Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

The sodium-glucose cotransporter isoform 1 (SGLT-1) is important for sperm energetics, motility, and fertility†.

Glucose is a key substrate for supporting sperm energy production and function. Previous studies have demonstrated that sperm glucose uptake is facilitated by several isoforms of the glucose transporters (GLUT). Here, we report that sperm also expresses the Na+-dependent sodium glucose cotransporter (SGLT). This was first suggested by our observation that genetic deletion of the testis-specific Na,K-ATPase α4, which impairs the sperm plasma membrane Na+ gradient, reduces glucose uptake and ATP production. Immunoblot analysis revealed the presence of an SGLT in sperm, with specific expression of isoform 1 (SGLT-1), but not of isoform 2 (SGLT-2). Immunocytochemistry identified SGLT-1 in the mid- and principal piece of the sperm flagellum. Inhibition of SGLT-1 with the isotype-selective inhibitor phlorizin significantly reduced glucose uptake, glycolytic activity, and ATP production in noncapacitated and capacitated sperm from wild-type mice. Phlorizin also decreased total sperm motility, as well as other parameters of sperm movement. In contrast, inhibition of SGLT-1 had no significant effect on sperm hyperactivation, protein tyrosine phosphorylation, or acrosomal reaction. Importantly, phlorizin treatment impaired the fertilizing capacity of sperm. Altogether, these results demonstrate that mouse sperm express a functional SGLT transport system that is important for supporting sperm energy production, motility, and fertility.

Multi-Electrode Array of Sensory Neurons as an In Vitro Platform to Identify the Nociceptive Response to Pharmaceutical Buffer Systems of Injectable Biologics.

PURPOSE: Pharmaceutical buffer systems, especially for injectable biologics such as monoclonal antibodies, are an important component of successful FDA-approved medications. Clinical studies indicate that buffer components may be contributing factors for increased injection site pain. METHODS: To determine the potential nociceptive effects of clinically relevant buffer systems, we developed an in vitro multi-electrode array (MEA) based recording system of rodent dorsal root ganglia (DRG) sensory neuron cell culture. This system monitors sensory neuron activity/firing as a surrogate of nociception when challenged with buffer components used in formulating monoclonal antibodies and other injectable biologics. RESULTS: We show that citrate salt and citrate mannitol buffer systems cause an increase in mean firing rate, burst frequency, and burst duration in DRG sensory neurons, unlike histidine or saline buffer systems at the same pH value. Lowering the concentration of citrate leads to a lower firing intensity of DRG sensory neurons. CONCLUSION: Increased activity/firing of DRG sensory neurons has been suggested as a key feature underlying nociception. Our results support the utility of an in vitro MEA assay with cultured DRG sensory neurons to probe the nociceptive potential of clinically relevant buffer components used in injectable biologics.

Na,K-ATPase α4, and Not Na,K-ATPase α1, is the Main Contributor to Sperm Motility, But its High Ouabain Binding Affinity Site is Not Required for Male Fertility in Mice.

Mammalian sperm express two Na,K-ATPase (NKA) isoforms, Na,K-ATPase α4 (NKAα4) and Na,K-ATPase α1 (NKAα1). While NKAα4 is critical to sperm motility, the role of NKAα1 in sperm movement remains unknown. We determined this here using a genetic and pharmacological approach, modifying the affinity of NKAα1 and NKAα4 for the inhibitor ouabain to selectively block the function of each isoform. Sperm from wild-type (WT) mice (naturally containing ouabain-resistant NKAα1 and ouabain-sensitive NKAα4) and three newly generated mouse lines, expressing both NKAα1 and NKAα4 ouabain resistant (OR), ouabain sensitive (OS), and with their ouabain affinity switched (SW) were used. All mouse lines produced normal sperm numbers and were fertile. All sperm types showed NKAα isoform expression levels and activity comparable to WT, and kinetics for ouabain inhibition confirming the expected changes in ouabain affinity for each NKA isoform. Ouabain at 1 µM, which only block ouabain-sensitive NKA, significantly inhibited total, progressive, and hyperactivated sperm motility in WT and OS, but had no significant effect on OR or SW sperm. Higher ouabain (1 mM), which inhibits both ouabain-sensitive and ouabain-resistant NKA, had little additional effect on sperm motility in all mouse lines, including the OR and SW. A similar pattern was found for the effect of ouabain on sperm intracellular sodium ([Na+]i). These results indicate that NKAα4, but not NKAα1 is the main contributor to sperm motility and that the ouabain affinity site in NKA is not an essential requirement for male fertility.

Na,K-ATPase Atp1a4 isoform is important for maintaining sperm flagellar shape.

PURPOSE: The aim of this study is to investigate the mechanisms by which the testis specific Na,K-ATPase ion transport system (Atp1a4) controls sperm morphology and shape. METHODS: Sperm from wild-type (WT) and Atp1a4 knockout (Atp1a4 KO) mice were analyzed morphologically, using light, transmission, and scanning electron microscopy; and functionally, applying sperm osmotic challenge and viability tests. In addition, a sperm proteomic study was performed. RESULTS: Light microscopy confirmed that sperm lacking Atp1a4 present a bend at the junction of the mid- and principal piece of the flagellum. This bend had different degrees of angulation, reaching occasionally a complete flagellar retroflexion. The defect appeared in sperm collected from the cauda epididymis, but not the epididymal caput or the testis. Transmission and scanning electron microscopy revealed a dilation of the cytoplasm at the site of the bend, with fusion of the plasma membrane in overlapping segments of the flagellum. This was accompanied by defects in the axoneme and peri-axonemal structures. Sperm from Atp1a4 KO mice showed an abnormal response to hypoosmotic challenge with decreased viability, suggesting reduced capacity for volume regulation. Exposure to Triton X-100 only partially recovered the flagellar bend of Atp1a4 KO sperm, showing that factors other than osmotic regulation contribute to the flagellar defect. Interestingly, several key sperm structural proteins were expressed in lower amounts in Atp1a4 KO sperm, with no changes in their localization. CONCLUSIONS: Altogether, our results show that Atp1a4 plays an important role in maintaining the proper shape of the sperm flagellum through both osmotic control and structurally related mechanisms.

Antitussive effects of NaV 1.7 blockade in Guinea pigs.

Our previous studies implicated the voltage-gated sodium channel subtype NaV 1.7 in the transmission of action potentials by the vagal afferent nerves regulating cough and thus identified this channel as a rational therapeutic target for antitussive therapy. But it is presently unclear whether a systemically administered small molecule inhibitor of NaV 1.7 conductance can achieve therapeutic benefit in the absence of side effects on cardiovascular function, gastrointestinal motility or respiration. To this end, we have evaluated the antitussive effects of the NaV 1.7 selective blocker Compound 801 administered systemically in awake guinea pigs or administered topically in anesthetized guinea pigs. We also evaluated the antitussive effects of ambroxol, a low affinity NaV blocker modestly selective for tetrodotoxin resistant NaV subtypes. Both Compound 801 and ambroxol dose-dependently inhibited action potential conduction in guinea pig vagus nerves (assessed by compound potential), with ambroxol nearly 100-fold less potent than the NaV 1.7 selective Compound 801 in this and other NaV 1.7-dependent guinea pig and human tissue-based assays. Both drugs also inhibited citric acid evoked coughing in awake or anesthetized guinea pigs, with potencies supportive of an NaV 1.7-dependent mechanism. Notably, however, the antitussive effects of systemically administered Compound 801 were accompanied by hypotension and respiratory depression. Given the antitussive effects of topically administered Compound 801, we speculate that the likely insurmountable side effects on blood pressure and respiratory drive associated with systemic dosing make topical formulations a viable and perhaps unavoidable therapeutic strategy for targeting NaV 1.7 in cough.

Stimulus intensity-dependent recruitment of NaV1 subunits in action potential initiation in nerve terminals of vagal C-fibers innervating the esophagus.

We investigated voltage-gated sodium channel (NaV1) subunits that regulate action potential initiation in the nerve terminals of vagal nodose C-fibers innervating the esophagus. Extracellular single fiber recordings were made from the nodose C-fibers, with mechanically sensitive nerve terminals in the isolated innervated guinea pig esophagus. NaV1 inhibitors were selectively delivered to the tissue-containing nerve terminals. Graded esophageal distention was used for mechanical stimulation. The NaV1.7 inhibitor PF-05089771 nearly abolished action potential initiation in response to low levels of esophageal distention but only partially inhibited the response to higher levels of esophageal distention. The PF-05089771-insensitive component of the response progressively increased (up to ≈50%) with increasing esophageal distention and was abolished by tetrodotoxin (TTX). In addition to NaV1.7, nodose C-fiber [transient receptor potential channel-vanilloid subfamily member 1 (TRPV1)-positive] neurons retrogradely labeled from the esophagus expressed mRNA for multiple TTX-sensitive NaV1s. The group NaV1.1, NaV1.2, and NaV1.3 inhibitor ICA-121431 inhibited but did not abolish the PF-05089771-insensitive component of the response to high level of esophageal distention. However, combination of ICA-121431 with compound 801, which also inhibits NaV1.7 and NaV1.6, nearly abolished the response to the high level of esophageal distention. Our data indicate that the action potential initiation in esophageal nodose C-fibers evoked by low (innocuous) levels of esophageal distention is mediated by NaV1.7. However, the response evoked by higher (noxious) levels of esophageal distention has a progressively increasing NaV1.7-independent component that involves multiple TTX-sensitive NaV1s. The stimulus intensity-dependent recruitment of NaV1s may offer novel opportunities for strategic targeting of NaV1 subunits for inhibition of nociceptive signaling in visceral C-fibers.NEW & NOTEWORTHY We report that pharmacologically distinguishable voltage-gated sodium channels (NaV1) mediate action potential initiation at low (innocuous) versus high (noxious) intensity of esophageal distention in nerve terminals of vagal nodose C-fibers. Action potential initiation at low intensity is entirely dependent on NaV1.7; however, additional tetrodotoxin (TTX)-sensitive NaV1s are recruited at higher intensity of distention. This is the first demonstration that NaV1s underlying action potential initiation in visceral C-fibers depend on the intensity of the stimulus.

21-Benzylidene digoxin decreases proliferation by inhibiting the EGFR/ERK signaling pathway and induces apoptosis in HeLa cells.

Cardiotonic steroids (CTS) are agents traditionally known for their capacity to bind to the Na,K-ATPase (NKA), affecting the ion transport and the contraction of the heart. Natural CTS have been shown to also have effects on cell signaling pathways. With the goal of developing a new CTS derivative, we synthesized a new digoxin derivative, 21-benzylidene digoxin (21-BD). Previously, we have shown that this compound binds to NKA and has cytotoxic actions on cancer, but not on normal cells. Here, we further studied the mechanisms of actions of 21-BD. Working with HeLa cells, we found that 21-BD decreases the basal, as well as the insulin stimulated proliferation. 21-BD reduces phosphorylation of the epidermal growth factor receptor (EGFR) and extracellular-regulated kinase (ERK), which are involved in pathways that stimulate cell proliferation. In addition, 21-BD promotes apoptosis, which is mediated by the translocation of Bax from the cytosol to mitochondria and the release of mitochondrial cytochrome c to the cytosol. 21-BD also activated caspases-8, -9 and -3, and induced the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Altogether, these results show that the new compound that we have synthesized exerts cytotoxic actions on HeLa cells by inhibition of cell proliferation and the activation of both the extrinsic and intrinsic apoptotic pathways. These results support the relevance of the cardiotonic steroid scaffold as modulators of cell signaling pathways and potential agents for their use in cancer.

Extracellular signal-regulated kinases mediate the enhancing effects of inflammatory mediators on resurgent currents in dorsal root ganglion neurons.

Previously we reported that a group of inflammatory mediators significantly enhanced resurgent currents in dorsal root ganglion neurons. To understand the underlying intracellular signaling mechanism, we investigated the effects of inhibition of extracellular signal-regulated kinases and protein kinase C on the enhancing effects of inflammatory mediators on resurgent currents in rat dorsal root ganglion neurons. We found that the extracellular signal-regulated kinases inhibitor U0126 completely prevented the enhancing effects of the inflammatory mediators on both Tetrodotoxin-sensitive and Tetrodotoxin-resistant resurgent currents in both small and medium dorsal root ganglion neurons. U0126 substantially reduced repetitive firing in small dorsal root ganglion neurons exposed to inflammatory mediators, consistent with prevention of resurgent current amplitude increases. The protein kinase C inhibitor Bisindolylmaleimide I also showed attenuating effects on resurgent currents, although to a lesser extent compared to extracellular signal-regulated kinases inhibition. These results indicate a critical role of extracellular signal-regulated kinases signaling in modulating resurgent currents and membrane excitability in dorsal root ganglion neurons treated with inflammatory mediators. It is also suggested that targeting extracellular signal-regulated kinases-resurgent currents might be a useful strategy to reduce inflammatory pain.

The discovery of benzoxazine sulfonamide inhibitors of NaV1.7: Tools that bridge efficacy and target engagement.

The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.

Correction to "Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency and Pharmacokinetics to Enable in Vivo Target Engagement".

[This corrects the article DOI: 10.1021/acsmedchemlett.6b00243.].

Control of Neurotransmission by NaV1.7 in Human, Guinea Pig, and Mouse Airway Parasympathetic Nerves.

Little is known about the neuronal voltage-gated sodium channels (NaVs) that control neurotransmission in the parasympathetic nervous system. We evaluated the expression of the α subunits of each of the nine NaVs in human, guinea pig, and mouse airway parasympathetic ganglia. We combined this information with a pharmacological analysis of selective NaV blockers on parasympathetic contractions of isolated airway smooth muscle. As would be expected from previous studies, tetrodotoxin potently blocked the parasympathetic responses in the airways of each species. Gene expression analysis showed that that NaV 1.7 was virtually the only tetrodotoxin-sensitive NaV1 gene expressed in guinea pig and human airway parasympathetic ganglia, where mouse ganglia expressed NaV1.1, 1.3, and 1.7. Using selective pharmacological blockers supported the gene expression results, showing that blocking NaV1.7 alone can abolish the responses in guinea pig and human bronchi, but not in mouse airways. To block the responses in mouse airways requires that NaV1.7 along with NaV1.1 and/or NaV1.3 is blocked. These results may suggest novel indications for NaV1.7-blocking drugs, in which there is an overactive parasympathetic drive, such as in asthma. The data also raise the potential concern of antiparasympathetic side effects for systemic NaV1.7 blockers.

Targeting the Nerve Growth Factor (NGF) Pathway in Drug Discovery. Potential Applications to New Therapies for Chronic Pain.

The neurotrophin nerve growth factor (NGF) has been implicated as a key mediator of chronic pain. NGF binds the tropomysin receptor kinase A (TrkA) and p75, resulting in the activation of downstream signaling pathways that have been linked to pro-nociception. While anti-NGF antibodies have demonstrated analgesia both preclinically and in patients, the mechanism of action of these agents remains unclear. We describe ligands targeting NGF, its receptors, and downstream/related targets. This Perspective highlights large and small molecule approaches to targeting the NGF-TrkA pathway both extra- and intracellularly. In addition, we present a strategic framework for future drug discovery efforts in this pathway beyond the targeting of NGF or its receptors. While existing tools have greatly informed NGF-mediated signaling, ongoing and future pathway research may help focus new drug discovery efforts on key novel targets and mechanisms. This may result in highly differentiated therapeutics with greater efficacy and/or improved safety profiles.

Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency and Pharmacokinetics to Enable in Vivo Target Engagement.

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.

Cardiac ion channels.

Ion channels are critical for all aspects of cardiac function, including rhythmicity and contractility. Consequently, ion channels are key targets for therapeutics aimed at cardiac pathophysiologies such as atrial fibrillation or angina. At the same time, off-target interactions of drugs with cardiac ion channels can be the cause of unwanted side effects. This manuscript aims to review the physiology and pharmacology of key cardiac ion channels. The intent is to highlight recent developments for therapeutic development, as well as elucidate potential mechanisms for drug-induced cardiac side effects, rather than present an in-depth review of each channel subtype.

Inhibition of Inactive States of Tetrodotoxin-Sensitive Sodium Channels Reduces Spontaneous Firing of C-Fiber Nociceptors and Produces Analgesia in Formalin and Complete Freund's Adjuvant Models of Pain.

While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported "compound 52" aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund's adjuvant (CFA) model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.

Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the ß4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel ß4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the ß4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

The discovery of aminopyrazines as novel, potent Na(v)1.7 antagonists: hit-to-lead identification and SAR.

Herein the discovery of a novel class of aminoheterocyclic Na(v)1.7 antagonists is reported. Hit compound 1 was potent but suffered from poor pharmacokinetics and selectivity. The compact structure of 1 offered a modular synthetic strategy towards a broad structure-activity relationship analysis. This analysis led to the identification of aminopyrazine 41, which had vastly improved hERG selectivity and pharmacokinetic properties.

Discovery and hit-to-lead optimization of pyrrolopyrimidines as potent, state-dependent Na(v)1.7 antagonists.

Herein we describe the discovery, optimization, and structure-activity relationships of novel potent pyrrolopyrimidine Na(v)1.7 antagonists. Hit-to-lead SAR studies of the pyrrolopyrimidine core, head, and tail groups of the molecule led to the identification of pyrrolopyrimidine 48 as exceptionally potent Na(v)1.7 blocker with good selectivity over hERG and improved microsomal stability relative to our hit molecule and pyrazolopyrimidine 8 as a promising starting point for future optimization efforts.

Discovery and optimization of aminopyrimidinones as potent and state-dependent Nav1.7 antagonists.

Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.