Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel NaV1.8 with a Unique Mechanism of Action.

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.

Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.

Using voltage-sensor toxins and their molecular targets to investigate NaV 1.8 gating.

Voltage-gated sodium (NaV ) channel gating is a complex phenomenon which involves a distinct contribution of four integral voltage-sensing domains (VSDI, VSDII, VSDIII and VSDIV). Utilizing accrued pharmacological and structural insights, we build on an established chimera approach to introduce animal toxin sensitivity in each VSD of an acceptor channel by transferring in portable S3b-S4 motifs from the four VSDs of a toxin-susceptible donor channel (NaV 1.2). By doing so, we observe that in NaV 1.8, a relatively unexplored channel subtype with distinctly slow gating kinetics, VSDI-III participate in channel opening whereas VSDIV can regulate opening as well as fast inactivation. These results illustrate the effectiveness of a pharmacological approach to investigate the mechanism underlying gating of a mammalian NaV channel complex.

Crystallographic insights into sodium-channel modulation by the ß4 subunit.

Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, ß-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether ß-subunits also influence ligand interactions, we found that ß4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular ß4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of ß4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of ß4. Disrupting this bridge by introducing a ß1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts ß4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of ß-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant ß-subunit behavior.

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.

Animal toxins influence voltage-gated sodium channel function.

Voltage-gated sodium (Nav) channels are essential contributors to neuronal excitability, making them the most commonly targeted ion channel family by toxins found in animal venoms. These molecules can be used to probe the functional aspects of Nav channels on a molecular level and to explore their physiological role in normal and diseased tissues. This chapter summarizes our existing knowledge of the mechanisms by which animal toxins influence Nav channels as well as their potential application in designing therapeutic drugs.

Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target NaV1.7.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (NaV) channel 1.7 (NaV1.7), which has been identified as a primary pain target. However, in developing NaV1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other NaV subtypes that are critical for survival. Since spider venoms are an excellent source of NaV channel modulators, we screened a panel of spider venoms to identify selective NaV1.7 inhibitors. This led to identification of two novel NaV modulating venom peptides (ß/µ-theraphotoxin-Pe1a and ß/µ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (ß/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the NaV1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and NaV channel subtype selectivity. Several analogues had improved potency against NaV1.7, and altered specificity against other NaV channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of NaV1.7.

Non-invasive diagnosis of early pulmonary disease in PECAM-deficient mice using infrared pulse oximetry.

Pulse oximetry is a common tool for detecting reduced pulmonary function in human interstitial lung diseases. It has not previously been used in a mouse model of interstitial lung disease. Further, platelet endothelial cell adhesion molecule deficient mice rarely show symptoms until disease is advanced. Using blood oxygen saturation, different stages of disease could be identified in a non-invasive manner. These stages could be correlated to pathology. Collagen deposition, using Picrosirius Red, did correlate with blood oxygen saturation. These studies are the first to show the use of an infrared pulse oximetry system to analyze the progression of a fibrotic interstitial lung disease in a mouse model of the human diseases. Further, these studies show that an early alveolar damage/enlargement event precedes the fibrosis in this mouse model, a stage that represents the best targets for disease analysis and prevention. This stage does not have extensive collagen deposition. Most importantly, targeting this earliest stage of disease for therapeutic intervention may lead to novel treatment for human disease.

The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) formed by TMEM16A or TMEM16B are broadly expressed in the nervous system, smooth muscles, exocrine glands, and other tissues. With two calcium-binding sites and a pore within each monomer, the dimeric CaCC exhibits voltage-dependent calcium sensitivity. Channel activity also depends on the identity of permeant anions. To understand how CaCC regulates neuronal signaling and how CaCC is, in turn, modulated by neuronal activity, we examined the molecular basis of CaCC gating. Here, we report that voltage modulation of TMEM16A-CaCC involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment.

NaV1.1 inhibition can reduce visceral hypersensitivity.

Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.

Differential effect of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) on leukocyte infiltration during contact hypersensitivity responses.

BACKGROUND: 2'-4' Dinitrofluorobenzene (DNFB) induced contact hypersensitivity is an established model of contact sensitivity and leukocyte migration. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) deficient mice were used to examine the role of PECAM-1 in the migration capacity of several different leukocyte populations after primary and secondary application. RESULTS: γδ T lymphocytes, granulocytes, and Natural Killer cells were most affected by PECAM-1 deficiency at the primary site of application. γδ T lymphocytes, granulocytes, DX5+ Natural Killer cells, and, interestingly, effector CD4+ T lymphocytes were most affected by the loss of PECAM-1 at the secondary site of application. CONCLUSIONS: PECAM-1 is used by many leukocyte populations for migration, but there are clearly differential effects on the usage by each subset. Further, the overall kinetics of each population varied between primary and secondary application, with large relative increases in γδ T lymphocytes during the secondary response.

The tarantula toxin ß/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity.

Voltage-gated sodium (NaV) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe ß/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Binary architecture of the Nav1.2-ß2 signaling complex.

To investigate the mechanisms by which ß-subunits influence Nav channel function, we solved the crystal structure of the ß2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in ß2 with Nav1.2. We identified a flexible loop formed by (72)Cys and (75)Cys, a unique feature among the four ß-subunit isoforms. Moreover, we found that (55)Cys helps to determine the influence of ß2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that (55)Cys forms a disulfide bond with (910)Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 (912/918)Cys residues. The concepts emerging from this work will help to form a model reflecting the ß-subunit location in a Nav channel complex.

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix.

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami.

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

Fluorescence spectroscopy of Rhodamine 6G: concentration and solvent effects.

Rhodamine 6G (R6G), also known as Rhodamine 590, is one of the most frequently used dyes for application in dye lasers and as a fluorescence tracer, e.g., in the area of environmental hydraulics. Knowing the spectroscopic characteristics of the optical emission is key to obtaining high conversion efficiency and measurement accuracy, respectively. In this work, solvent and concentration effects are studied. A series of eight different organic solvents (methanol, ethanol, n-propanol, iso-propanol, n-butanol, n-pentanol, acetone, and dimethyl sulfoxide (DMSO)) are investigated at constant dye concentration. Relatively small changes of the fluorescence spectrum are observed for the different solvents; the highest fluorescence intensity is observed for methanol and lowest for DMSO. The shortest peak wavelength is found in methanol (568 nm) and the longest in DMSO (579 nm). Concentration effects in aqueous R6G solutions are studied over the full concentration range from the solubility limit to highly dilute states. Changing the dye concentration provides tunability between ∼550 nm in the dilute case and ∼620 nm at high concentration, at which point the fluorescence spectrum indicates the formation of R6G aggregates.

A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a.

ß-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1-S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides.

Nav1.1 modulation by a novel triazole compound attenuates epileptic seizures in rodents.

Here, we report the discovery of a novel anticonvulsant drug with a molecular organization based on the unique scaffold of rufinamide, an anti-epileptic compound used in a clinical setting to treat severe epilepsy disorders such as Lennox-Gastaut syndrome. Although accumulating evidence supports a working mechanism through voltage-gated sodium (Nav) channels, we found that a clinically relevant rufinamide concentration inhibits human (h)Nav1.1 activation, a distinct working mechanism among anticonvulsants and a feature worth exploring for treating a growing number of debilitating disorders involving hNav1.1. Subsequent structure-activity relationship experiments with related N-benzyl triazole compounds on four brain hNav channel isoforms revealed a novel drug variant that (1) shifts hNav1.1 opening to more depolarized voltages without further alterations in the gating properties of hNav1.1, hNav1.2, hNav1.3, and hNav1.6; (2) increases the threshold to action potential initiation in hippocampal neurons; and (3) greatly reduces the frequency of seizures in three animal models. Altogether, our results provide novel molecular insights into the rational development of Nav channel-targeting molecules based on the unique rufinamide scaffold, an outcome that may be exploited to design drugs for treating disorders involving particular Nav channel isoforms while limiting adverse effects.

Animal toxins can alter the function of Nav1.8 and Nav1.9.

Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.