The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

G-protein-gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel-phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

Mining the Nav1.7 interactome: Opportunities for chronic pain therapeutics.

The peripherally expressed voltage-gated sodium NaV1.7 (gene SCN9A) channel boosts small stimuli to initiate firing of pain-signaling dorsal root ganglia (DRG) neurons and facilitates neurotransmitter release at the first synapse within the spinal cord. Mutations in SCN9A produce distinct human pain syndromes. Widely acknowledged as a "gatekeeper" of pain, NaV1.7 has been the focus of intense investigation but, to date, no NaV1.7-selective drugs have reached the clinic. Elegant crystallographic studies have demonstrated the potential of designing highly potent and selective NaV1.7 compounds but their therapeutic value remains untested. Transcriptional silencing of NaV1.7 by a naturally expressed antisense transcript has been reported in rodents and humans but whether this represents a viable opportunity for designing NaV1.7 therapeutics is currently unknown. The demonstration that loss of NaV1.7 function is associated with upregulation of endogenous opioids and potentiation of mu- and delta-opioid receptor activities, suggests that targeting only NaV1.7 may be insufficient for analgesia. However, the link between opioid-dependent analgesic mechanisms and function of sodium channels and intracellular sodium-dependent signaling remains controversial. Thus, additional new targets - regulators, modulators - are needed. In this context, we mine the literature for the known interactome of NaV1.7 with a focus on protein interactors that affect the channel's trafficking or link it to opioid signaling. As a case study, we present antinociceptive evidence of allosteric regulation of NaV1.7 by the cytosolic collapsin response mediator protein 2 (CRMP2). Throughout discussions of these possible new targets, we offer thoughts on the therapeutic implications of modulating NaV1.7 function in chronic pain.

Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain.

We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.

Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

BACKGROUND AND PURPOSE: N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. EXPERIMENTAL APPROACH: Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. KEY RESULTS: The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. CONCLUSIONS AND IMPLICATIONS: These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Orexin Depolarizes Central Amygdala Neurons via Orexin Receptor 1, Phospholipase C and Sodium-Calcium Exchanger and Modulates Conditioned Fear.

Orexins (OX), also known as hypocretins, are excitatory neuropeptides with well-described roles in regulation of wakefulness, arousal, energy homeostasis, and anxiety. An additional and recently recognized role of OX is modulation of fear responses. We studied the OX neurons of the perifornical hypothalamus (PeF) which send projections to the amygdala, a region critical in fear learning and fear expression. Within the amygdala, the highest density of OX-positive fibers was detected in the central nucleus (CeA). The specific mechanisms underlying OX neurotransmission within the CeA were explored utilizing rat brain slice electrophysiology, pharmacology, and chemogenetic stimulation. We show that OX induces postsynaptic depolarization of medial CeA neurons that is mediated by OX receptor 1 (OXR1) but not OX receptor 2 (OXR2). We further characterized the mechanism of CeA depolarization by OX as phospholipase C (PLC)- and sodium-calcium exchanger (NCX)- dependent. Selective chemogenetic stimulation of OX PeF fibers recapitulated OXR1 dependent depolarization of CeA neurons. We also observed that OXR1 activity modified presynaptic release of glutamate within the CeA. Finally, either systemic or intra-CeA perfusion of OXR1 antagonist reduced the expression of conditioned fear. Together, these data suggest the PeF-CeA orexinergic pathway can modulate conditioned fear through a signal transduction mechanism involving PLC and NCX activity and that selective OXR1 antagonism may be a putative treatment for fear-related disorders.

Dissecting the role of the CRMP2-neurofibromin complex on pain behaviors.

Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.

Sensitization of Ion Channels Contributes to Central and Peripheral Dysfunction in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (Nf1) is a progressive, autosomal disorder with a large degree of variability and severity of manifestations including neurological, cutaneous, ocular/orbital, orthopedic, and vascular abnormalities. Nearly half of Nf1 patients presents with cognitive impairment, specifically spatial learning deficits. These clinical manifestations suggest a global impairment of both central and peripheral nervous system functions in neurofibromatosis. Nf1 encodes for neurofibromin, a Ras GTPase-activating protein (Ras GAP) that has been implicated in the regulation of long-term potentiation (LTP), Ras/ERK (extracellular signal-regulated kinase) signaling, and learning in mice. Over the last decades, mice with a targeted mutation in the Nf1 gene, Nf1 -/- chimeric mice, Nf1 exon-specific knockout mice, and mice with tissue-specific inactivation of Nf1 have been generated to model the human Nf1 disease. These studies have implicated neurofibromin in regulation of the release of the inhibitory neurotransmitter γ-amino butyric acid (GABA) in the hippocampus and frontal lobe, which can regulate memory. Mutations in neurofibromin thus lead to perturbed ERK signaling, which alters GABA release, LTP, and subsequently leads to learning deficits. In addition to these cognitive deficits, Nf1 patients also have defects in fine and gross motor coordination as well as decreased muscle strength. Although the mechanisms underlying these motor deficits are unknown, deficits in GABAergic neurotransmission in both the motor cortex and cerebellum have been suggested. In this review, we present evidence to support the hypothesis that alterations of ion channel activity in Nf1 underscore the dysregulated neuronal communication in non-neuronal and neuronal cells that likely contributes to the clinical cornucopia of Nf1.

Hierarchical CRMP2 posttranslational modifications control NaV1.7 function.

Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrin-dependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.

Chimeric derivatives of functionalized amino acids and α-aminoamides: compounds with anticonvulsant activity in seizure models and inhibitory actions on central, peripheral, and cardiac isoforms of voltage-gated sodium channels.

Six novel 3″-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant activities in animal seizure models and for their sodium channel activities. We found compounds with various substituents at the terminal aromatic ring that had excellent anticonvulsant activity. Of these compounds, (R)-N-4'-((3″-chloro)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-5) and (R)-N-4'-((3″-trifluoromethoxy)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-9) exhibited high protective indices (PI=TD50/ED50) comparable with many antiseizure drugs when tested in the maximal electroshock seizure test to mice (intraperitoneally) and rats (intraperitoneally, orally). Most compounds potently transitioned sodium channels to the slow-inactivated state when evaluated in rat embryonic cortical neurons. Treating HEK293 recombinant cells that expressed hNaV1.1, rNaV1.3, hNaV1.5, or hNaV1.7 with (R)-9 recapitulated the high levels of sodium channel slow inactivation.

Chimeric agents derived from the functionalized amino acid, lacosamide, and the α-aminoamide, safinamide: evaluation of their inhibitory actions on voltage-gated sodium channels, and antiseizure and antinociception activities and comparison with lacosamide and safinamide.

The functionalized amino acid, lacosamide ((R)-2), and the α-aminoamide, safinamide ((S)-3), are neurological agents that have been extensively investigated and have displayed potent anticonvulsant activities in seizure models. Both compounds have been reported to modulate voltage-gated sodium channel activity. We have prepared a series of chimeric compounds, (R)-7-(R)-10, by merging key structural units in these two clinical agents, and then compared their activities with (R)-2 and (S)-3. Compounds were assessed for their ability to alter sodium channel kinetics for inactivation, frequency (use)-dependence, and steady-state activation and fast inactivation. We report that chimeric compounds (R)-7-(R)-10 in catecholamine A-differentiated (CAD) cells and embryonic rat cortical neurons robustly enhanced sodium channel inactivation at concentrations far lower than those required for (R)-2 and (S)-3, and that (R)-9 and (R)-10, unlike (R)-2 and (S)-3, produce sodium channel frequency (use)-dependence at low micromolar concentrations. We further show that (R)-7-(R)-10 displayed excellent anticonvulsant activities and pain-attenuating properties in the animal formalin model. Of these compounds, only (R)-7 reversed mechanical hypersensitivity in the tibial-nerve injury model for neuropathic pain in rats.

Carbamazepine potentiates the effectiveness of morphine in a rodent model of neuropathic pain.

Approximately 60% of morphine is glucuronidated to morphine-3-glucuronide (M3G) which may aggravate preexisting pain conditions. Accumulating evidence indicates that M3G signaling through neuronal Toll-like receptor 4 (TLR4) may be central to this proalgesic signaling event. These events are known to include elevated neuronal excitability, increased voltage-gated sodium (NaV) current, tactile allodynia and decreased opioid analgesic efficacy. Using an in vitro ratiometric-based calcium influx analysis of acutely dissociated small and medium-diameter neurons derived from lumbar dorsal root ganglion (DRG), we observed that M3G-sensitive neurons responded to lipopolysaccharide (LPS) and over 35% of these M3G/LPS-responsive cells exhibited sensitivity to capsaicin. In addition, M3G-exposed sensory neurons significantly increased excitatory activity and potentiated NaV current as measured by current and voltage clamp, when compared to baseline level measurements. The M3G-dependent excitability and potentiation of NaV current in these sensory neurons could be reversed by the addition of carbamazepine (CBZ), a known inhibitor of several NaV currents. We then compared the efficacy between CBZ and morphine as independent agents, to the combined treatment of both drugs simultaneously, in the tibial nerve injury (TNI) model of neuropathic pain. The potent anti-nociceptive effects of morphine (5 mg/kg, i.p.) were observed in TNI rodents at post-injury day (PID) 7-14 and absent at PID21-28, while administration of CBZ (10 mg/kg, i.p.) alone failed to produce anti-nociceptive effects at any time following TNI (PID 7-28). In contrast to either drug alone at PID28, the combination of morphine and CBZ completely attenuated tactile hyperalgesia in the rodent TNI model. The basis for the potentiation of morphine in combination with CBZ may be due to the effects of a latent upregulation of NaV1.7 in the DRG following TNI. Taken together, our observations demonstrate a potential therapeutic use of morphine and CBZ as a combinational treatment for neuropathic pain.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

CRMP2 protein SUMOylation modulates NaV1.7 channel trafficking.

Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.

(Biphenyl-4-yl)methylammonium chlorides: potent anticonvulsants that modulate Na+ currents.

We have reported that compounds containing a biaryl linked unit (Ar-X-Ar') modulated Na(+) currents by promoting slow inactivation and fast inactivation processes and by inducing frequency (use)-dependent inhibition of Na(+) currents. These electrophysiological properties have been associated with the mode of action of several antiepileptic drugs. In this study, we demonstrate that the readily accessible (biphenyl-4-yl)methylammonium chlorides (compound class B) exhibited a broad range of anticonvulsant activities in animal models, and in the maximal electroshock seizure test the activity of (3'-trifluoromethoxybiphenyl-4-yl)methylammonium chloride (8) exceeded that of phenobarbital and phenytoin upon oral administration to rats. Electrophysiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8 promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na(+) currents.

Discovery of lacosamide affinity bait agents that exhibit potent voltage-gated sodium channel blocking properties.

Lacosamide ((R)-1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)-1 modulates voltage-gated Na(+) channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)-1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-8), (S)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((S)-8), (R)-N-(3″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-9), and (R)-N-(3″-acrylamidobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that (R)-8 and (R)-9 are potent inhibitors of Na(+) channel function and function by a pathway similar to that observed for (R)-1. We further demonstrate that (R)-8 function is stereospecific. The calculated IC50 values determined for Na(+) channel slow inactivation for (R)-1, (R)-8, and (R)-9 were 85.1, 0.1, and 0.2 µM, respectively. Incubating (R)-9 with the neuronal-like CAD cells led to appreciable levels of Na(+) channel slow inactivation after cellular wash, and the level of slow inactivation only modestly decreased with further incubation and washing. Collectively, these findings have identified a promising structural template to investigate the voltage-gated Na(+) channel slow inactivation process.

Identification of the benzyloxyphenyl pharmacophore: a structural unit that promotes sodium channel slow inactivation.

Four compounds that contained the N-benzyl 2-amino-3-methoxypropionamide unit were evaluated for their ability to modulate Na(+) currents in catecholamine A differentiated CAD neuronal cells. The compounds differed by the absence or presence of either a terminal N-acetyl group or a (3-fluoro)benzyloxy moiety positioned at the 4'-benzylamide site. Analysis of whole-cell patch-clamp electrophysiology data showed that the incorporation of the (3-fluoro)benzyloxy unit, to give the (3-fluoro)benzyloxyphenyl pharmacophore, dramatically enhanced the magnitude of Na(+) channel slow inactivation. In addition, N-acetylation markedly increased the stereoselectivity for Na(+) channel slow inactivation. Furthermore, we observed that Na(+) channel frequency (use)-dependent block was maintained upon inclusion of this pharmacophore. Confirmation of the importance of the (3-fluoro)benzyloxyphenyl pharmacophore was shown by examining compounds where the N-benzyl 2-amino-3-methoxypropionamide unit was replaced by a N-benzyl 2-amino-3-methylpropionamide moiety, as well as examining a series of compounds that did not contain an amino acid group but retained the pharmacophore unit. Collectively, the data indicated that the (3-fluoro)benzyloxyphenyl unit is a novel pharmacophore for the modulation of Na(+) currents.

Inhibition of transmitter release and attenuation of anti-retroviral-associated and tibial nerve injury-related painful peripheral neuropathy by novel synthetic Ca2+ channel peptides.

N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca(2+) channel complex. Nat. Med. 17, 822-829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388-402), "L1") and the distal C terminus (CaV1.2(2014-2028) "Ct-dis") that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2'-3'-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.