An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections.

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Esculentin-1a-Derived Peptides Promote Clearance of Pseudomonas aeruginosa Internalized in Bronchial Cells of Cystic Fibrosis Patients and Lung Cell Migration: Biochemical Properties and a Plausible Mode of Action.

Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 µM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all-l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.

Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia.

The TMEM16A protein has a potential role as a Ca(2+)-activated Cl(-) channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o-). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short-interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca(2+)-dependent Cl(-) transport. Following treatment for 24-72 h with interleukin-4 (IL-4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL-4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A-positive cells when epithelia were treated with IL-4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl(-) channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL-4, and that TMEM16A upregulation by IL-4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

A minimal isoform of the TMEM16A protein associated with chloride channel activity.

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.

The anoctamin family: TMEM16A and TMEM16B as calcium-activated chloride channels.

The Ca(2+)-activated Cl(-) channels (CaCCs) are involved in a variety of physiological functions, such as transepithelial anion transport, smooth muscle contraction and olfaction. Recently, the question of the molecular identity of CaCCs has apparently been resolved with the identification of TMEM16A protein (also known as anoctamin-1). Expression of TMEM16A is associated with the appearance of Ca(2+)- and voltage-dependent Cl(-) currents with properties similar to those of native CaCCs. The putative structure of TMEM16A consists of eight transmembrane domains, with both the amino- and the carboxy-terminus protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. A close paralogue of TMEM16A, TMEM16B (anoctamin-2), is also associated with CaCC activity, although with different properties. The TMEM16B-dependent channels require higher intracellular Ca(2+) concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin-6), may have different functions.

The VRAC blocker DCPIB directly gates the BK channels and increases intracellular Ca2+ in melanoma and pancreatic duct adenocarcinoma cell lines.

BACKGROUND AND PURPOSE: The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma. EXPERIMENTAL APPROACH: We used patch-clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc-1 and MiaPaCa-2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line. KEY RESULTS: DCPIB markedly increased whole-cell currents in Panc-1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa 1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+ -dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding. CONCLUSION AND IMPLICATIONS: DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+ . Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB-based modulators of ion channels and other membrane proteins.

Pulmonary Safety Profile of Esc Peptides and Esc-Peptide-Loaded Poly(lactide-co-glycolide) Nanoparticles: A Promising Therapeutic Approach for Local Treatment of Lung Infectious Diseases.

In recent years, we have discovered Esc(1-21) and its diastereomer (Esc peptides) as valuable candidates for the treatment of Pseudomonas lung infection, especially in patients with cystic fibrosis (CF). Furthermore, engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were revealed to be a promising pulmonary delivery system of antimicrobial peptides. However, the "ad hoc" development of novel therapeutics requires consideration of their stability, tolerability, and safety. Hence, by means of electrophysiology experiments and preclinical studies on healthy mice, we demonstrated that neither Esc peptides or Esc-peptide-loaded PLGA NPs significantly affect the integrity of the lung epithelium, nor change the global gene expression profile of lungs of treated animals compared to those of vehicle-treated animals. Noteworthy, the Esc diastereomer endowed with the highest antimicrobial activity did not provoke any pulmonary pro-inflammatory response, even at a concentration 15-fold higher than the efficacy dosage 24 h after administration in the free or encapsulated form. The therapeutic index was ≥70, and the peptide was found to remain available in the bronchoalveolar lavage of mice, after two days of incubation. Overall, these studies should open an avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease.

TMEM16A protein: a new identity for Ca(2+)-dependent Cl⁻ channels.

Ca(+)-dependent Cl⁻ channels (CaCCs) play a variety of physiological roles in different organs and tissues, including transepithelial Cl⁻ secretion, smooth muscle contraction, regulation of neuronal excitability, and transduction of sensory stimuli. The recent identification of TMEM16A protein as an important component of CaCCs should allow a better understanding of their physiological role, structure-function relationship, and regulatory mechanisms.

I112M SOD1 mutation causes ALS with rapid progression and reduced penetrance in four Mediterranean families.

We evaluated a possible genotype-phenotype correlation and looked for a founder effect in four Mediterranean families carrying the I112M SOD1 mutation. The structural characteristics of the mutated protein were also analysed. Clinical data of FALS subjects from four families were evaluated. Mutational analysis of the SOD1 gene was carried out by direct sequencing. A haplotype study was carried out using 11 polymorphic markers flanking the SOD1 gene. Structural analysis was performed by means of homology modelling and molecular graphics methods. The clinical pattern of 17 FALS patients was characterized by prevalent spinal onset, mean age at onset of 47.1 years and mean duration of 20.7 months. Several obligate carriers were observed. These findings indicate that the I112M mutation is consistently associated with a uniform, fast-progressing phenotype with reduced penetrance of the disease. The haplotype analysis did not show a common haplotype among the Spanish families and the Italian family; however, a possible common founder could be hypothesized for Spanish families. From a structural viewpoint, mutation at codon 112 seems to confer a severe phenotype, probably related to altered protein functionality.

The Application of Bicarbonate Recovers the Chemical-Physical Properties of Airway Surface Liquid in Cystic Fibrosis Epithelia Models.

Cystic fibrosis (CF) is a genetic disease associated with the defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes obstructive disease and chronic bacterial infections in airway epithelia. Deletion of phenylalanine at position 508, p.F508del, the most frequent mutation among CF patients, causes a folding and traffic defect, resulting in a dramatic reduction in the CFTR expression. To investigate whether the direct application of bicarbonate could modify the properties of the airway surface liquid (ASL), we measured the micro-viscosity, fluid transport and pH of human bronchial epithelial cells monolayers. We have demonstrated that the treatment of a CF-epithelia with an iso-osmotic solution containing bicarbonate is capable of reducing both, the ASL viscosity and the apical fluid re-absorption. We suggest the possibility of design a supportive treatment based on topical application of bicarbonate, or any other alkaline buffer.

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines.

Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents.

Regulation of TMEM16A chloride channel properties by alternative splicing.

Expression of TMEM16A protein is associated with the activity of Ca(2+)-activated Cl(-) channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca(2+) sensitivity by nearly 4-fold, resulting in Cl(-) currents requiring lower Ca(2+) concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca(2+)-activated Cl(-) channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca(2+) dependence of the TMEM16A-dependent Cl(-) channels in a tissue-specific manner.

Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel.

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Lumacaftor-rescued F508del-CFTR has a modified bicarbonate permeability.

Deletion of phenylalanine at position 508, F508del, the most frequent mutation among Cystic fibrosis (CF) patients, destabilizes the protein, thus causing both a folding and a trafficking defect, resulting in a dramatic reduction in expression of CFTR. In vitro treatment with lumacaftor produces an enhancement of anion transport in cells. We studied the permeability properties of the CFTR mutant F508del treated with the corrector lumacaftor, showing that the rescued protein has selectivity properties different than the wild type CFTR, showing an augmented bicarbonate permeability. This difference would indicate a diverse conformation of the rescued F508del-CFTR, that is plausibly reflected on an improper regulation of the airway surface liquid, lessening the efficacy of the corrector. Our findings rather support the idea that a combination of correctors would be required to address the CFTR-dependent bicarbonate permeability.

Bronchial epithelium repair by Esculentin-1a-derived antimicrobial peptides: involvement of metalloproteinase-9 and interleukin-8, and evaluation of peptides' immunogenicity.

The airway epithelium is seriously damaged upon pulmonary Pseudomonas aeruginosa infection, especially in cystic fibrosis (CF) sufferers. Therefore, the discovery of novel anti-infective agents accelerating healing of infected injured tissues is crucial. The antipseudomonal peptides esculentin-1a(1-21)NH2 and its diastereomer Esc(1-21)-1c (Esc peptides) hold promise in this respect. In fact, they stimulate airway epithelial wound repair, but no mechanistic insights are available. Here we demonstrated that this process occurs through promotion of cell migration by an indirect activation of epidermal growth factor receptor mediated by metalloproteinases. Furthermore, we showed an increased expression of metalloproteinase 9, at both gene and protein levels, in peptide-treated bronchial epithelial cells with a functional or mutated form of CF transmembrane conductance regulator. In addition, the two peptides counteracted the inhibitory effect of Pseudomonas lipopolysaccharide (mimicking an infection condition) on the wound healing activity of the airway epithelium, and they enhanced the production of interleukin-8 from both types of cells. Finally, no immunogenicity was discovered for Esc peptides, suggesting their potential safety for clinical usage. Besides representing a step forward in understanding the molecular mechanism underlying the peptide-induced wound healing activity, these studies have contributed to highlight Esc peptides as valuable therapeutics with multiple functions.

Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.