Cationic Serine Protease Research Articles

Substituted 4H-3,1-benzoxazine-4-one Derivatives as Inhibitors of Cathepsin G.

Cathepsin G (CatG) is a cationic serine protease with a wide substrate specificity. CatG has been reported to play a role in several pathologies, including rheumatoid arthritis, ischemic reperfusion injury, acute respiratory distress syndrome, and cystic fibrosis, among others. We aim to develop a new class of CatG inhibitors and evaluate their potency and selectivity against a series of serine proteases. We exploited chemical synthesis as well as chromogenic substrate hydrolysis assays to construct and evaluate the new inhibitors. In this communication, we report on a new class of CatG inhibitors of 4H-3,1-benzoxazin- 4-one derivatives. We constructed a small library of seven substituted 4H-3,1-benzoxazin-4-one derivatives and identified their inhibition potential against CatG. Five molecules were identified as CatG inhibitors with values of 0.84-5.5 μM. Inhibitor 2 was the most potent, with an IC50 of 0.84 ± 0.11 μM and significant selectivity over representative serine proteases of thrombin, factor XIa, factor XIIa, and kallikrein. Thus, we propose this inhibitor as a lead molecule to guide subsequent efforts to develop clinically relevant potent and selective CatG inhibitors for use as anti-inflammatory agents.

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities. Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and SDS-PAGE were exploited to decipher the mechanism of CatG inhibition by SPGG. Molecular modelling was also used to identify a plausible binding site. SPGG displayed an inhibition potency of 57 nM against CatG, which was substantially selective over other proteases. SPGG protected fibronectin and laminin against CatG-mediated degradation. SPGG reduced VMAX of CatG hydrolysis of a chromogenic substrate without affecting KM, suggesting an allosteric mechanism. Resolution of energy contributions indicated that non-ionic interactions contribute ~91% of binding energy, suggesting a substantial possibility of specific recognition. Molecular modelling indicated that SPGG plausibly binds to an anion-binding sequence of 109SRRVRRNRN117. We present the discovery of SPGG as the first small molecule, potent, allosteric glycosaminoglycan mimetic inhibitor of CatG. SPGG is expected to open a major route to clinically relevant allosteric CatG anti-inflammatory agents.

Microscale coiling in bis-imidazolium supramolecular hydrogel fibres induced by the release of a cationic serine protease inhibitor.

Gels formed by a gemini dicationic amphiphile incorporate a serine protease inhibitor, which could be used in a new approach to the treatment of Rosacea, within the fibres as well as in the space between them, affecting a number of gel properties but most importantly inducing remarkable fibre coiling at the microscopic level as a result of drug release from the gel. Drug release and skin permeation experiments show its potential for topical administration.

A Lack of Secretory Leukocyte Protease Inhibitor (SLPI) Causes Defects in Myeloid Differentiation

AbstractAbstract 10Previously, we demonstrated severe diminished mRNA and protein levels of secretory leukocyte protease inhibitor (SLPI) in CD33+ myeloid cells in PMNs and in plasma of patients with severe congenital neutropenia (CN), as compared to G-CSF treated healthy individuals. We further analysed whether diminished levels of SLPI are associated with the “maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI in the CD34+ bone marrow hematopoietic progenitor cells and in the acute myelid leukemia cell line NB4 using lentivirus-based transduction with SLPI-specific shRNA and analysed G-CSF- or ATRA-triggered myeloid differentiation, respectively. We found that G-CSF-triggered myeloid differentiation of CD34+ cells transduced with SLPI-specific shRNA was significantly diminished, in comparison to ctrl shRNA transduced cells (on day 14 of differentiation 53.8 % of CD11b+ cells in SLPI shRNA group vs 86.3 % in the ctrl shRNA group (ttest: p = 0.005); 13.1 % of CD16+ cells in SLPI shRNA group vs 48.8 % in the ctrl shRNA group (ttest: p = 0.0006)). This was accompanied by G0/G1 cell cycle arrest and elevated apoptosis of SLPI shRNA transduced cells: on day 7 of differentiation 83.9 % of SLPI shRNA transduced cells and 66.6 % of the ctrl shRNA transduced cells were in G0/G1 phase (ttest: p = 0.03); 11 % of annexin V positive cells in SLPI shRNA transduced group vs 6.5 % in ctrl shRNA group (ttest: p = 0.004). Similar effects on the ATRA-induced myeloid differentiation were observed after inhibition of SLPI in the NB4 cell line.We next analysed the mechanisms of diminished myeloid differentiation due to diminished SLPI levels. SLPI is a cationic serine protease inhibitor with antiprotease, primarily anti-neutrophil elastase, activities. Previously we showed regulation of neutrophil elastase by SLPI in myeloid cells. However, SLPI is also known to modulate intracellular signal transduction pathways such as NF-kB and Erk. SLPI is involved in the phosphorylation of Erk1/2 protein. Erk1/2 protein plays a role in myeloid differentiation. We found that ATRA treatment of NB4 cells transduced with ctrl shRNA led to the transient time-dependent phosphorylation of Erk1/2 on day 2 of culture with subsequent decline on day 4 of treatment. Interestingly, Erk1/2 phosphorylation was almost completely abolished on day 2 of ATRA treatment of NB4 cells transduced with SLPI shRNA. Recently, we demonstrated an essential role of LEF-1 in the granulocytic differentiation and severe diminished LEF-1 expression levels in myeloid cells of CN patients. It has been shown that Erk1/2 phosphorylates LEF-1 transcription factor. In line with defective phosphorylation of Erk1/2 we measured severe diminished levels of phosphorylated LEF-1 in SLPI shRNA transduced cells, in comparison to ctrl shRNA samples. Therefore, we concluded that SLPI regulates myeloid differentiation by regulation of Erk1/2 dependent LEF-1 phosphorylation and autoregulation.To evaluate additional intracellular signaling pathways which are affected during G-CSF-triggered myeloid differentiation if SLPI is downregulated, we performed microarray analysis of RNA from CD34+ cells transduced with SLPI or ctrl shRNA and subsequently treated with G-CSF for 24 hours. Analysis of the microarray data using Ingenuity Systems Pathway Analysis (IPA) revealed severe significant defects in the signaling cascades involved in the differentiation (p-Value = 0.0093), proliferation (p-Value = 0.015), survival (p-Value = 0.012) and chemotaxis (p-Value = 0.0093) of myeloid progenitor cells and neutrophils which was correlated with significant downregulation of FPR2, MMP8, XCL1, S100A9, IL2 and OLFM4 mRNA expression. Moreover, signaling systems regulating DNA replication checkpoint and arrest in Go/G1 phase were also significantly affected (p-Value = 0.0009 and 0.0065, respectively) due to downregulation of mRNA expression of CHEK1, CCNE2, CDC6, MYBL1 and MYC.Taken together, SLPI is a new candidate factor playing an important role in myelopoiesis by controlling proliferation, differentiation and cell cycle of myeloid cells not only by inhibition of neutrophil elastase but also by regulation of intracellular signaling pathways. Disclosures:No relevant conflicts of interest to declare.

MRNA and Protein Expression Levels of Secretory Leukocyte Protease Inhibitor (SLPI) are Severely Reduced in Patients with Severe Congenital Neutropenia (CN)

Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-NeutrophilELastase (NE), activities. The molecular interaction and the balance between NE and SLPI is tightly regulated.

MRNA and Protein Expression Levels of Secretory Leukocyte Protease Inhibitor (SLPI) Are Severely Reduced in Patients with Severe Congenital Neutropenia (CN)

Abstract 2165Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-Neutrophil ELastase (NE), activities. Moreover, SLPI modulates intracellular signal transduction pathways such as NF-kB and Erk. The molecular interaction and the balance between NE and SLPI is tightly regulated. On the one side, NE upregulates the SLPI expression and at the other hand SLPI inhibits the NE-induced degradation of proteins.We identified severe diminished levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN) harbouring either ELANE or HAX1 mutations, as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. SLPI protein levels in plasma of CN patients were also significantly reduced. We further analysed whether diminished levels of SLPI are associated with the „maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI using lentivirus-based transduction of the myeloid cell line NB4 with SLPI-specific shRNA and analysed ATRA-triggered myeloid differentiation. Indeed, myeloid differentiation was severely affected in NB4 cells transduced with SLPI-specific shRNA, as compared to control shRNA transduced cells.Further, we analysed the mechanisms leading to SLPI downregulation. Previously, we identified severely reduced mRNA and protein levels of NE in myeloid cells and in plasma of CN patients with either ELANE or HAX1 mutations, as compared to healthy individuals. Knowing that NE induces SLPI expression, we assumed that diminished NE levels may be responsible for the low SLPI expression in CN patients. Indeed, inhibition of NE in the myeloid cell line NB4 using NE-specific shRNAs led to diminished expression of SLPI mRNA, as compared to ctrl shRNA transduced cells. At the same time, we also found that transduction of the myeloid cell line NB4 with wild type (WT) NE resulted in the increased expression of SLPI mRNA but mutated (MUT) forms of NE as found in CN patients were not able to induce SLPI mRNA, as compared to ctrl transduced cells. Taken together, both diminished NE levels and mutations in ELANE gene may cause downregulation of SLPI.In summary, SLPI is severely downregulated in CN patients due to defective NE protein levels and ELANE mutations. As a consequence, the anti-microbial and antiinflammatory activities of SLPI are diminished in CN patients. Disclosures:No relevant conflicts of interest to declare.

Protease inhibition by oleic acid transfer from chronic wound dressings to albumin

High elastase and cathepsin G activities have been observed in chronic wounds to inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid is a non-toxic elastase inhibitor. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of oleic acid by albumin under conditions mimicking chronic wounds. The mechanism of oleic acid uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin–oleic acid complexes under liquid equilibrium conditions revealed fully saturated albumin–oleic acid complexes with a 1:1 weight ratio of albumin:oleic acid. Liquid–solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of oleic acid per mole albumin. Comparing oleic acid formulated wound dressings for dose dependent ability to lower elastase activity, we found cotton gauze > hydrogel > hydrocolloid. In contrast, the cationic serine protease cathepsin G was inhibited by oleic acid within a narrow range of oleic acid–cotton formulations. 2% albumin was sufficient to transfer quantities of oleic acid necessary to achieve a significant elastase-lowering effect. Oleic acid bound to cotton wound dressings may have promise in the selective lowering of cationic serine protease activity useful in topical application for chronic inflammatory pathogenesis.

Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor.

Secretory leukocyte protease inhibitor (SLPI) is a cationic serine protease inhibitor with anti-microbial and anti-inflammatory properties found in large quantities in mucosal fluids, including saliva. SLPI is expressed during cutaneous wound healing, however, its role in oral wound repair is unknown. We have used a novel approach involving a murine buccal mucosal acute wound model to investigate the role of SLPI in oral healing. In parallel to the observed cutaneous healing phenotype, an absence of SLPI results in markedly impaired oral wound healing associated with increased inflammation and raised elastase activity. Moreover, matrix deposition was decreased, while MMP activity was enhanced in the oral SLPI null wounds suggesting deregulated proteolysis. Intriguingly, regardless of genotype, reduced collagen deposition was observed in oral compared to dermal wounds, associated with reduced TGF-beta expression and decreased fibroblast collagen expression in vitro. We propose that SLPI is a pivotal endogenous factor necessary for optimal tissue repair including intra-oral wound healing. In addition, our model provides a unique opportunity to delineate the cellular and molecular mechanisms underlying the differences between dermal scarring and oral scar-free healing.

Tailoring an antibacterial peptide of human lysosomal cathepsin G to enhance its broad-spectrum action against antibiotic-resistant bacterial pathogens.

Neutrophils contain several cationic antimicrobial proteins or peptides (CAPs) that exert antibiotic-like action against bacteria. These host-derived antibiotics kill susceptible bacteria by oxygen-independent mechanisms. Considerable interest in their activity has been generated in recent years due not only to their likely important role in innate host defense against infection, but also their possible use as therapeutic agents in treating infections caused by antibiotic-resistant pathogens. We have studied the antibacterial properties of human lysosomal cathepsin G (cat G). This highly cationic serine protease contains at least three antibacterial regions that by themselves can exert antibacterial action against Gram-negative bacteria, such as Pseudomonas aeruginosa. Only one of these peptides, defined by residues 117-136 of full-length cat G, has bactericidal action against Gram-positive pathogens, such as Staphylococcus aureus. Due to the broad-spectrum antibacterial action of this peptide, we have sought to define the amino acids within its primary sequence required for this activity and have developed variants with improved activity. This review emphasizes the importance of both cationicity and hydrophobicity as necessary characteristics for the antibacterial action of CAPs. It also proposes the strategy that naturally occurring large human CAPs can be dissected to smaller CAPs and then modified to enhance their activity in vitro. This approach could prove beneficial to those interested in developing antimicrobial peptides as therapeutic agents.