Human Neutrophil Elastase Inhibitors Research Articles

Novel Sulfonamide Analogs of Sivelestat as Potent Human Neutrophil Elastase Inhibitors.

Human neutrophil elastase (HNE) is involved in a number of essential physiological processes and has been identified as a potential therapeutic target for treating acute and chronic lung injury. Nevertheless, only one drug, Sivelestat, has been approved for clinical use and just in Japan and the Republic of Korea. Thus, there is an urgent need for the development of low-molecular-weight synthetic HNE inhibitors, and we have developed a wide variety of HNE inhibitors with various chemical scaffolds. We hypothesized that substitution of the active fragment of Sivelestat into these HNE inhibitor scaffolds could modulate their inhibitory activity, potentially resulting in higher efficacy and/or improved chemical stability. Here, we report the synthesis, biological evaluation, and molecular modeling studies of novel compounds substituted with the 4-(sulfamoyl)phenyl pivalate fragment necessary for Sivelestat activity. Many of these compounds were potent HNE inhibitors with activity in the nanomolar range (IC50 = 19–30 nM for compounds 3a, 3b, 3f, 3g, and 9a), confirming that the 4-(sulfamoyl)phenyl pivalate fragment could substitute for the N-CO group at position 1 and offer a different point of attack for Ser195. Results of molecular docking of the these pivaloyl-containing compounds into the HNE binding site supported the mechanism of inhibitory activity involving a nucleophilic attack of Ser195 from the catalytic triad onto the pivaloyl carbonyl group. Furthermore, some compounds (e.g., 3a and 3f) had a relatively good stability in aqueous buffer (t1/2 > 9 h). Thus, this novel approach led to the identification of a number of potent HNE inhibitors that could be used as leads for the further development of new therapeutics.

Novel and Modified Neutrophil Elastase Inhibitor Loaded in Topical Formulations for Psoriasis Management.

Human neutrophil elastase (HNE) is a serine protease that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-β-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-β-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and serine protease assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

Sulfonated Arylurea Derivatives are Potent Inhibitors of Human Neutrophil Elastase

Human neutrophil elastase (HNE) is a serine protease in the chymotrypsin superfamily of enzymes. HNE has been reported to play important roles in inflammation, innate immune response, and tissue remodeling processes. Therefore, HNE has been actively pursued as a drug target, particularly for the treatment of cardiopulmonary diseases. Although thousands of molecules have been reported to inhibit HNE, yet very few have been tested in early clinical trials and with only marginal success. We report here a novel chemotype of heparin‐like sulfonated arylurea derivatives as potent and noncompetitive inhibitors of HNE. Using a chromogenic substrate hydrolysis assay, 14 heparin‐like sulfonated molecules were tested for their inhibitory activity against HNE. Only 12 molecules inhibited HNE with IC50 values of 0.22 – 88.3 μM. The inhibition of HNE by these molecules was NaCl‐dependent. Interestingly, a specific hexa‐sulfonated arylurea derivative inhibited HNE with an IC50 value of 0.22 μM via noncompetitive mechanism as demonstrated by Michaelis‐Menten kinetics. The hexa‐sulfonated derivative demonstrated 455‐, 221‐, 21‐fold selectivity indices over other heparin‐binding serine proteases including thrombin, factor Xa, and factor XIa, respectively. The molecule also demonstrated 381‐fold selectivity over factor XIIIa, a transglutaminase clotting factor with a potential heparin‐binding domain. The molecule did not show heparin‐like effects on clotting times of human plasma as determined by partial activated thromboplastin time and prothrombin time assays. A computational approach was exploited to identify the putative binding site for this novel class of HNE inhibitors. Overall, reported sulfonated arylurea derivatives serve as a new platform to develop potent, selective, and noncompetitive inhibitors of HNE for therapeutic purposes.Support or Funding InformationThis work was supported by NIGMS/NIH under award number SC3GM131986 and by IDeA program from NIGMS/NIH under grant number P20 GM103424 to RAAH. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.

Molecular Docking Analysis of Triazole Analogues as Inhibitors of Human Neutrophil Elastase (HNE), Matrix Metalloproteinase (MMP 2 and MMP 9) and Tyrosinase

Triazole analogues are well known to have desirable properties for medicinal chemistry such as hydrogen bonding capability, moderate dipole character, rigidity and stability under in vivo conditions etc. This prompts us to carry out the present study, we have selected 20 triazole analogues and evaluated on the docking behavior of four targeted enzymes such as HNE, MMP 2, MMP 9 and tyrosinase. The molecular physicochemical analysis revealed that all the tested ligands (expect ligand 5 and 18) showed nil violation and complied well with the Lipinski's rule of five. ADME analysis showed that ligand 15 alone predicated to have low gastrointestinal (GI) absorption effect. Docking studies revealed that ligand 18 [2-(2,4-difluorophenyl)-1-(4-((1-(4-ethylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol] had exhibited the maximum binding energy for three target enzymes such as HNE, MMP 2 and MMP 9. The present study has paved a new insight in understanding 20 triazole analogues as potential inhibitors against HNE, MMP 2, MMP 9 and tyrosinase.

New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation.

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. Here, we report the further elaboration of our previously reported 3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bearing different substituents at the 4-phenyl ring. The most potent compounds 3a, 3g, and 3h, had IC50 values of 16, 11, and 18 nM, respectively. Molecular modeling studies and molecular dynamic (MD) simulations demonstrated no substantial differences between the 3-methylisoxazole derivatives previously tested and the corresponding 3-unsubstituted derivatives in the snapshot conformations sampled during the MD simulations, which is consistent with their similar levels of HNE inhibitory activity. Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold requirements, and the resulting derivatives are quite potent HNE inhibitors.

Neutrophil elastase inhibitor purification strategy from cowpea seeds.

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.

Single dose escalation studies with inhaled POL6014, a potent novel selective reversible inhibitor of human neutrophil elastase, in healthy volunteers and subjects with cystic fibrosis

BackgroundPOL6014 is a novel, orally inhaled neutrophil elastase (NE) inhibitor in development for cystic fibrosis (CF). MethodsTwo studies, one in healthy volunteers (HVs, doses 20 to 960 mg) and one in subjects with CF (doses 80 to 320 mg) were conducted to evaluate the safety, tolerability and pharmacokinetics (PK) of single ascending doses of inhaled POL6014 with a Pari eFlow® nebuliser. PK was evaluated over a period of 24 h. In addition, NE activity in CF sputum was measured. ResultsAfter single doses, POL6014 was safe and well tolerated up to 480 mg in HVs and at all doses in subjects with CF. POL6014 showed a dose-linear PK profile in both populations with Cmax between 0.2 and 2.5 μM in HVs and between 0.2 and 0.5 μM in subjects with CF. Tmax was reached at approximately 2–3 h. Mean POL6014 levels in CF sputum rapidly reached 1000 μM and were still above 10 μM at 24 h. >1-log reduction of active NE was observed at 3 h after dosing. ConclusionInhalation of POL6014 can safely lead to high concentrations within the lung and simultaneously low plasma concentrations, allowing for a clear inhibition of NE in the sputum of subjects with CF after single dosing.Trial Registration: European Medicines Agency EudraCT-Nr. 2015-001618-83 and 2016-000493-38.

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Human neutrophil elastase (HNE) inhibitory polyprenylated acylphloroglucinols from the flowers of Hypericum ascyron

In the course of an investigation of human neutrophil elastase (HNE) associated with inflammation, the extract of the flower parts of Hypericum ascyron showed a significant influence to HNE. The responsible metabolites to HNE inhibition were found to be eight polyprenylated acylphloroglucinols, PPAPs (1–8) which showed IC50 ranges between 2.4 and 19.9 μM. This is the first report to demonstrate that PPAP skeleton exhibits potent HNE inhibition. The compounds 1–3 were characterized and newly named as ascyronone E (IC50 = 4.3 μM), ascyronone F (IC50 = 19.9 μM), ascyronone G (IC50 = 4.5 μM) based on 2D-NMR spectroscopic data. In the kinetic analysis of double reciprocal plots, all the compounds showed noncompetitive behaviors to HNE enzyme with the remaining of Km and the increase of Vmax. The binding affinity levels (KSV) by using fluorescence were sufficient to be able to prove that PPAPs (1–8) had compliant interaction with inhibitory potencies.

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.

Further modifications of 1H-pyrrolo[2,3-b]pyridine derivatives as inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes and is considered to be a multifunctional enzyme. HNE is also involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrrolo[2,3-b]pyridine derivatives of our previously reported potent HNE inhibitors. Our results show that position 2 of the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted, and modifications of this position resulted in loss of HNE inhibitory activity. Conversely, the introduction of certain substituents at position 5 was tolerated, with retention of HNE inhibitory activity (IC50 = 15-51 nM) after most substitutions, indicating that bulky and/or lipophilic substituents at position 5 probably interact with the large pocket of the enzyme site and allow Michaelis complex formation. The possibility of Michaelis complex formation between Ser195 and the ligand carbonyl group was assessed by molecular docking, and it was found that highly active HNE inhibitors are characterized by geometries favorable for Michaelis complex formation and by relatively short lengths of the proton transfer channel via the catalytic triad.

Pharmacological activities of Nepenthes miranda extracts

Carnivorous Nepenthes plants exhibit many ethnobotanical uses and their extracts possess many pharmacological activities. In this study, we prepared different extracts from leaves, stems, pitchers, and flowers of N. miranda (Nm) and investigated the antioxidant, antibacterial, anticancer, and anti‐inflammatory properties. To evaluate extraction efficiency, different solvents such as methanol, ethanol, acetone, and distilled water were used individually for Nm extract preparations. Through DPPH radical scavenging assay, Folin‐Ciocalteu method, and aluminium chloride calorimetric analysis, the stems extract obtained by 100% acetone (Nm‐s‐a) showed the highest total antioxidant activity, total phenolic content, and total flavonoid content, respectively. In addition, we found that the same extract also exhibited the highest antibacterial activity against three pathogens, namely, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The inhibition zones of Nm‐s‐a determined by agar‐well diffusion assay ranged from 9–29 mm and were in the following order: E. coli > S. aureus > P. aeruginosa. To demonstrate the cytotoxicity effect of Nm‐s‐a, human embryonic kidney 293 cells (HEK 293) and melanoma cells were cultured with various concentrations of Nm‐s‐a. Furthermore, inhibition of human neutrophil elastase by Nm‐s‐a was also found, suggesting the anti‐inflammatory activity. These results indicated that N. miranda may have some medicinal potentials. To further elucidate the structure‐activity relationship, our laboratory is currently using LC‐MS to isolate and identify the active compounds from Nm‐s‐a.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Discovery of a Plant‐derived Cell‐penetrating Proteasome Inhibitor

Plant‐derived cysteine‐rich peptides (CRPs) are known to play a role in plant defence against fungi and insects [1]. An increasing body of evidence, however, showed that certain CRPs display cell‐penetrating functions, and could be exploited for therapeutics targeting intracellular proteins [2]. Hibiscus sabdariffa, commonly known as roselle, is a shrub cultivated in South‐east Asia. We previously reported the discovery of CRPs from roselle, which we designated as roseltides (rT1‐rT7) [3]. Roseltides possess an evolutionary conserved six‐cysteine motif typical of hevein‐like peptides, but without a chitin‐binding domain. Roseltide rT1 is a human neutrophil elastase inhibitor, which in part, correlates to the ethnomedicinal use of roselle. Here we report that roseltide rT7, the second most abundant CRP in roselle, is a cell‐penetrating peptide and a proteasome inhibitor. To prepare the synthetic roseltide rT7, we used a stepwise solid‐phase peptide synthesis, followed by oxidative folding, to afford a native‐folded rT7. We showed that the synthetic and native roseltide rT7 are indistinguishable using reversed‐phase high‐performance liquid chromatography and nuclear magnetic resonance spectroscopy. To gain insight into its cell‐penetrating properties, we selectively labeled roseltide rT7 at its N‐terminus with a fluorescent probe (TAMRA‐rT7). Using flow cytometry and live‐cell confocal microscopy, we showed that TAMRA‐rT7 is cell membrane permeable via the endocytotic pathway. Roseltide rT7 was found to inhibit the chymotrypsin‐like activities of human 20S‐proteasome. Taken together, this study highlights the discovery of roseltide rT7 as the first plant‐derived cell‐penetrating proteasome inhibitor that can be potentially useful in the development of therapeutic biologics for cancer, inflammatory, and heart diseases.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).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.

Neutrophil elastase inhibitory effects of pentacyclic triterpenoids from Eriobotrya japonica (loquat leaves)

Ethnopharmacological relevanceEriobotrya japonica, a traditional herbal medicine in China and Japan, has long been used to treat chronic bronchitis and coughs. Aim of the studyPentacyclic triterpenoids (PTs), especially ursolic acid (UA), have been found as reversibly and competitively human neutrophil elastase (HNE) inhibitors. However, the limited solubility and poor bioavailability of PTs hinder their clinical use. Crude plant extracts may have a greater activity than isolated constituents of the equivalent dosage. In this study, an Eriobotrya japonica (loquat leaves) extract (triterpenoid composition of loquat leaves, TCLL) with enriched PTs such as UA was prepared. The study aims to compare the HNE inhibitory (HNEI) effect in vitro and the therapeutic effect on acute lung injury (ALI) in vivo between TCLL and UA. Materials and methodsAn HNEI activity bioassay was performed with Sivelestat sodium hydrate as a positive control. A lipopolysaccharide (LPS)-induced lung inflammatory model was established to evaluate TCLL's therapeutic effect on ALI in vivo. The absorption of UA in TCLL and in UA alone was determined using a Caco-2 cell uptake model and LC-MS. ResultsThe IC50 values of TCLL and UA for the HNEI effect were 3.26 ± 0.56 μg/mL and 8.49 ± 0.42 μg/mL (P < 0.01), respectively. TCLL significantly improved the inflammatory cells and inflammatory cytokine production in mice compared with the LPS group (P < 0.05). Additionally, it performed better than the UA alone group (P < 0.05). Moreover, the uptake by Caco-2 cells of UA in TCLL was higher than that in UA alone (P < 0.05). ConclusionTCLL has a significant HNEI effect in vitro and a therapeutic effect on LPS-induced inflammation in a mouse model. Both the effects are more efficient than UA. Improved absorption of PTs in TCLL may be one explanation for these results.

A potential contribution of trappin-2 to the development of vasculopathy in systemic sclerosis.

Trappin-2/pre-elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological process of systemic sclerosis (SSc). To clarify the potential role of trappin-2 in SSc. Serum trappin-2 levels were determined by enzyme-linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin-2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription-real-time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation. Since serum trappin-2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin-2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin-2 levels than in those with normal levels. Furthermore, serum trappin-2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin-2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin-2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin-2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter. Endothelial trappin-2 up-regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.

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.

Investigation of Human Neutrophil Elastase Inhibition by Staphylococcus aureus EapH1: The Key Role Played by Arginine 89.

Staphylococcus aureus secretes a family of potent, noncovalent inhibitory proteins that selectively target the neutrophil serine proteases neutrophil elastase, cathepsin-G, and proteinase-3. A majority of our understanding of these so-called EAP domain proteins has come from structure/function studies on EapH1 and its effects on human neutrophil elastase (hNE). Inspection of the EapH1/hNE cocrystal structure suggested that EapH1 residues R89, E94, and K95 are positioned near the EapH1/hNE interface and might contribute to the potent inhibition of hNE by EapH1. In this study, we used site directed mutagenesis, kinetic evaluation, and surface plasmon resonance to probe the individual contributions of R89, E94, and K95 to EapH1 function. We found that the wild-type EapH1/hNE complex is characterized by a fast association rate (2.0 × 106 M-1 s-1) and a very slow dissociation rate (4.3 × 10-5 s-1), yielding an apparent inhibition constant of 21 pM. The slow dissociation rate of EapH1 from hNE resulted in a time-dependent inhibition pattern. Although conservative mutants E94Q and K95M, as well as the E94Q/K95M double mutant, had on- and off-rates comparable to wild-type EapH1, mutation of R89 to methionine resulted in a 15,000-fold decrease in inhibition (321 nM) and loss of the time-dependent inhibition characteristic. The double mutants R89M/E94Q and R89M/K95M, as well as the triple mutant R89M/E94Q/K95M were similarly perturbed. Mutation of R89 to lysine restored a portion of the inhibition of hNE (27 nM). Given these observations, we conclude that R89 is a primary contributor to EapH1 function vis-à-vis time-dependent inhibition of hNE.

1H-pyrrolo[2,3-b]pyridine: A new scaffold for human neutrophil elastase (HNE) inhibitors.

Human neutrophil elastase (HNE) is a potent serine protease belonging to the chymotrypsin family. It is an important target for the development of novel and selective inhibitors for the treatment of inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis and biological evaluation of a new series of HNE inhibitors with a pyrrolo[2,3-b]pyridine scaffold, which is an isomer of our previously reported indazoles, in order to assess how a shift of the nitrogen from position 2 to position 7 influences activity. The majority of new compounds were effective HNE inhibitors and had IC50 values in the micromolar/submicromolar range, with some compounds active in low nanomolar levels. For example, 2a and 2b inhibited HNE with IC50 values of 15 and 14 nM, respectively. Molecular modeling of compounds differing in the position of heteroatom(s) in the bicyclic moiety and in the oxadiazole ring demonstrated that the calculated geometries of enzyme-inhibitor complexes were in agreement with the observed biological activities. Docking experiments showed that orientation of the active pyrrolo[2,3-b]pyridines in the HNE catalytic triad Ser195-His57-Asp102 correlated with effectiveness of the inhibitor interaction with the enzyme. Thus, the pyrrolo[2,3-b]pyridine scaffold represents a novel scaffold for the development of potent HNE inhibitors.