KCNQ2/Q3 channels as a molecular target for retigabine and suggest that activation of KCNQ2/Q3 channels may be responsible for at least some of the anticonvulsant activity of this agent.

We present the cloning and characterization of two novel calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4, that are enriched in the testis and brain, respectively. We compare and contrast the steady state and kinetic properties of these beta subunits with the previously cloned mouse beta1 (mKCNMB1) and the human beta2 subunit (hKCNMB2). Once inactivation is removed, we find that hKCNMB2 has properties similar to mKCNMB1. hKCNMB2 slows Hslo1 channel gating and shifts the current-voltage relationship to more negative potentials. hKCNMB3 and hKCNMB4 have distinct effects on slo currents not observed with mKCNMB1 and hKCNMB2. Although we found that hKCNMB3 does interact with Hslo channels, its effects on Hslo1 channel properties were slight, increasing Hslo1 activation rates. In contrast, hKCNMB4 slows Hslo1 gating kinetics, and modulates the apparent calcium sensitivity of Hslo1. We found that the different effects of the beta subunits on some Hslo1 channel properties are calcium-dependent. mKCNMB1 and hKCNMB2 slow activation at 1 microM but not at 10 microM free calcium concentrations. hKCNMB4 decreases Hslo1 channel openings at low calcium concentrations but increases channel openings at high calcium concentrations. These results suggest that beta subunits in diverse tissue types fine-tune slo channel properties to the needs of a particular cell.

Exposure to zinc can cause pain and inflammation and can be harmful to human health. New evidence suggests that activation of the irritant receptor, TRPA1, which is expressed on pain-sensing neurons, may be responsible for some of these symptoms of zinc toxicity.

Action potential prolongation is a common finding in human heart failure and in animal models of cardiac hypertrophy. The mechanism of action potential prolongation involves altered expression of a variety of depolarising and hyperpolarising currents in the myocardium. In particular, decreased density of the transient outward potassium current seems to play a prominent role, regardless of species, precipitating factors or the severity of hypertrophy. The decreased density of the transient outward current appears to be caused by reduced transcription of Kv4.2 and Kv4.3 and may be caused in part by an inhibitory effect of alpha-adrenoceptor stimulation. During the early stage of the disease process, action potential prolongation may increase the amplitude of the intracellular calcium transient, causing positive inotropy. We argue therefore, that action prolongation may be a compensatory response which may acutely support the compromised cardiac output. In severe hypertrophy and end-stage heart failure however, despite continued action potential prolongation, the amplitude of the calcium transient becomes severely reduced. The mechanism underlying this event appears to involve reduced expression of calcium handling proteins, and these late events may herald the onset of failure. At present the events leading to the late changes in calcium handling are poorly understood. However, chronic activation of compensatory mechanisms including action potential prolongation may trigger these late events. In the present article we outline a hypothesis which describes a potential role for action potential prolongation, and the associated elevation in the levels of intracellular calcium, in maladaptive gene expression and the progression toward cardiac failure.