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A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca V 2.2) as therapeutic targets for chronic pain. A state-dependent Ca V 2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca V 2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca V 2 inhibitor (3 R )-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1 H -1,2,4-triazol-3-yl)-1,3-dihydro-2 H -indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca V 2.2 channels with an IC 50 of 0.11 μM. The voltage dependence of Ca V 2.2 inhibition was examined using automated electrophysiology. TROX-1 IC 50 values were 4.2, 0.90, and 0.36 μM at −110, −90, and −70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca V 2.2 with a 10-fold IC 50 separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca V 2.2 channels with an IC 50 of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca V 2 subfamily. Depolarized IC 50 values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca V 2.1, Ca V 2.2, and Ca V 2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective Ca V 2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca V 2.2-selective inhibitor ziconotide in preclinical models of chronic pain.

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50)20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.

A series of 3-amino-1,5-benzodiazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of this series displayed subnanomolar, state-dependent sodium channel block, and was orally efficacious in a mouse model of epilepsy.

Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In a high-throughput functional assay, a novel sodium channel (Na(V)) blocker, N-[[2'-(aminosulfonyl)biphenyl-4-yl]methyl]-N'-(2,2'-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of Na(V)1.2, Na(V)1.5, and Na(V)1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of Na(V)1.2 was 1.2 and 0.14 microM, respectively. BPBTS blocked Na(V)1.7 and Na(V)1.2 with similar potency, whereas block of Na(V)1.5 was slightly more potent. The slow tetrodotoxin-resistant Na(+) current in small-diameter DRG neurons was also potently blocked by BPBTS. [(3)H]BPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K(d) = 6.1 nM), and in membranes derived from HEK cells stably expressing Na(V)1.5 (K(d) = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.

Nodulisporic acid (NA) is an indole diterpene fungal product with insecticidal activity. NA activates a glutamate-gated chloride channel (GluCl) in grasshopper neurons and potentiates channel opening by glutamate. The endectocide ivermectin (IVM) induces a similar, but larger current than NA. Using Drosophila melanogaster head membranes, a high affinity binding site for NA was identified. Equilibrium binding studies show that an amide analogue, N-(2-hydroxyethyl-2,2-(3)H)nodulisporamide ([(3)H]NAmide), binds to a single population of sites in head membranes with a K(D) of 12 pM and a B(max) of 1.4 pmol/mg of protein. A similar K(D) is determined from the kinetics of ligand binding and dissociation. Four lines of evidence indicate that the binding site is a GluCl. First, NA potentiates opening of a glutamate-gated chloride current in grasshopper neurons. Second, glutamate inhibits the binding of [(3)H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM potently inhibits the binding of [(3)H]NAmide and IVM binds to GluCls. Finally, the binding of [(3)H]IVM is inhibited by NA. The B(max) of [(3)H]IVM is twice that of [(3)H]NAmide, and about half of the [(3)H]IVM binding sites are inhibited by NA with high affinity (K(I) = 25 pM). In contrast, [(3)H]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and there are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophila IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identifies subpopulations of IVM binding sites.

Previously, we have described the properties of recombinant baculoviruses expressing three chimeric genes, mag4, sat2, and ssh1, that encode secretable insect selective sodium channel toxins, μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus, respectively. We now show that μ-Aga-IV and As II act at distinct sites on voltage-sensitive sodium channels of insects and synergistically promote channel opening. We also show that these toxins have synergistic insecticidal activity against the blowfly Lucilia sericata and the fall armyworm Spodoptera frugiperda. To determine if toxin synergy also occurs in the context of virus replication, we inserted the chimeric toxin genes into nonessential sites of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of either a modified promoter, P synXIV , or an insect derived promoter, P hsp70 . Comparative analyses showed that viruses expressing toxin genes under the control of the P hsp70 promoter were more effective as biopesticides than under the control of P synXIV . Two toxins, μ-Aga-IV and As II, exerted the most potent effects in S. frugiperda and Trichoplusia ni larvae, respectively. A virus simultaneously expressing two P hsp70 -promoted toxin genes, mag4 and sat2, exhibited properties similar to the two viruses expressing each of the toxin genes individually except that larval feeding time (FT 50 ) was reduced an additional 10%, indicating a small advantage to coproducing synergistic toxins.

We report the investigation of sulfonamide-derived Cav2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Cav2.2 potency without the liability of the circulating sulfonamide metabolite.

The voltage-gated calcium channel Ca(v)2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca(v)2.2. In particular, 19 is an inhibitor of Ca(v)2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca(v)2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta is the only known agent that blocks L-type and N-type Ca channels with equal high potency (IC50 < or = 1 nM). From the same venom, we have purified and sequenced a family of peptides which are homologous to omega-Aga-IIIA but vary over 100-fold in their relative affinity for L-type versus N-type Ca channels. One of these, omega-Aga-IIIB, is 76 amino acids long and identical to omega-Aga-IIIA in 66 positions. We identified two other similar peptides, omega-Aga-IIIC and omega-Aga-IIID, as well as one single amino acid variant of omega-Aga-IIIA and two of omega-Aga-IIIB. The type III omega-agatoxins exhibit similar but distinct activities on voltage-gated Ca channels. omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID are nearly indistinguishable in their actions at the insect neuromuscular junction (no effect at 0.1 microM), on atrial T-type Ca channels (no effect at 0.5 microM), and in two assays for synaptosomal Ca channels: they are nearly equipotent inhibitors of 125I-omega-conotoxin GVIA binding to rat brain synaptic membranes (IC50 = 0.17-0.33 nM) and blockers of the K(+)-induced 45Ca2+ influx into chick brain synaptosomes (omega-Aga-IIIB, 1.2 nM; omega-Aga-IIIA, 2.4 nM). In contrast, omega-Aga-IIIA is a better blocker of locust Ca channels (IC50 approximately 10-50 nM) than is omega-Aga-IIIB. Finally, although omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID all block atrial L-type Ca channels, omega-Aga-IIIA is over 100-fold more potent. Thus, although type III omega-agatoxins appear to recognize a binding site common to L- and N-type Ca channels, omega-Aga-IIIB and omega-Aga-IIID identify differences between the two channels.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Nav1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. With a goal to develop potent peripherally active sodium channel blockers, a series of low molecular weight biaryl substituted imidazoles, oxazoles, and thiazole carboxamides were identified with good in vitro and in vivo potency.

A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.

The tarantula venom peptides ProTx-I and ProTx-II inhibit voltage-gated sodium channels by shifting their voltage dependence of activation to a more positive potential, thus acting by a mechanism similar to that of potassium channel gating modifiers such as hanatoxin and VSTX1. ProTx-I and ProTx-II inhibit all sodium channel (Nav1) subtypes tested with similar potency and represent the first potent peptidyl inhibitors of TTX-resistant sodium channels. Like gating modifiers of potassium channels, ProTx-I and ProTx-II conform to the inhibitory cystine knot motif, and ProTx-II was demonstrated to bind to sodium channels in the closed state. Both toxins have been synthesized chemically, and ProTx-II, produced by recombinant means, has been used to map the interaction surface of the peptide with the Nav1.5 channel. In comparison, β-scorpion toxins activate sodium channels by shifting the voltage dependence of activation to more negative potentials, and together these peptides represent valuable tools for exploring the gating mechanism of sodium channels.

Delayed-rectifier K+ currents (I(DR)) in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltage-gated K+ channel, K(V)2.1, is expressed in beta-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I(DR) in rodent beta-cells. A novel peptidyl inhibitor of K(V)2.1/K(V)2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration approximately 1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to beta-cell physiology. In mouse beta-cells, GxTX-1 inhibits 90% of I(DR) and, as for K(V)2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the beta-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the beta-cell I(DR), which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.

The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.