Human Neutrophil Elastase Activity Research Articles

Terpenoids as Human Neutrophil Elastase (HNE) Inhibitors: A Comprehensive Review of Natural Anti-inflammatory Isoprenoids.

Human neutrophil elastase (HNE) is an enzyme that plays a key role in the body's inflammatory response. It has been linked to several diseases such as chronic obstructive pulmonary disease (COPD), emphysema, and cystic fibrosis. As potential treatments for these diseases, HNE inhibitors are of great interest. Metabolites derived from plants, particularly terpenoids such as β-caryophyllene found in black pepper and other plants, and geraniol present in several essential oils, are recognized as significant sources of inhibitors for HNE. Because of their ability to inhibit HNE, terpenoids are considered promising candidates for developing novel therapies to treat inflammatory conditions such as COPD and emphysema. Furthermore, nature can serve as an excellent designer, and it may offer a safer drug candidate for inhibiting HNE production and activity in the future. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were searched to get relevant and up-to-date literature on terpenoids as human neutrophil elastase inhibitors. This review focuses on the isolation, chemical diversity, and inhibition of human neutrophil elastase (HNE) of various terpenoids reported from natural sources up to 2022. A total of 251 compounds from various terpenoids classes have been reported. Further, it also provides a summary of HNE inhibitors and includes a thorough discussion on the structure-activity relationship.

Could the Oxidation of α1-Antitrypsin Prevent the Binding of Human Neutrophil Elastase in COVID-19 Patients?

Human neutrophil elastase (HNE) is involved in SARS-CoV-2 virulence and plays a pivotal role in lung infection of patients infected by COVID-19. In healthy individuals, HNE activity is balanced by α1-antitrypsin (AAT). This is a 52 kDa glycoprotein, mainly produced and secreted by hepatocytes, that specifically inhibits HNE by blocking its activity through the formation of a stable complex (HNE-AAT) in which the two proteins are covalently bound. The lack of this complex, together with the detection of HNE activity in BALf/plasma samples of COVID-19 patients, leads us to hypothesize that potential functional deficiencies should necessarily be attributed to possible structural modifications of AAT. These could greatly diminish its ability to inhibit neutrophil elastase, thus reducing lung protection. The aim of this work was to explore the oxidation state of AAT in BALf/plasma samples from these patients so as to understand whether the deficient inhibitory activity of AAT was somehow related to possible conformational changes caused by the presence of abnormally oxidized residues.

Identification and characterization of aptameric inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation and tissue remodelling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used systematic evolution of ligands by exponential enrichment (SELEX) to develop single-stranded DNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency, and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.

Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios

In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 μg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.

The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against Moraxella catarrhalis.

Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen's existence in the airways under inflammation.

Effect of Cuban Porcine Pulmonary Surfactant (Surfacen) and rCmPI-II Protease Inhibitor on Neutrophil Elastase Activity.

In inflammatory respiratory diseases, the imbalance between proteases and endogenous protease inhibitors leads to an exacerbated activity of human neutrophil elastase (a protease that destroys the extracellular matrix and stimulates proinflammatory cytokine release). Elastase is considered a target in the search for therapeutic treatments for inflammatory respiratory diseases. Pulmonary surfactant is a promising product for this purpose, because in addition to its biophysical function, it has anti-inflammatory properties. Evaluate effect of the Cuban porcine pulmonary surfactant (Surfacen), the rCmPI-II elastase inhibitor, and the Surfacen/rCmPI-II combination on activated neutrophil elastase activity in vitro, and determine if Surfacen's interface property changes in the presence of the inhibitor. The anti-elastase effect of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination was evaluated in an in vitro model of activated neutrophils, previously purified from the blood of healthy subjects. The cells were stimulated with LPS/fMLP and were incubated with different concentrations of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination. Elastase activity was measured. The interface property was determined on a Langmuir surface balance. The new index, called the abdominal adipose deposit index, was obtained by multiplying the subcutaneous fat thickness by visceral fat thickness, both measured by ultrasound. A cutoff point was established that facilitated discernment of an unhealthy phenotype: normal weight but metabolically obese, a cardiometabolic risk factor. Surfacen at 10 mg/mL inhibited 71% of stimulated neutrophil elastase activity. rCmPI-II at 0.1 μM reduced 20% of elastase activity; at 200 μM-the maximum concentration evaluated-inhibition was 68%. Both products had a dose-dependent effect. The Surfacen/inhibitor combination (0.5 mg/mL/80 µM) did not affect the surfactant interface property or the inhibitory activity of rCmPI-II against human neutrophil elastase. Surfacen and the rCmPI-II inhibitor have an anti-elastase effect on an activated neutrophil model. rCmPI-II does not affect Surfacen's interface property and, therefore, both can be evaluated for combined use in treating inflammatory lung diseases.

Investigating the Link between Alpha-1 Antitrypsin and Human Neutrophil Elastase in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Neutrophils play a pathogenic role in COVID-19 by releasing Neutrophils Extracellular Traps (NETs) or human neutrophil elastase (HNE). Given that HNE is inhibited by α1-antitrypsin (AAT), we aimed to assess the content of HNE, α1-antitrypsin (AAT) and HNE–AAT complexes (the AAT/HNE balance) in 33 bronchoalveolar lavage fluid (BALf) samples from COVID-19 patients. These samples were submitted for Gel-Electrophoresis, Western Blot and ELISA, and proteins (bound to AAT or HNE) were identified by Liquid Chromatography-Mass Spectrometry. NETs’ release was analyzed by confocal microscopy. Both HNE and AAT were clearly detectable in BALf at high levels. Contrary to what was previously observed in other settings, the formation of HNE–AAT complex was not detected in COVID-19. Rather, HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free AAT to bind to histones. While confirming this binding, AAT was not able to inhibit NET formation. In conclusion, despite the finding of a high burden of free and bound HNE, the lack of the HNE–AAT inhibitory complex in COVID-19 BALf demonstrates that AAT is not able to block HNE activity. Furthermore, while binding to histones, AAT does not prevent NET formation nor their noxious activity.

Cystic Fibrosis Airway Mucus Hyperconcentration Produces a Vicious Cycle of Mucin, Pathogen, and Inflammatory Interactions that Promotes Disease Persistence.

The dynamics describing the vicious cycle characteristic of cystic fibrosis (CF) lung disease, initiated by stagnant mucus and perpetuated by infection and inflammation, remain unclear. Here we determine the effect of the CF airway milieu, with persistent mucoobstruction, resident pathogens, and inflammation, on the mucin quantity and quality that govern lung disease pathogenesis and progression. The concentrations of MUC5AC and MUC5B were measured and characterized in sputum samples from subjects with CF (N = 44) and healthy subjects (N = 29) with respect to their macromolecular properties, degree of proteolysis, and glycomics diversity. These parameters were related to quantitative microbiome and clinical data. MUC5AC and MUC5B concentrations were elevated, 30- and 8-fold, respectively, in CF as compared with control sputum. Mucin parameters did not correlate with hypertonic saline, inhaled corticosteroids, or antibiotics use. No differences in mucin parameters were detected at baseline versus during exacerbations. Mucin concentrations significantly correlated with the age and sputum human neutrophil elastase activity. Although significantly more proteolytic cleavages were detected in CF mucins, their macromolecular properties (e.g., size and molecular weight) were not significantly different than control mucins, likely reflecting the role of S-S bonds in maintaining multimeric structures. No evidence of giant mucin macromolecule reflecting oxidative stress-induced cross-linking was found. Mucin glycomic analysis revealed significantly more sialylated glycans in CF, and the total abundance of nonsulfated O-glycans correlated with the relative abundance of pathogens. Collectively, the interaction of mucins, pathogens, epithelium, and inflammatory cells promotes proteomic and glycomic changes that reflect a persistent mucoobstructive, infectious, and inflammatory state.

Engineering Molecular Probes for In Vivo Near-Infrared Fluorescence/Photoacoustic Duplex Imaging of Human Neutrophil Elastase.

Early detection of human neutrophil elastase (HNE), the potential biomarker of lung cancer, is crucial for the accurate diagnosis and evaluation of lung cancer. Currently, little progress of HNE-activated probes has been made for in vivo imaging. Herein, assisted by probe-active pocket match engineering, we synthesized a series of near-infrared fluorescence (NIRF) and photoacoustic (PA) duplex imaging probes by conjugating diverse fluorinated amide chains onto hemi-cyanine. Finally, we identified that probe 2 (denoted as LET-8), with the pentafluoroethyl group, is a superior probe to detect HNE with the best selectivity as well as good response ability and thus successfully realized NIRF/PA duplex imaging of HNE activity both in vitro and in vivo.

1,5,6,7-Tetrahydro-4H-indazol-4-ones as human neutrophil elastase (HNE) inhibitors

Human neutrophil elastase (HNE) is a serine protease that is expressed in polymorphonuclear neutrophils. It has been recognized as an important therapeutic target for treating inflammatory diseases, especially related to the respiratory system, but also for various types of cancer. Thus, compounds able to inhibit HNE are of great interest in medicinal chemistry. In the present paper, we report the synthesis and biological evaluation of a new series of HNE inhibitors with an innovative 1,5,6,7-tetrahydro-4H-indazol-4-one core that was developed as a molecular modification of our previously reported indazole-based HNE inhibitors. Since the 1,5,6,7-tetrahydro-4H-indazol-4-one scaffold can occur in two possible tautomeric forms, the acylation/alkylation reactions resulted in a mixture of the two isomers, often widely unbalanced in favor of one form. Using analytical techniques and NMR spectroscopy, we characterized and separated the isomer pairs and confirmed the compounds used in biological testing. Analysis of the compounds for HNE inhibitory activity showed that they were potent inhibitors, with Ki values in the low nanomolar range (6–35 nM). They also had reasonable stability in aqueous buffer, with half-lives over 1 h. Overall, our results indicate that the 1,5,6,7-tetrahydro-4H-indazol-4-one core is suitable for the synthesis of potent HNE inhibitors that could be useful in the development of new therapeutics for treating diseases involving excessive HNE activity.

MO1046DOPING POLYSULFONE DIALYSIS MEMBRANES WITH HUMAN NEUTROPHIL ELASTASE INHIBITORS - A PILOT STUDY

Abstract Background and Aims During hemodialysis (HD) therapy, the long-term intradialytic contact of blood with large surfaced artificial materials (HD membranes) leads to continuous neutrophil activation, with the release of neutrophil elastase (NE), among other products. NE appears to contribute to enhance inflammation favoring the development of atherosclerosis, which is the main cause of mortality and morbidity in End-Stage Renal Disease patients. The modification of polysulfone (PSF) HD membranes by incorporating selective human NE inhibitors (NEIs) might reduce the inflammatory response and prevent HD associated complications. Thus, the present study aimed to dope PSF membranes with NEIs and assess their bioactivity and biocompatibility. Method As NEIs, it was used the commercially available Sivelestat (SIV), from Abcam, and an in house synthetized 4-oxo-β-lactam (D4L-3) based compound, selected from our library, and prepared as described elsewhere [1]. The PSF membranes were prepared according to [2] and further doped with each NEI by adsorption. Three independent assays were performed (in triplicates) where PSF membrane circles (2 cm in diameter) were incubated with ultrapure water (blank), NEIs vehicle (2.5% DMSO) or with 10 - 2000 nM SIV or D4L-3. The bioactivity of these modified PSF membranes was evaluated by a human NE activity assay [1]. The same method was used to determine the IC50 of both NEIs in solution. Biocompatibility assays (n = 3) were carried-out using duplicates of modified PSF membranes circles with Ø 2 cm incubated with 1.0 mL of whole-blood. After incubation, platelet (PLT) poor and PLT rich plasma were used to assess the levels of plasma hemoglobin (Hb) and PLT activation, respectively [2]. Results The IC50 of SIV and D4L-3 in solution were 30.9 and 87.6 nM, respectively. For PSF membranes doped with NEIs, their bioactivity increased in a concentration-dependent manner, with the highest NE inhibition of 44 and 22 % at 2000 nM SIV and D4L-3, respectively. The blank membranes showed the highest hemolytic capacity, whereas SIV and D4L-3 PSF membranes presented lower plasma Hb levels when compared with the blank or the vehicle; on average, SIV-PSF membranes presented 31% less plasma Hb than vehicle, while in D4L-3-PSF this decrease was of 51%. Regarding the thrombotic potential of these biomaterials, blank membranes presented slightly increased PLT activation levels when compared to vehicle, and modified SIV-PSF membranes showed, on average, 58% more PLT activation than the vehicle, while D4L-3-PSF membranes displayed 25% less, on average. Conclusion The successful adsorption of NEIs into PSF membranes was achieved and the NE inhibition ability was directly dependent on the concentration of the inhibitor utilized. Moreover, the bioactivity of SIV and D4L-3 when immobilized into PSF membranes it appears to follow their inhibitory capacity occurring in solution, with SIV showing a greater affinity for NE. However, concerning biocompatibility, D4L-3 displayed a greater safety performance than SIV, especially regarding the potential for triggering platelet activation. To ensure the applicability of these modified PSF membranes as a medical device, extensive further studies have to be carried-out by tweaking the conditions of the immobilization process in order to obtain an optimal equilibrium between bioactivity and biocompatibility, as well test new NEIs. Acknowledgments This work was supported by UIDB/50006/2020 and UIDB/04378/2020 with funding from FCT/MCTES through national funds, and by the project Dial4Life co-financed by FCT/MCTES (PTDC/MEC-CAR/31322/2017) and FEDER/COMPETE 2020 (POCI-01-0145-FEDER-031322).

Ahp-Cyclodepsipeptides as tunable inhibitors of human neutrophil elastase and kallikrein 7: Total synthesis of tutuilamide A, serine protease selectivity profile and comparison with lyngbyastatin 7

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors.

Human Neutrophil Elastase Activated Fluorescent Probe for Pulmonary Diseases Based on Fluorescence Resonance Energy Transfer Using CdSe/ZnS Quantum Dots.

There is an increasing demand for effective noninvasive diagnosis against common pulmonary diseases, which are rising sharply due to the serious air pollution. Human neutrophil elastase (HNE), a typical protease highly involved in pulmonary inflammatory diseases and lung cancer, is a potential predictor for disease progression. Currently, few of the HNE-targeting probes are applicable in vivo due to the limitation in sensitivity and biocompatibility. Herein, we reported the achievement of in vitro detection and in vivo imaging of HNE by incorporating the HNE-specific peptide substrate, quantum dots (QDs), and organic dyes into the fluorescence resonance energy transfer (FRET) system. The refined nanoprobe, termed QDP, could specifically measure the HNE with excellent sensitivity of 7.15 pM in aqueous solution and successfully image the endogenous and exogenous HNE in living cells. In addition, this nanoprobe enabled HNE imaging in mouse models of lung cancer and acute lung injury, and the HNE activity at high temporal and spatial resolution was continuously monitored. Most importantly, QDP successfully discriminated the serums of patients with lung diseases from those of the healthy controls based on the HNE activity determination. Overall, this study demonstrates the advantages of a FRET-system-based nanoprobe in imaging performance and provides an applicable tool for in vivo HNE detection and pulmonary disease diagnosis.

New coumarins isolated from the stem bark of Fraxinus rhynchophylla inhibit human neutrophil elastase and LPS-induced inflammation in RAW 264.7 cells

Two new coumarins (1 and 2), together with six known compounds (3–8), were isolated from the stem bark of Fraxinus rhynchophylla. The structures of these compounds were established by extensive spectroscopic studies and chemical evidence. The inhibitory activities of the isolated compounds (1–8) against human neutrophil elastase (HNE) were evaluated in vitro; all compounds tested exhibited inhibitory effects against HNE activity, with IC50 values ranging from 3.6–74.3 μM. Among the compounds tested, compounds 2 and 8 exhibited considerable HNE inhibitory activity, with IC50 values of 3.6 and 9.6 μM, respectively. The mechanism by which the two active compounds (2 and 8) inhibited HNE was noncompetitive inhibition, with inhibition constant (Ki) values of 2.2 and 6.8 μM, respectively. Furthermore, compounds 2 and 8 dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. Consistent with these findings, they also suppressed LPS-induced expression of iNOS and COX-2 at the protein level in RAW 264.7 cells.

A patenting perspective on human neutrophil elastase (HNE) inhibitors (2014-2018) and their therapeutic applications

ABSTRACTIntroduction: Human neutrophil elastase (HNE) is involved in a variety of serious chronic diseases, especially cardiopulmonary pathologies. For this reason, the regulation of HNE activity represents a promising therapeutic approach, which is evident by the development of a number of new and selective HNE inhibitors, both in the academic and pharmaceutical environments.Areas covered: The present review analyzes and summarizes the patent literature regarding human neutrophil elastase inhibitors for the treatment of cardiopulmonary diseases over 2014–2018.Expert opinion: HNE is an interesting and defined target to treat various inflammatory diseases, including a number of cardiopulmonary pathologies. The research in this field is quite active, and a number of HNE inhibitors are currently in various stages of clinical development. In addition, new opportunities for HNE inhibitor development stem from recent studies demonstrating the involvement of HNE in many other inflammatory pathologies, including rheumatoid arthritis, inflammatory bowel disease, skin diseases, and cancer. Furthermore, the development of dual HNE/proteinase 3 inhibitors is being pursued as an innovative approach for the treatment of neutrophilic inflammatory diseases. Thus, these new developments will likely stimulate new and increased interest in this important therapeutic target and for the development of novel and selective HNE inhibitors.

Glycosaminoglycans and Glycosaminoglycan Mimetics as Human Neutrophil Elastase Inhibitors for Cystic Fibrosis Management

Cystic fibrosis (CF), a genetic disease, affects multiple organs, especially the lungs. Human neutrophil elastase (HNE), a serine protease vital for host defense mechanism, is markedly elevated in CF and plays a key role in disease progression. In fact, it is a biomarker of disease progression. Yet, there are no FDA‐approved small molecule inhibitors for HNE. The current CF therapy employs a host of symptom‐alleviating agents including DNAse to lyse mucous, hypertonic saline to aid mucous clearance, and antibiotics to combat infection. CF therapy would benefit greatly from HNE inhibitors.Previous work has shown that HNE is potently inhibited by negatively charged molecules such as heparin. Yet, heparin's powerful anticoagulant property limits its usefulness in CF. To identify selective glycosaminoglycan (GAG)‐based inhibitors of HNE, we studied the anti‐HNE activity of various GAG species of different sulfation patters, molecular weights and chain lengths. Our studies indicate that GAG sulfation patterns different from those required for anti‐coagulant property drive HNE inhibition. The results also show that sequences longer than hexasaccharides are required for anti‐HNE activity. Michaelis‐Menten kinetics suggests an allosteric mechanism of inhibition, which provides an advantage for the development of selective agents. Additionally, salt dependence studies of HNE inhibition points to the involvement of both ionic and non‐ionic interactions for HNE inhibition.Based on these results, we screened a library of 60 diverse, non‐saccharide GAG mimetics (NSGMs) against HNE, of which 10 molecules were found to be nanomolar inhibitors. Since hypertonic saline is an integral part of current CF therapy, we studied the effect of high salt levels on these inhibitors and identified six NSGMs that retained excellent potency, probably arising from considerable specific, non‐ionic interactions in binding to HNE. Studies on inhibition of HNE degradation of macromolecular substrate elastin led to further winnowing of inhibitors to two promising NSGMs. Finally, studies on a panel of serine proteases and coagulation assays led to the selection of a tetrasulfated diflavonoid NSGM for further testing in sputum from CF patients. This agent showed a dose‐dependent decrease in HNE activity of CF sputum when used in combination with DNAse. These studies indicate that GAG‐based inhibition of HNE is a promising avenue for developing anti‐CF agents. More specifically, the results suggest that a tetra‐sulfated diflavonoid NSGM could add value to the trio of DNAse, hypertonic saline and antibiotics used currently in the clinic.Support or Funding InformationThis work is funded by NHLBI grant 1P01HL107152 to Dr. Umesh R. Desai.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Safety and efficacy of the human neutrophil elastase inhibitor BAY 85-8501 for the treatment of non-cystic fibrosis bronchiectasis: A randomized controlled trial

BackgroundThere are only limited treatment options for patients with non-cystic fibrosis bronchiectasis (non-CF BE). Human neutrophil elastase (HNE) is a mediator of tissue destruction in non-CF BE. BAY 85-8501, a selective and reversible HNE inhibitor, could represent a new treatment option for this disease. MethodsThis was a phase 2a, randomized, placebo-controlled, double-blind, parallel-group study. The primary objective was to assess the safety and tolerability of 1 mg BAY 85-8501 once daily (OD) for 28 days compared with placebo in patients with non-CF BE. Secondary objectives were to investigate the effects of 4 weeks of treatment with BAY 85-8501 on health-related quality of life, pulmonary function, and inflammatory and tissue damage biomarkers in sputum, blood and/or urine, and to evaluate the pharmacokinetics of BAY 85-8501. ResultsOverall, 94 patients (mean age, 66 years; 53% male) were randomized (n = 47 per group), and 82 completed the study (BAY 85-8501, n = 37; placebo, n = 45). Treatment-emergent adverse events (TEAEs) occurred in 31 patients (66%) taking BAY 85-8501 and in 36 patients (77%) taking placebo, and were mostly mild or moderate. The serious TEAEs (BAY 85-8501, n = 3; placebo, n = 1) were not considered to be study-drug related.There were no changes in pulmonary function parameters from baseline to end of treatment, and health-related quality of life did not improve in any group. HNE activity in blood after zymosan challenge decreased significantly with BAY 85-8501 treatment (P = 0.0250 versus placebo). There were no significant differences in other biomarkers between treatment groups, with the exception of a small increase in interleukin-8 levels in sputum in the BAY 85-8501 group. Trough plasma concentrations of BAY 85-8501 plateaued after 2 weeks. Conclusions1 mg BAY 85-8501 OD had a favourable safety and tolerability profile when administered for 28 days to patients with non-CF BE. Further studies with a longer treatment duration are needed to evaluate the potential clinical efficacy in this study population.

Inhibition of Human Neutrophil Elastase by Sesquiterpene Lactone Dimers from the Flowers of Inula britannica.

A new sesquiterpene lactone dimer [1], together with five known compounds (2-6), was isolated from the flowers of Inula britannica. The structures of these compounds were established by extensive spectroscopic studies and chemical evidence. The inhibitory activities of these isolated compounds (1-6) against human neutrophil elastase (HNE) were also evaluated in vitro; compounds 1 and 6 exhibited significant inhibitory effects against HNE activity, with IC50 values of 8.2 and 10.4 μM, respectively, comparable to that of epigallocatechin gallate (EGCG; IC50 = 10.9 μM). In addition, compounds 3 and 5 exhibited moderate HNE inhibitory effects, with IC50 values of 21.9 and 42.5 μM, respectively. In contrast, compounds 2 and 4 exhibited no such activity (IC50 ＞ 100 μM). The mechanism by which 1 and 3 inhibited HNE was noncompetitive inhibition, with inhibition constant (Ki) values of 8.0 and 22.8 μM, respectively.

Identification and Quantification of Human Neutrophil Elastase Inhibitory Caffeoylquinic Acids in the Leaves of Aster koraiensis

A method for identifying and quantifying caffeoylquinic acids (CQAs) in aqueous extracts of the leaves of Aster koraiensis (AKAE) using high-performance liquid chromatography coupled with diode-array detection and electrospray ionization mass spectrometry was developed. Six CQAs (5-CQA, 3-CQA, 4-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in AKAE were identified by comparing their retention times and ultraviolet spectra with those of the corresponding standards, and the results were confirmed by ESI/MS. The developed HPLC method was applied to quantitative analyses of the six CQAs in AKAE, whose contents in AKAE were in the range 2.04–4.00 mg/g. The inhibitory activities of the CQAs against human neutrophil elastase (HNE) were also evaluated; all tested di-CQA isomers showed considerable inhibition of HNE activity, with IC50 values ranging from 2.09 to 14.20 μM, while three mono-CQAs exhibited no such activity (IC50 > 100 μM). 3,5-diCQA and 4,5-diCQA inhibited HNE in a mixed-type noncompetitive manner, with inhibition constant ( Ki) values of 0.47 and 1.4 μM, respectively.

Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an insilico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.