Investigating Polypharmacology through Targeting Known Human Neutrophil Elastase Inhibitors to Proteinase 3.

Using a combination of multisite λ-dynamics (MSλD) together with in vitro IC50 assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. The affinities we observe suggest that the dihydropyrimidinone scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.

Human neutrophil elastase inhibitors: Classification, biological-synthetic sources and their relevance in related diseases.

BACKGROUND: Human neutrophil elastase is a multifunctional protease enzyme whose function is to break the bonds of proteins and degrade them to polypeptides or amino acids. In addition, it plays an essential role in the immune mechanism against bacterial infections and represents a key mediator in tissue remodeling and inflammation. However, when the extracellular release of this enzyme is dysregulated in response to low levels of its physiological inhibitors, it ultimately leads to the degradation of proteins, in particular elastin, as well as other components of the extracellular matrix, producing injury to epithelial cells, which can promote sustained inflammation and affect the innate immune system, and, therefore, be the basis for the development of severe inflammatory diseases, especially those associated with the cardiopulmonary system. OBJECTIVE: This review aims to provide an update on the elastase inhibitory properties of several molecules, either synthetic or biological sources, as well as their classification and relevance in related pathologies since a clear understanding of the function of these molecules with the inhibitory capacity of this protease can provide valuable information for the development of pharmacological therapies that manage to modify the prognosis and survival of various inflammatory diseases. METHODS: Collected data from scientific databases, including PubMed, Google Scholar, Science Direct, Nature, Wiley, Scopus, and Scielo. Articles published in any country and language were included. RESULTS: We reviewed and included 132 articles conceptualizing neutrophil elastase activity and known inhibitors. CONCLUSION: Understanding the mechanism of action of elastase inhibitors based on particular aspects such as their kinetic behavior, structure-function relationship, chemical properties, origin, pharmacodynamics, and experimental progress has allowed for a broad classification of HNE inhibitors.

Design and synthesis of sirtinol analogs as human neutrophil elastase inhibitors.

Human neutrophil elastase (HNE) overexpression has a crucial role in most acute inflammation and alpha1-antitrypsin deficiency syndromes observed in humans, triggering neutrophil invasion and activation of macrophage inflammatory and proteolytic effects, leading to tissue damage. Manipulating HNE level homeostasis could potentially help treat neutrophilic inflammation. Previous studies have shown that sirtinol (1) has a specific influence on HNE and potently attenuates acute lung injury and hepatic injury mediated by lipopolysaccharide or trauma hemorrhage. Therefore, 1 was chosen as the model structure to obtain more potent anti-HNE agents. In the present study, we synthesized a series of sirtinol analogues and determined their inhibitory effects on HNE. Structure-activity relationship (SAR) studies showed that swapping the imine and methyl groups of the sirtinol scaffold with diazene and carboxyl groups, respectively, enhances the HNE inhibiting potency. Compound 29 exhibited the highest potency in the SAR study and showed dual inhibitory effects on HNE and proteinase 3 with IC50 values of 4.91 and 20.69 µM, respectively. Furthermore, 29 was confirmed to have dual impacts on inhibiting O2•- generation and elastase release in cell-based assays with IC50 values of 0.90 and 1.86 µM, respectively. These findings suggest that 29 is a promising candidate for developing HNE inhibitors in the treatment of neutrophilic inflammatory diseases.

ITIH5 inhibits proliferation, adipogenic differentiation, and secretion of inflammatory cytokines of human adipose stem cells-A new key in treating obesity?

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is widely expressed in the human body, and it is detected to be particularly abundant in adipose tissue. ITIH5 expression is increased in people with obesity compared to lean persons and is decreased by diet-induced weight loss. This suggests that ITIH5 may be involved in the development of adiposity and clinical metabolic variables, although its exact function remains unknown. We measured the protein concentration of ITIH5 in adipose samples from patients undergoing abdominoplasty and tested for correlation with the subjects' BMI as well as inflammatory mediators. We stimulated human adipose stem cells (ASCs) with recombinant (r)ITIH5 protein and tested for an effect on proliferation, differentiation, and immunosuppressive properties when the cells were exposed to an artificial inflammatory environment. We found positive correlations between ITIH5 levels and the BMI (p < .001) as well as concentrations of inflammatory cytokines (TNF-α, IL-6, and MCP-1) in adipose tissue (p < .01). Application of the rITIH5 protein inhibited both proliferation (p < .001) and differentiation of ASCs. Especially, the development of mature adipocytes was reduced by over 50%. Moreover, rITIH5 decreased the release of IL-6 and MCP-1 when the cells were exposed to TNF-α and IL-1ß (p < .001). Our data suggest that ITIH5 is an adipokine that is increasingly released during human adipose tissue development, acting as a regulator that inhibits proliferation and adipogenic differentiation of ASCs. ITIH5 thus presents itself as a positive regulator of adipose tissue homeostasis, possibly protecting against both hyperplasia and hypertrophy of adipose tissue and the associated chronic inflammation.

Early-onset tufting enteropathy in HAI-2-deficient mice is independent of matriptase-mediated cleavage of EpCAM.

Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.

Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects.

Aberrantly up-regulated activity of the type II transmembrane protease Matriptase-1 has been associated with the development and progression of a range of epithelial-derived carcinomas, and a variety of signaling pathways can mediate Matriptase-dependent tumorigenic events. During mammalian carcinogenesis, gain of Matriptase activity often results from imbalanced ratios between Matriptase and its cognate transmembrane inhibitor Hai1. Similarly, in zebrafish, unrestrained Matriptase activity due to loss of hai1a results in epidermal pre-neoplasms already during embryogenesis. Here, based on our former findings of a similar tumor-suppressive role for the Na+/K+-pump beta subunit ATP1b1a, we identify epithelial polarity defects and systemic hypotonic stress as another mode of aberrant Matriptase activation in the embryonic zebrafish epidermis in vivo. In this case, however, a different oncogenic pathway is activated which contains PI3K, AKT and NFkB, rather than EGFR and PLD (as in hai1a mutants). Strikingly, epidermal pre-neoplasm is only induced when epithelial polarity defects in keratinocytes (leading to disturbed Matriptase subcellular localization) occur in combination with systemic hypotonic stress (leading to increased proteolytic activity of Matriptase). A similar combinatorial effect of hypotonicity and loss of epithelial polarity was also obtained for the activity levels of Matriptase-1 in human MCF-10A epithelial breast cells. Together, this is in line with the multi-factor concept of carcinogenesis, with the notion that such factors can even branch off from one and the same initiator (here ATP1a1b) and can converge again at the level of one and the same mediator (here Matriptase). In sum, our data point to tonicity and epithelial cell polarity as evolutionarily conserved regulators of Matriptase activity that upon de-regulation can constitute an alternative mode of Matriptase-dependent carcinogenesis in vivo.

ITIH5, as a predictor of prognosis and immunotherapy response for P53-like bladder cancer, is related to cell proliferation and invasion.

p53-like bladder cancer (BLCA) is a bladder cancer subtype that is resistant to cisplatin-based chemotherapy. The ideal treatment modality for such tumors remains poorly defined, and immunotherapy seems to be a potential approach. Therefore, it is significant to understand the risk stratification of p53-like BLCA and identify novel therapeutic targets. ITIH5 is a member of the inter-α-trypsin inhibitory (ITI) gene family, and the effect of ITIH5 on p53-like BLCA remains elusive. In this study, TCGA data and in vitro experiments were used to explore the prognostic value of ITIH5 for p53-like BLCA and its effect on tumor cell proliferation, migration, and invasion. The impact of ITIH5 on the level of immune cell infiltration was explored using seven different algorithms, and the predictive value of ITIH5 on the efficacy of immunotherapy for p53-like BLCA was explored in combination with an independent immunotherapy cohort. The results showed that patients with high ITIH5 expression had a better prognosis, and overexpression of ITIH5 could inhibit the proliferation, migration, and invasion of tumor cells. Two or more algorithms consistently showed that ITIH5 promoted the infiltration of antitumor immune cells, such as B cells, CD4+ T cells, and CD8+ T cells. In addition, ITIH5 expression was positively correlated with the expression levels of many immune checkpoints, and the high ITIH5 expression group showed better response rates to PD-L1 and CTLA-4 therapies. In short, ITIH5 is a predictor of prognosis and the immunotherapy response for p53-like BLCA and is correlated with tumor immunity.

Secretory leukocyte protease inhibitor modulates FcεRI-dependent but not Mrgprb2-dependent mastocyte function in psoriasis.

Psoriasis, which involves mast cells, is a chronic inflammatory skin disorder whose pathophysiology is still not fully understood. We investigated the role of secretory leukocyte protease inhibitor (SLPI), a potential inhibitor of mastocyte serine proteases, on mast cell-dependent processes of relevance to the skin barrier defense in psoriasis. Here, we demonstrate that the dermal mast cells of patients with psoriasis express SLPI but not those of healthy donors. Moreover, SLPI transcripts were found to be markedly upregulated in murine mast cells by mediators derived from psoriasis skin explant cultures. Using mast cells from SLPI-deficient mice and their SLPI+ wild-type controls, we show that SLPI inhibits the activity of serine protease chymase in mastocytes. SLPI was also found to enhance the degranulation of mast cells activated via anti-IgE Abs but not Mrgprb2 ligands. Finally, we demonstrate that the expression and function of Mrgprb2 in mast cells are suppressed by a normal and, to a larger extent, psoriatic skin environment. Together, these findings reveal mechanisms underlying FcεRI- and Mrgprb2-dependent mast cell function that have not been described previously.

Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.

HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1 ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

The neutrophil elastase inhibitor, sivelestat, attenuates acute lung injury in patients with cardiopulmonary bypass.

Background: The sivelestat is a neutrophil elastase inhibitor thought to have an effect against acute lung injury (ALI) in patients after scheduled cardiac surgery. However, the beneficial effect of sivelestat in patients undergoing emergent cardiovascular surgery remains unclear. We aim to evaluate the effect of sivelestat on pulmonary protection in patients with ALI after emergent cardiovascular surgery. Methods: Firstly, a case-control study in 665 patients undergoing emergent cardiovascular surgery from January 1st, 2020 to October 26th, 2022 was performed. 52 patients who received sivelestat (0.2mg/kg/h for 3 days) and 613 age- and sex-matched controls. Secondly, a propensity-score matched cohort (sivelestat vs control: 50 vs 50) was performed in these 665 patients. The primary outcome was a composite of adverse outcomes, including 30-day mortality, ECMO, continuous renal replacement therapy (CRRT) and IABP, etc. The secondary outcome included pneumonia, ventricular arrhythmias and mechanical ventilation time, etc. Results: In propensity-matched patients, the 30-day mortality (16% vs 24%, P=0.32), stroke (2% vs 8%, P=0.17), ECMO(6% vs 10%, P=0.46), IABP(4% vs 8%, P=0.40) and CRRT(8% vs 20%, P=0.08) had no differences between sivelestat and control group; sivelestat could significantly decrease pneumonia (40% vs 62%, P=0.03), mechanical ventilation time (median: 96hours, IQR:72-120hours vs median:148hours, IQR:110-186hours, P<0.01), bilateral pulmonary infiltrates (P<0.01), oxygen index (P<0.01), interleukin-6(P=0.02), procalcitonin(P<0.01) and C-reactive protein(P<0.01). Conclusion: Administration of sivelestat might improve postoperative outcomes in patients with ALI after emergent cardiovascular surgery. Our results show that sivelestat may be considered to protect pulmonary function against inflammatory injury by CPB. Registration: http://www.chictr.org.cn/showproj.aspx?proj=166643, identifier ChiCTR2200059102.

Possible role of combined therapy targeting MET and pro-HGF activation for renal cell carcinoma: analysis by human HGF-producing SCID mice.

MET is a high-affinity receptor tyrosine kinase of HGF (hepatocyte growth factor). HGF is secreted as an inactive single-chain precursor (pro-HGF), which requires proteolytic activation for conversion to an active form. HGF activator inhibitor (HAI)-2 is a transmembrane Kunitz-type serine protease inhibitor, which inhibits all pro-HGF-activating enzymes. In RCC, increased expression of MET and decreased expression of HAI-2 were reported to be poor prognostic factors. In the current study, we tried to inhibit the growth of RCC cells by dual inhibition of both MET phosphorylation and pro-HGF-activation using MET inhibitor and HAI-2 overexpression. A transgenic mouse model which expressed human HGF (HGF mouse) was used for in vivo analysis to evaluate the HGF/MET signaling axis accurately. Initially, doxycycline-induced HAI-2 overexpression RCC cells (786-O-HAI2) were prepared. The cells were cultured with pro-HGF, and inhibitory effect of MET inhibitor (SCC244) and HAI-2 was evaluated by phosphorylation of MET and cell proliferation. Next, the cells were subcutaneously implanted to HGF mice and the growth inhibition was determined by SCC244 and HAI-2. Single use of each inhibitor showed significant inhibition in MET phosphorylation, migration and proliferation of 786-O-HAI2 cells; however, the strongest effect was observed by combined use of both inhibitors. Although in vivo analysis also showed apparent downregulation of MET phosphorylation and growth inhibition in combined treatment, statistical significance was not observed compared with single use of MET inhibitor. Combined treatment with MET-TKI and HAI-2 suggested to consider as a candidate for new strong therapy for RCC.

The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells.

BACKGROUND: Multiple myeloma (MM) is an incurable malignancy of plasma cells. The serine protease matriptase is frequently dysregulated in human carcinomas, which facilitates tumor progression and metastatic dissemination. The importance of matriptase in hematological malignancies is yet to be clarified. In this study, we aimed to characterize the role of matriptase in MM. MATERIALS AND METHODS: mRNA expression of matriptase and its inhibitors hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 was studied in primary MM cells from patient samples and human myeloma cell lines (HMCLs). We further investigated the effect of matriptase on migration and proliferation of myeloma cells in vitro. By use of the CoMMpass database, we assessed the clinical relevance of matriptase in MM patients. RESULTS: Matriptase was expressed in 96% of patient samples and all HMCLs tested. Overexpression of matriptase in vitro reduced proliferation, and significantly decreased cytokine-induced migration. Conversely, matriptase knockdown significantly enhanced migration. Mechanistically, overexpression of matriptase inhibited activation of Src kinase. CONCLUSIONS: Our findings may suggest a novel role of matriptase as a tumor suppressor in MM pathogenesis.

Establishment of a mouse model of Netherton syndrome based on CRISPR/Cas9 technology

Background: Netherton syndrome is a rare but severe autosomal recessive disorder with dominant impaired skin barrier function, caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, which encodes LEKTI (lymphoepithelial Kazal-type-related inhibitor). Objectives: To establish a murine model of Netherton syndrome based on CRISPR/Cas9 gene editing technology. Materials & Methods: Spink5-sgRNA was designed to target exon 3 of the mouse Spink5 gene. Cas9 mRNA and sgRNA were microinjected into the zygotes of C57BL/6J mice. Spink5 homozygous knockout mice were born from a heterozygous intercross, and the phenotype of these mice was compared with wild-type regarding gross morphology, histopathology and immunofluorescent detection of LEKTI. Results: Following microinjection of zygotes using the CRISPR/Cas9 system, sequencing demonstrated a 22-bp deletion at exon 3 of the mouse Spink5 gene. Histological investigation revealed complete detachment of the stratum corneum from the underlying granular layer and an absence of LEKTI in skin from Spink5 homozygous knockout mice. Conclusion: The 22-bp deleted Spink5 transgenic mouse model demonstrates the clinical phenotype and genotype of human Netherton syndrome, representing a useful tool for future gene correction and skin barrier/inflammation studies.

LncRNA SPINT1-AS1/miR-433-3p/E2F3 positive feedback loop promotes the KRAS-mutant colorectal cancer cell proliferation, migration and invasion.

Colorectal cancer (CRC) features high prevalence and mortality. Long non-coding RNAs (lncRNAs) exert nonnegligible roles in human cancer development. Nevertheless, the functions of most lncRNAs still remain unexplored. We currently focused on detecting the influence of SPINT1 antisense RNA 1 (SPINT1-AS1) on CRC development and investigating into the potential regulatory mechanism. RT-qPCR analysis first confirmed that SPINT1-AS1 exhibited high expression in KRAS-mutant (KRASMUT) CRC cells. Through series of functional experiments, we observed that knockdown of SPINT1-AS1 weakened KRASMUT CRC cell proliferation, migration and invasion. Afterwards, the implementation of mechanism assays help to verify that SPINT1-AS1 sequestered microRNA-433-3p (miR-433-3p) to regulate the expression of E2F transcription factor 3 (E2F3). Besides, E2F3 was validated to activate the transcription of SPINT1-AS1 in turn. Rescue experiments confirmed the functional influence of SPINT1-AS1/miR-433-3p/E2F3 on CRC cells. In summary, the molecular axis of SPINT1-AS1/miR-433-3p/E2F3 forms a positive loop which might become a potential biomarker in CRC.

Diversity oriented clicking delivers ß-substituted alkenyl sulfonyl fluorides as covalent human neutrophil elastase inhibitors.

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.

Lobelia chinensis Extract and Its Active Compound, Diosmetin, Improve Atopic Dermatitis by Reinforcing Skin Barrier Function through SPINK5/LEKTI Regulation.

The skin acts as a mechanical barrier that protects the body from the exterior environment, and skin barrier function is attributed to the stratum corneum (SC), which is composed of keratinocytes and skin lipids. Skin barrier homeostasis is maintained by a delicate balance between the differentiation and exfoliation of keratinocytes, and keratinocyte desquamation is regulated by members of the serine protease kalikrein (KLK) family and their endogenous inhibitor SPINK5/LEKTI (serine protease inhibitor Kazal type 5/lympho-epithelial Kazal-type-related inhibitor). Furthermore, SPINK5/LEKTI deficiency is involved in impaired skin barrier function caused by KLK over-activation. We sought to determine whether increased SPINK5/LEKTI expression ameliorates atopic dermatitis (AD) by strengthening skin barrier function using the ethanol extract of Lobelia chinensis (LCE) and its active compound, diosmetin, by treating human keratinocytes with UVB and using a DNCB-induced murine model of atopic dermatitis. LCE or diosmetin dose-dependently increased the transcriptional activation of SPINK5 promoter and prevented DNCB-induced skin barrier damage by modulating events downstream of SPINK5, that is, KLK, PAR2 (protease activated receptor 2), and TSLP (thymic stromal lymphopoietin). LCE or diosmetin normalized immune response in DNCB treated SKH-1 hairless mice as determined by reductions in serum immunoglobulin E and interleukin-4 levels and numbers of lesion-infiltrating mast cells. Our results suggest that LCE and diosmetin are good candidates for the treatment of skin barrier-disrupting diseases such as Netherton syndrome or AD, and that they do so by regulating SPINK5/LEKTI.

Novel benzoxazinone derivative as potent human neutrophil elastase inhibitor: Potential implications in lung injury.

Neutrophil elastase, a powerful physiological defence tool, may serve as drug target for diverse diseases due to its bystander effect on host cells like chronic obstructive pulmonary disease (COPD). Here, we synthesised seven novel benzoxazinone derivatives and identified that these synthetic compounds are human neutrophil elastase inhibitor that was demonstrated by enzyme substrate kinetic assay. One such compound, PD05, emerged as the most potent inhibitor with lower IC50 as compared to control drug sivelestat. While this inhibition is competitive based on substrate dilution assay, PD05 showed a high binding affinity for human neutrophil elastase (Kd = 1.63 nM) with faster association and dissociation rate compared to notable elastase inhibitors like ONO 6818 and AZD9668, and its interaction with human neutrophil elastase was fully reversible.Preclinical pharmacokinetic studies were performed in vitro where protein binding was found to be 72% with a high recovery rate, aqueous solubility of 194.7 µM, low permeability along with a favourable hERG. Experiments with cell line revealed that the molecule successfully prevented elastase induced rounding and retracted cell morphology and cell cytotoxicity. In mouse model PD05 is able to reduce the alveolar collapse induced by neutrophil elastase. In summary, we demonstrate the in situ, in vitro and in vivo anti-elastase potential of the newly synthesised benzoxazinone derivative PD05 and thus this could be promising candidate for further investigation as a drug for the treatment of COPD.

Urine inter-alpha-trypsin inhibitor family-related proteins may serve as biomarkers for disease activity of lupus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease involving multiple tissues. Inter-Alpha-Trypsin Inhibitor (ITI) family proteins have a role in maintaining tissue homeostasis, but their possible clinical significance in the SLE patients has not been reported. The aim of this study was to analyze and verify the expression of ITI-related proteins in the urine of SLE patients, further explore the features of these proteins in disease activity. METHODS: Based on label-free proteomics technology and bioinformatics technology, we analyzed the expression of ITI family-related proteins in the urine of lupus. Subsequently, Western-blot and targeted proteomics were used to qualitatively and quantitatively verify the expression of these proteins, respectively. RESULTS: A total of seven ITI family-related proteins were screened and identified; and six of these proteins were differentially expressed in the urine of SLE patients. Further quantitative analysis showed that the expressions of ITIH2, ECM1, and ITIH5 in urine between active SLE group and stable SLE group were consistent with the preliminary screening results. The expression of ITIH2 and ECM1 in the renal damage group were also consistent with the screening results. Moreover, ITIH2 and ECM1 have a good correlation with disease activity and have a certain correlation with renal damage. CONCLUSIONS: In this exploratory study, we evaluated the expression of ITI family-related proteins in the urine of SLE and found that urine ITIH2 and ECM1 were closely related to SLE activity, especially kidney damage, providing an experimental basis for further exploration of the potential roles in monitoring lupus and lupus nephritis activity.