The Epithelial Sodium Channel-An Underestimated Drug Target.

Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents.

Stapled peptides as potential inhibitors of SARS-CoV-2 binding to the hACE2 receptor.

Stapled peptides are synthetic peptidomimetics of bioactive sites in folded proteins which carry chemical links, introduced during peptide synthesis, designed to retain the secondary structure in the native protein molecule. Stapled peptides have been investigated as potential modulators of protein-protein interactions for over two decades. The potential use of stapled peptides as inhibitors of viral entry, and therefore as antiviral therapeutics, has been established for several important viruses causing disease in humans, such as the human immunodeficiency virus type 1 (HIV-1), respiratory syncytial virus (RSV), and Middle East Respiratory Syndrome (MERS) coronavirus. Several independent research initiatives have investigated the inhibitory effect of stapled peptides on binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, to its receptor, angiotensin-converting-enzyme 2 (ACE2). These stapled peptides, which mimic Helix 1 of the human ACE2 receptor, have demonstrated mixed ability to prevent infection with SARS-CoV-2 in cell-based studies.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel.

AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, ß, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αßγ- or δßγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).

AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride-sensitive Na(+) current in primary dog, pig and rat alveolar type II cells.

Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung conditions, such as ALI and ARDS. Apart from ventilation strategies, no specific therapy yet exists for treatment of these potentially fatal illnesses. The oedema-reducing capacity of the lectin-like domain of TNF (TIP) and of synthetic peptides, mTIP and hTIP, which mimic the TIP domain of mouse and human TNF, have been demonstrated in various studies in rodents. Cell-based electrophysiological studies have revealed that the alveolar fluid clearing capacity of TNF and the TIP peptides is due to activation of the amiloride-sensitive Na(+) current in alveolar epithelial cells and that the primary site of action is on the apical side of these cells. AP301, a synthetic cyclic peptide mimicking the TIP domain of human TNF is currently undergoing clinical trials as a therapy for pulmonary permeability oedema. AP301 has been shown to improve alveolar liquid clearance and lung function in a porcine model of ALI. For non-clinical regulatory assessment, dog, pig and rat are standard animal models; accordingly, pre-clinical toxicological and pharmacological safety studies have been conducted with AP301 in dogs and rats. Hitherto, no studies have assessed the pharmacodynamic effect of AP301 on primary canine or porcine type II AEC. The current study describes the effect of AP301 on the amiloride-sensitive Na(+) current in type II AEC isolated from dog, pig and rat lungs. In whole cell patch clamp experiments with dog type II AEC, an increase in the amiloride-sensitive Na(+) current from 3.7 pA to 49.4 pA was observed in the presence of AP301; in pig type II AEC, an increase from 10.0 pA to 159.6 pA was observed, and in rat AEC, from 6.9 pA to 62.4 pA. In whole cell patch clamp experiments in A549 cells, AP301-induced enhancement of the amiloride-sensitive current was eliminated when Na(+) in the bath solution was replaced with N-methyl-d-glucamine (NMDG), and when the cells were pre-incubated with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn(2+) or l-cis-diltiazem prior to AP301. These results provide strong evidence that AP301 activates the amiloride-sensitive Na(+) current through ENaC in type II AEC from dog, pig and rat. To our knowledge, this is the first cell-based analysis of the oedema-clearing effect of AP301 observed in the porcine model of pulmonary oedema. Furthermore, the results validate the dog and pig models in non-clinical assessment of AP301.

Conformational ensemble of the TNF-derived peptide solnatide in solution.

Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and, as such, recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have analyzed its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities.

Restoration of Epithelial Sodium Channel Function by Synthetic Peptides in Pseudohypoaldosteronism Type 1B Mutants.

The synthetically produced cyclic peptides solnatide (a.k.a. TIP or AP301) and its congener AP318, whose molecular structures mimic the lectin-like domain of human tumor necrosis factor (TNF), have been shown to activate the epithelial sodium channel (ENaC) in various cell- and animal-based studies. Loss-of-ENaC-function leads to a rare, life-threatening, salt-wasting syndrome, pseudohypoaldosteronism type 1B (PHA1B), which presents with failure to thrive, dehydration, low blood pressure, anorexia and vomiting; hyperkalemia, hyponatremia and metabolic acidosis suggest hypoaldosteronism, but plasma aldosterone and renin activity are high. The aim of the present study was to investigate whether the ENaC-activating effect of solnatide and AP318 could rescue loss-of-function phenotype of ENaC carrying mutations at conserved amino acid positions observed to cause PHA1B. The macroscopic Na+ current of all investigated mutants was decreased compared to wild type ENaC when measured in whole-cell patch clamp experiments, and a great variation in the membrane abundance of different mutant ENaCs was observed with Western blotting experiments. However, whatever mechanism leads to loss-of-function of the studied ENaC mutations, the synthetic peptides solnatide and AP318 could restore ENaC function up to or even higher than current levels of wild type ENaC. As therapy of PHA1B is only symptomatic so far, the peptides solnatide and AP318, which directly target ENaC, are promising candidates for the treatment of the channelopathy-caused disease PHA1B.

TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B.

Previous in vitro studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only ß-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αßγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αßγ-ENaC, whereas membrane abundance varied between mutants. Solnatide restored function in α-ENaC frameshift mutants to current density levels of wild type ENaC or higher despite their lacking a binding site for solnatide, previously located to the region between TM2 and the C-terminus of the α-subunit. TNF similarly restored current density to wild type levels in the mutant αR448fs. Activation of ßγ-ENaC may contribute to this moderate current enhancement, but whatever the mechanism, experimental data indicate that solnatide could be a new strategy to treat PHA1B.

Glycosylation-dependent activation of epithelial sodium channel by solnatide.

Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xßγ-hENaC or δN166,311,384Q,D552Xßγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xßγ-hENaC- and δD552Xßγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

A FIM study to assess safety and exposure of inhaled single doses of AP301-A specific ENaC channel activator for the treatment of acute lung injury.

AP301 is an activator of ENaC-mediated Na(+) uptake for the treatment of pulmonary permeability edema in acute respiratory distress syndrome (ARDS). The purpose of this "first-in-man" study was to examine local and systemic safety and systemic exposure of ascending single doses of AP301, when inhaled by healthy male subjects. In a double-blind, placebo-controlled study, 48 healthy male subjects were randomized to 6 ascending dose groups (single doses up to 120 mg) of 8 subjects each (3:1 randomization of AP301: placebo). Serial assessments included spirometry, exhaled nitric oxide (eNO), vital signs, ECG, safety laboratory, adverse events (AE), and blood samples for the quantification of AP301 in plasma. Descriptive statistics was applied. All 48 subjects received treatment, and completed the study as per protocol. No serious, local (e.g., hoarseness, cough, bronchospasm), or dose-limiting AEs were noted. None of the assessments indicated notable dose or time-related alterations of safety outcomes. Observed AP301 systemic exposure levels were very low, with mean Cmax values of <2.5 ng/mL in the highest dose groups. Inhaled AP301 single doses up to 120 mg were safe and well tolerated by healthy male subjects. Distribution of inhaled AP301 was largely confined to the lung, as indicated by very low AP301 systemic exposure levels.

Amyloidogenic sequences in native protein structures.

Numerous short peptides have been shown to form beta-sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 proteins containing 45 sequences known to form amyloid, to see how the proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen-bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form beta structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a beta structure. Secondly, amyloidogenic sequences that are found in beta structure are usually buried within the protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to protein aggregation via assembly of the amyloidogenic regions. The use of alpha-helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for beta structure, is thus a widespread mechanism to avoid protein aggregation.

Essential structural features of TNF-α lectin-like domain derived peptides for activation of amiloride-sensitive sodium current in A549 cells.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a prominent role in sodium uptake from alveolar fluid and is the major component in alveolar fluid clearance in normal and diseased lungs. The lectin-like domain of TNF-α has been shown to activate amiloride-sensitive sodium uptake in type II alveolar epithelial cells. Therefore, several synthetic peptides that mimic the lectin-like domain of TNF-α (TIP) were synthesized and their ability to enhance sodium current through ENaC was studied in A549 cells with the patch clamp technique. Our data suggest that a free positively charged N-terminal amino group on residue 1 and/or a free negatively charged carboxyl group on residue 17 of the TIP peptide is essential for the ENaC-activating effect. Ventilation strategies apart, no standard treatment exists for pulmonary permeability edema. Therefore, novel therapies activating sodium uptake from the alveolar fluid via ENaC could improve clinical outcome.

Mechanisms of quinolone resistance in clinical isolates of Enterobacter cloacae.

OBJECTIVE: To study and evaluate changes in the gyrA gene and the outer-membrane protein patterns in relation to evolution of resistance against the quinolones in Enterobacter cloacae. METHODS: Strains expressing gyrA-mediated quinolone resistance become susceptible to quinolones upon insertion of the plasmid pNJR3-2. This plasmid (containing wild-type Escherichia coli quinolone-susceptible DNA gyrase A subunits) and pLA2917 (the vector) were introduced into 10 resistant or moderately susceptible clinical isolates of Enterobacter cloacae by conjugation. The transconjugants, the original isolates, the plasmid and the vector control were screened for susceptibility to ofloxacin, ciprofloxacin and sparfloxacin. Additionally, examinations of the outer-membrane proteins were performed. RESULTS: A reduction of MICs by a factor of 8--32 was found for the transconjugants of five Enterobacter cloacae isolates in the presence of the gene probe, suggesting that these isolates harbored mutations in gyrA. No discernible difference in the patterns of outer-membrane proteins of sensitive and resistant strains could be detected. CONCLUSIONS: It seems that changes in the target site such as alterations in gyrA are important factors leading to a change in the susceptibility of bacteria to the quinolones, whereas there were no evident changes in the outer-membrane proteins to account for evolution of resistance.