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Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting., Liu et al study the molecular and structural mechanism of the modulation of ASIC1 channel gating using functional, crystallographic, computational and mutational approaches. They identify small molecules that allosterically stabilize the closed state by binding at the acidic pocket of the channel.

Measurement of intracellular calcium in live cells is a key component of a wide range of basic life science research, and crucial for many high-throughput assays used in modern drug discovery. Synthetic calcium indicators have become the industry standard, due their ease of use, high reliability, wide dynamic range, and availability of a large variety of spectral and chemical properties. Genetically-encoded calcium indicators (GECIs) have been optimized to the point where their performance rivals that of synthetic calcium indicators in many applications. Stable expression of a GECI has distinct advantages over synthetic calcium indicators in terms of reagent cost and simplification of the assay process. We generated a clonal cell line constitutively expressing GCaMP6s; high expression of the GECI was driven by coupling to a blasticidin resistance gene with a self-cleaving cis-acting hydrolase element (CHYSEL) 2A peptide. Here, we compared the performance of the GECI GCaMP6s to the synthetic calcium indicator fluo-4 in a variety of assay formats. We demonstrate that the pharmacology of ion channel and GPCR ligands as determined using the two indicators is highly similar, and that GCaMP6s is viable as a direct replacement for a synthetic calcium indicator.

[Image: see text] This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure–activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

[Image: see text] Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC(50) = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED(50) = 0.6 mg/kg, plasma EC(50) = 9 ng/mL).

Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects.

Members of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediate the majority of fast synaptic transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic intervention. Here, we describe novel AMPA receptor modulators that require the presence of the accessory protein CACNG8, also known as transmembrane AMPA receptor regulatory protein γ8 (TARP-γ8). Using calcium flux, radioligand binding, and electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these compounds possess a novel mechanism of action consistent with a partial disruption of the interaction between the TARP and the pore-forming subunit of the channel. One of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. This molecule showed strong, dose-dependent inhibition of neurotransmission within the hippocampus, and a strong anticonvulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electroencephalogram, transient hyperlocomotion, no motor impairment on rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool for reversible AMPA receptor inhibition, particularly within the hippocampus, with potential therapeutic utility as an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism of action demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential number of druggable targets.

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.

Utilization of a tetrahydro-pyrimdoazepine core as a bioisosteric replacement for a piperazine-urea resulted in the discovery a novel series of potent antagonists of TRPV1. The tetrahydro-pyrimdoazepines have been identified as having good in vitro and in vivo potency and acceptable physical properties.

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.

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.

We have identified and synthesized a series of 2,7-diamino-thiazolo[5,4-d]pyrimidines as TRPV1 antagonists. An exploration of the structure-activity relationships at the 2-, 5-, and 7-positions of the thiazolo[5,4-d]pyrimidine led to the identification of several potent TRPV1 antagonists, including 3, 29, 51, and 57. Compound 3 was orally bioavailable and afforded a significant reversal of carrageenan-induced thermal hyperalgesia with an ED(50)=0.5mg/kg in rats.

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.

Transient receptor potential vanilloid 1 (TRPV1) plays an integral role in modulating the cough reflex, and it is an attractive antitussive drug target. The purpose of this study was to characterize a TRPV1 antagonist, 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), against the guinea pig TRPV1 receptor in vitro followed by a proof-of-principle study in an acid-induced model of cough. The affinity of JNJ17203212 for the recombinant guinea pig TRPV1 receptor was estimated by radioligand binding, and it was functionally characterized by antagonism of low-pH and capsaicin-induced activation of the ion channel (fluorometric imaging plate reader and electrophysiology). The nature of antagonism was further tested against the native channel in isolated guinea pig tracheal rings. Following pharmacokinetic characterization of JNJ17203212 in guinea pigs, pharmacodynamic and efficacy studies were undertaken to establish the antitussive efficacy of the TRPV1 antagonist. The pK(i) of JNJ17203212 for recombinant guinea pig TRPV1 was 7.14 +/- 0.06. JNJ17203212 inhibited both pH (pIC(50) of 7.23 +/- 0.05) and capsaicin (pIC(50) of 6.32 +/- 0.06)-induced channel activation. In whole-cell patch clamp, the pIC(50) for inhibition of guinea pig TRPV1 was 7.3 +/- 0.01. JNJ17203212 demonstrated surmountable antagonism in isolated trachea, with a pK(B) value of 6.2 +/- 0.1. Intraperitoneal administration of 20 mg/kg JNJ17203212 achieved a maximal plasma exposure of 8.0 +/- 0.4 microM, and it attenuated capsaicin evoked coughs with similar efficacy to codeine (25 mg/kg). Last, JNJ17203212 dose-dependently produced antitussive efficacy in citric acid-induced experimental cough in guinea pigs. Our data provide preclinical support for developing TRPV1 antagonists for the treatment of cough.

High throughput functional assays are increasingly relied upon to generate early and novel discovery leads for drug development. Ion transport proteins including channels, transporters, and pumps play central roles in cellular bioenergetics, excitability, and a multitude of other biological functions. Facile, robust methods for detecting ion transport activity in both native and heterologous systems is desirable for rapid functional analysis and drug discovery for these difficult but important targets. Here we discuss cell-compatible fluorescent probes, functional assays, and VIPR instrumentation that are used to monitor real-time target activity and screen large chemical libraries for potent and selective modulators. Advances and issues for both exogenously applied and fluorescent protein probes of cellular membrane potential, Ca2+, Cl-, and pH are addressed. High throughput screening (HTS) compatible, rapid kinetic and fluorescence resonance energy transfer (FRET) assays are emphasized, in particular the use of voltage-sensitive FRET probes to assay ion channel activity in single cells and 96/384-well formats.

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.

A description is given of a functional silicon micromachined device that permits non-invasive, bidirectional, highly specific communication with cultured mammalian neurons. The heart of the system is a well structure that holds the cell in close proximity to a metal extracellular electrode while permitting normal outgrowth of axons and dendrites. An iterative approach is used to create a design that allows normal growth of the neurons while preventing their escape. An array of 16 such neurowells makes it possible to perform studies of biological neural network development and function with unprecedented detail.

Thank you for the opportunity to reply to the comment by [Witkin and Gardinier (2016)][1] regarding our recent article, “Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8” ([Maher et al., 2016][2]). It is scientifically reassuring when two independent lines of

Recent experiments in charge-density-wave systems suggest that thermally activated phase slip is responsible for current flow. We present a quantitative theory that explains the nonlinear current-voltage relationship

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.

Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Forster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell.

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).

High throughput functional assays are increasingly relied upon to generate early and novel discovery leads for drug development. Ion transport proteins including channels, transporters, and pumps play central roles in cellular bioenergetics, excitability, and a multitude of other biological functions. Facile, robust methods for detecting ion transport activity in both native and heterologous systems is desirable for rapid functional analysis and drug discovery for these difficult but important targets. Here we discuss cell-compatible fluorescent probes, functional assays, and VIPR instrumentation that are used to monitor real-time target activity and screen large chemical libraries for potent and selective modulators. Advances and issues for both exogenously applied and fluorescent protein probes of cellular membrane potential, Ca2+, Cl-, and pH are addressed. High throughput screening (HTS) compatible, rapid kinetic and fluorescence resonance energy transfer (FRET) assays are emphasized, in particular the use of voltage-sensitive FRET probes to assay ion channel activity in single cells and 96/384-well formats.

Work in progress toward the creation of a "cultured neuron probe" is described. This is a silicon structure similar to that for a multielectrode probe, but with embedded neurons which are intended to grow out and integrate with the neurons of a host nervous system into which the probe has been inserted. Each probe neuron is in close proximity to an electrode which is designed for extracellular estimation and recording from the probe neuron. In this way the probe neurons may provide a highly specific long-term communication link between external electronics and the host. Initial experiments to establish the feasibility of the concept will be done in rat hippocampus.

A new type of multielectrode array for studies of cultured neural networks is described. It is a silicon microstructure, a "neurochip", which embodies wells on the surface of a chip that are designed to contain single neurons. Each well has a platinized gold electrode which can be used for stimulating a neuron in the well or for recording from it. Each well is covered with a structure which provides tunnels through which axons and dendrites can grow out and a central hole through which the neurons can be loaded into the well. The processes thus trap the cells in the wells. The first application of this method is described: A4/spl times/4 array of wells spaced 100 /spl mu/m apart. Results are described which show long term survival and outgrowth of neurons from the wells, and the use of well electrodes to both record and stimulate electrical activity of individual neurons.

The neurochip is a silicon micromachined device upon which cultured mammalian neurons can be continuously and individually monitored and stimulated. The neurochip is based upon a 4×4 array of metal electrodes, each of which has a caged well structure designed to hold a single mature cell body while permitting normal outgrowth of neural processes. We demonstrate that this device is capable of maintaining cell survival, and that the electrodes can both record and stimulate electrical activity in individual cells with no crosstalk between channels.

Voltage-gated sodium channel 1.7 (Nav1.7) plays a role in the conduction of action potentials and is involved in the sensation of pain. Spider toxins such as Huwentoxin IV (HwTx-IV) are potent inhibitors of Nav1.7, functioning as a gating modifier trapping the voltage sensor in an inward closed conformation. HwTx-IV interacts with specific residues in the voltage sensor S3-S4 region of domain II. The purpose of the present study was to understand the residues important for interaction between HwTx-IV and Nav1.7. Computationally, molecular dynamics was used to study wild type HwTx-IV along with various alanine and cystine mutations to determine residues important for the stability of toxin and to obtain a model of the interacting surface. A homology model of Nav1.7 was built and the toxin docked to determine key interactions. Experimentally, alanine mutants of HwTx-IV were tested for functional activity using FLIPR, QPatch, and manual patch. Computational and experimental results suggest key residues critical for binding of the toxin to Nav1.7, particularly S25, W30 and K32. These studies provide a more clear idea of the nature of interaction between HwTx-IV and Nav1.7 and may therefore be a useful guide in designing novel peptides with improved selectivity for sodium channel subtypes.