A computational approach yields selective inhibitors of human excitatory amino acid transporter 2 (EAAT2).

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.

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7.

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 Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 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 Nav1.7-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 Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 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 Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

State-Dependent Allosteric Inhibition of the Human SLC13A5 Citrate Transporter by Hydroxysuccinic Acids, PF-06649298 and PF-06761281.

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.

Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8.

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.

The discovery and preclinical characterization of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists.

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-1ß P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1ß release in a mouse ex vivo model with a 50mg/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.

Targeting the ion channel Kv1.3 with scorpion venom peptides engineered for potency, selectivity, and half-life.

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.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

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.

Discovery of a novel series of selective HCN1 blockers.

The discovery of a series of novel, potent, and selective blockers of the cyclic nucleotide-modulated channel HCN1 is disclosed. Here we report an SAR study around a series of selective blockers of the HCN1 channel. Utilization of a high-throughput VIPR assay led to the identification of a novel series of 2,2-disubstituted indane derivatives, which had moderate selectivity and potency at HCN1. Optimization of this hit led to the identification of the potent, 1,1-disubstituted cyclohexane HCN1 blocker, 2-ethoxy-N-((1-(4-isopropylpiperazin-1-yl)cyclohexyl)methyl)benzamide. The work leading to the discovery of this compound is described herein.

Selective Targeting of Nav1.7 with Engineered Spider Venom-Based Peptides.

A fundamental mechanism that drives the propagation of electrical signals in the nervous system is the activation of voltage-gated sodium channels. The sodium channel subtype Nav1.7 is critical for the transmission of pain-related signaling, with gain-of-function mutations in Nav1.7 resulting in various painful pathologies. Loss-of-function mutations cause complete insensitivity to pain and anosmia in humans that otherwise have normal nervous system function, rendering Nav1.7 an attractive target for the treatment of pain. Despite this, no Nav1.7 selective therapeutic has been approved for use as an analgesic to date. Here we present a summary of research that has focused on engineering peptides found in spider venoms to produce Nav1.7 selective antagonists. We discuss the progress that has been made on various scaffolds from different venom families and highlight the challenges that remain in the effort to produce a Nav1.7 selective, venom-based analgesic.

Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrimido-[4,5-d]azepines as novel TRPV1 antagonists.

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.

1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines as TRPV1 antagonists with improved properties.

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.

Functional α6ß4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6ß4 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 α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 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 α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.

Aryl sulfonamido tetralin inhibitors of the Kv1.5 ion channel.

Aryl sulfonamido tetralins based on lead compound 2a were synthesized and evaluated for Kv1.5 inhibitory activity. Several compounds having IC(50) values less then 0.1 microM were identified. Kv1.5 inhibitors have the potential to be atrium-selective agents for the treatment of atrial fibrillation.

Identification and synthesis of 2,7-diamino-thiazolo[5,4-d]pyrimidine derivatives as TRPV1 antagonists.

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.

Activation of TRPA1 by farnesyl thiosalicylic acid.

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.

In vivo profile of ICA-27243 [N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide], a potent and selective KCNQ2/Q3 (Kv7.2/Kv7.3) activator in rodent anticonvulsant models.

Openers or activators of neuronal KCNQ2/Q3 potassium channels decrease neuronal excitability and may provide benefit in the treatment of disorders of neuronal excitability such as epilepsy. In the present study, we evaluate the effects of ICA-27243 [N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide], an orally bioavailable, potent, and selective KCNQ2/Q3 opener, in a broad range of rodent seizure models. ICA-27243 was effective against maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in both rats (MES, ED(50) = 1.5 mg/kg p.o.; PTZ, ED(50) = 2.2 mg/kg p.o.) and mice (MES, ED(50) = 8.6 mg/kg p.o.; PTZ, ED(50) = 3.9 mg/kg p.o.) in the rat amygdala kindling model of partial seizures (full protection from seizure at 9 mg/kg p.o.) and in the 6-Hz model of psychomotor seizures in mice (active at 10 mg/kg i.p.). Antiseizure efficacy in all models was observed at doses significantly less than those shown to effect open-field locomotor activity (rat ED(50) = 40 mg/kg p.o.) or ability to remain on a Rotorod (no effect in rat at doses up to 100 mg/kg p.o.). There was no evidence of cognition impairment as measured in the Morris water maze in the rat (10 and 30 mg/kg p.o.), nor was there evidence of the development of tolerance after multiple doses of ICA-27243. Our findings suggest that selective KCNQ2/Q3 opening activity in the absence of effects on KCNQ3/Q5 or GABA-activated channels may be sufficient for broad-spectrum antiepileptic activity in rodents.

Potentiating SLC transporter activity: Emerging drug discovery opportunities.

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.

T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers.

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.

4-Methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate.

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.