Efficacy of host cell serine protease inhibitor MM3122 against SARS-CoV-2 for treatment and prevention of COVID-19.

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

The Long-Acting Serine Protease Inhibitor mPEG-SPA-MDSPI16 Alleviates LPS-Induced Acute Lung Injury.

Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with other drugs to treat fever and viral diseases caused by various factors. In our previous study, a novel serine protease inhibitor-encoding gene (MDSPI16) with improved anti-inflammatory activity was selected from a constructed suppressive subducted hybridization library of housefly larvae. This protein could easily induce an immune response in animals and had a short half-life, which limited its wide application in the clinic. Thus, in this study, mPEG-succinimidyl propionate (mPEG-SPA, Mw = 5 kDa) was used to molecularly modify the MDSPI16 protein, and the modified product mPEG-SPA-MDSPI16, which strongly inhibited elastase production, was purified. It had good stability and safety, low immunogenicity, and a long half-life, and the IC50 for elastase was 86 nM. mPEG-SPA-MDSPI16 effectively inhibited the expression of neutrophil elastase and decreased ROS levels. Moreover, mPEG-SPA-MDSPI16 exerted anti-inflammatory effects by inhibiting activation of the NF-κB signaling pathway and the MAPK signaling pathway in neutrophils. It also exerted therapeutic effects on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. In summary, mPEG-SPA-MDSPI16 is a novel anti-inflammatory protein modified with PEG that has the advantages of safety, nontoxicity, improved stability, and strong anti-inflammatory activity in vivo and in vitro and is expected to become an effective anti-inflammatory drug.

The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

Identification and functional analysis of a serine protease inhibitor using machine learning strategy.

In the intricate realm of animal biology, a multitude of vital processes heavily rely on precisely orchestrated proteinase cascades, but the potential for havoc makes proteinase inhibitors indispensable, with serine proteinase inhibitors (serpins) at the forefront, serving as custodians of homeostasis and participating in various critical biological processes. Importantly, there are still many unexplored facets of serpin functionality. In this study, we focused on the serpin family proteins from Marsupenaeus japonicus, utilizing a fine-tuned pretrained protein language model. This approach led to the identification and evolutionary validation of 28 serpins, one of which, referred to as Mjserpin-1, was both computationally and experimentally demonstrated to show potential as an antiviral and apoptosis inhibitor. Our research unveils exciting prospects for the fusion of state-of-the-art artificial intelligence and rich bioinformatics, holding the promise of significant discoveries that could pave the way for future therapeutic advancements.

Attachment of Proteolytic Enzyme Inhibitors to Vascular Prosthesis-An Analysis of Binding and Antimicrobial Properties.

Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.

Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Nanoengineered Red Blood Cells Loaded with TMPRSS2 and Cathepsin L Inhibitors Block SARS-CoV-2 Pseudovirus Entry into Lung ACE2+ Cells.

The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.

Therapeutic Intervention of Serine Protease Inhibitors against Hepatitis C Virus.

Hepatitis C virus (HCV) is a globally prevalent and hazardous disorder that is responsible for inducing several persistent and potentially fatal liver diseases. Current treatment strategies offer limited efficacy, often accompanied by severe and debilitating adverse effects. Consequently, there is an urgent and compelling need to develop novel therapeutic interventions that can provide maximum efficacy in combating HCV while minimizing the burden of adverse effects on patients. One promising target against HCV is the NS3-4A serine protease, a complex composed of two HCV-encoded proteins. This non-covalent heterodimer is crucial in the viral life cycle and has become a primary focus for therapeutic interventions. Although peginterferon, combined with ribavirin, is commonly employed for HCV treatment, its efficacy is hampered by significant adverse effects that can profoundly impact patients' quality of life. In recent years, the development of direct-acting antiviral agents (DAAs) has emerged as a breakthrough in HCV therapy. These agents exhibit remarkable potency against the virus and have demonstrated fewer adverse effects when combined with other DAAs. However, it is important to note that there is a potential for developing resistance to DAAs due to alterations in the amino acid position of the NS3-4A protease. This emphasizes the need for ongoing research to identify strategies that can minimize the emergence of resistance and ensure long-term effectiveness. While the combination of DAAs holds promise for HCV treatment, it is crucial to consider the possibility of drug-drug interactions. These interactions may occur when different DAAs are used concurrently, potentially compromising their therapeutic efficacy. Therefore, carefully evaluating and monitoring potential drug interactions are vital to optimize treatment outcomes. In the pursuit of novel therapeutic interventions for HCV, the field of computational biology and bioinformatics has emerged as a valuable tool. These advanced technologies and methodologies enable the development and design of new drugs and therapeutic agents that exhibit maximum efficacy, reduced risk of resistance, and minimal adverse effects. By leveraging computational approaches, researchers can efficiently screen and optimize potential candidates, accelerating the discovery and development of highly effective treatments for HCV, treatments.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

A Serine Protease Inhibitor, Camostat Mesilate, Suppresses Urinary Plasmin Activity and Alleviates Hypertension and Podocyte Injury in Dahl Salt-Sensitive Rats.

In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions.

A Genome-Wide Analysis of Serine Protease Inhibitors in Cydia pomonella Provides Insights into Their Evolution and Expression Pattern.

Serine protease inhibitors (serpins) appear to be ubiquitous in almost all living organisms, with a conserved structure and varying functions. Serpins can modulate immune responses by negatively regulating serine protease activities strictly and precisely. The codling moth, Cydia pomonella (L.), a major invasive pest in China, can cause serious economic losses. However, knowledge of serpin genes in this insect remain largely unknown. In this study, we performed a systematic analysis of the serpin genes in C. pomonella, obtaining 26 serpins from the C. pomonella genome. Subsequently, their sequence features, evolutionary relationship, and expression pattern were characterized. Comparative analysis revealed the evolution of a number of serpin genes in Lepidoptera. Importantly, the evolutionary relationship and putative roles of serpin genes in C. pomonella were revealed. Additionally, selective pressure analysis found amino acid sites with strong evidence of positive selection. Interestingly, the serpin1 gene possessed at least six splicing isoforms with distinct reactive-center loops, and these isoforms were experimentally validated. Furthermore, we observed a subclade expansion of serpins, and these genes showed high expression in multiple tissues, suggesting their important roles in C. pomonella. Overall, this study will enrich our knowledge of the immunity of C. pomonella and help to elucidate the role of serpins in the immune response.

Upamostat: a serine protease inhibitor for antiviral, gastrointestinal, and anticancer indications.

INTRODUCTION: Serine proteases are involved in many normal metabolic processes but also contribute to diseases of several organ systems, including viral and gastrointestinal diseases and oncology. Upamostat is an orally bioavailable prodrug of WX-UK1, which is most active against trypsins and closely related enzymes. AREAS COVERED: Research over the past two decades suggests several diseases in the three areas noted above which upamostat may be active. Upamostat has been studied clinically against several cancers and for outpatient treatment of COVID-19. Preclinical and clinical pharmacokinetic and metabolism studies demonstrate good bioavailability, sustained tissue levels, and high concentrations of the active moiety, WX-UK1, in stool, potentially important for treatment of gastrointestinal diseases. Clinical studies suggest activity against SARS-CoV-2; results against pancreatic cancer are also encouraging, though studies in both indications are not definitive. The drug was very well tolerated for periods of 2 weeks to several months. EXPERT OPINION: Upamostat is an orally bioavailable serine protease inhibitor with an excellent safety profile and favorable pharmacokinetic properties. It has demonstrated preliminary evidence of efficacy against COVID-19, and nonclinical data suggest potential applicability against other viral illnesses, gastrointestinal diseases, and cancer.

In vitro testing of host-targeting small molecule antiviral matriptase/TMPRSS2 inhibitors in 2D and 3D cell-based assays.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic strongly stimulated the development of small molecule antivirals selectively targeting type II transmembrane serine proteases (TTSP), required for the host-cell entry of numerous viruses. A set of 3-amidinophenylalanine derivatives (MI-21, MI-472, MI-477, MI-485, MI-1903 and MI-1904), which inhibit the cleavage of certain viral glycoproteins was characterized in 2D and 3D primary human hepatocyte models on collagen- and Matrigel-coating using a CCK-8 assay to evaluate their cytotoxicity, a resorufin-based method to detect redox imbalances, fluorescence and ultrafiltration experiments to evaluate their interactions with human serum albumin (HSA) and α-acidic glycoprotein (AGP), and luminescence measurement to assess CYP3A4 modulation. For elucidation of selectivity of the applied compounds towards matriptase, transmembrane serine protease 2 (TMPRRS2), thrombin and factor Xa (FXa) Ki values were determined. It was proven that cell viability was only deteriorated by inhibitor MI-1903, and redox status was not influenced by administration of the selected inhibitors at 50 µM for 24 h. MI-472 and MI-477 formed relatively stable complexes with AGP. CYP3A4 inhibition was found to be strong in PHHs exposed to all inhibitors with the exception of MI-21, which seems to be a promising drug candidate also due to its better selectivity towards matriptase and TMPRSS2 over the blood clotting proteases thrombin and FXa. Our in vitro pharmacokinetic screening with these inhibitors helps to select the compounds with the best selectivity and safety profile suitable for a further preclinical characterization without animal sacrifice.

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.

Biological activities of Vipegrin, an anti-adhesive Kunitz-type serine proteinase inhibitor purified from Russell's viper venom.

Vipegrin is a 6.8 kDa Kunitz-type serine proteinase inhibitor purified from Russell's viper (Vipera russelii russelii) venom. Kunitz-type serine proteinase inhibitors are non-enzymatic proteins and are ubiquitous constituents of viper venoms. Vipegrin could significantly inhibit the catalytic activity of trypsin. It also posseses disintegrin-like properties and could inhibit collagen and ADP-induced platelet aggregation in a dose-dependent manner. Vipegrin is cytotoxic to MCF7 human breast cancer cells and restricts its invasive property. Confocal microscopic analysis revealed that Vipegrin could induce apoptosis in MCF7 cells. Vipegrin disrupts cell to cell adhesion of MCF7 cells through its disintegrin-like activity. It also causes disruption of attachment of MCF7 cells to synthetic (poly L-lysine) and natural (fibronectin, laminin) matrices. Vipegrin did not cause cytotoxicity on non-cancerous HaCaT, human keratinocytes. The observed properties indicate that Vipegrin may help the development of a potent anti-cancer drug in future.

Identification and characterization of aptameric inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. 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 the systematic evolution of ligands by exponential enrichment to develop ssDNA 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.

Enteral administration of the protease inhibitor gabexate mesilate preserves vascular function in experimental trauma/hemorrhagic shock.

Preserving vascular function is crucial for preventing multiorgan failure and death in ischemic and low-pressure states such as trauma/hemorrhagic shock (T/HS). It has recently been reported that inhibiting circulating proteases released from the bowel to the circulation during T/HS may preserve vascular function and improve outcomes following T/HS. This study aimed to evaluate the role of the serine protease inhibitor gabexate mesilate (GM) in preserving vascular function during T/HS when given enterally. We studied the vascular reactivity of mesenteric arteries from male Wistar rats treated with enteral GM (10 mg/kg) (GM-treated, n = 6) or control (Shock-control, n = 6) following (T/HS) using pressure myography. Concentration-response curves of endothelial-dependent and endothelial-independent agonists (e.g., acetylcholine, sodium nitroprusside) ranging from 10-10 to 10-5 M were performed. In a second set of experiments, ex-vivo arteries from healthy rats were perfused with plasma from shocked animals from both groups and vascular performance was similarly measured. Arteries from the GM-treated group demonstrated a preserved concentration-response curve to the α1 adrenergic agonist phenylephrine compared to arteries from Shock-control animals (- logEC50: - 5.73 ± 0.25 vs. - 6.48 ± 0.2, Shock-control vs. GM-treated, p = 0.04). When perfused with plasma from GM-treated rats, healthy arteries exhibited an even greater constriction and sensitivity to phenylephrine (- logEC50: - 6.62 ± 0.21 vs. - 7.13 ± 0.21, Shock-control vs. GM-treated, p = 0.02). Enteral GM also preserved the endothelium-dependent vascular response to agonists following T/HS and limited syndecan-1 shedding as a marker of glycocalyx compromise (41.84 ± 9 vs. 17.63 ± 3.97 ng/mL, Shock-control vs. GM-treated, p = 0.02). Syndecan-1 cleavage was correlated with plasma trypsin-like activity (r2 = 0.9611). Enteral gabexate mesilate was able to maintain vascular function in experimental T/HS, which was reflected by improved hemodynamics (mean arterial pressure 50.39 ± 7.91 vs. 64.95 ± 3.43 mmHg, Shock-control vs. GM treated, p = 0.0001). Enteral serine protease inhibition may be a potential therapeutic intervention in the treatment of T/HS.

SerpinB1 is required for Rev-erbα-mediated protection against acute lung injury induced by lipopolysaccharide-in mice.

BACKGROUND AND PURPOSE: Acute lung injury (ALI) is a serious, life-threatening inflammation of the lungs that still lacks effective treatment. We previously showed that serine protease inhibitor B1 (SerpinB1) protects against ALI induced by orthotopic autologous liver transplantation. However, the role of SerpinB1 in lipopolysaccharide (LPS)-induced ALI and its regulatory mechanisms are not known. EXPERIMENTAL APPROACH: Wild-type (WT) and SerpinB1 knockout (KO) mice were treated with intratracheal LPS stimulation to induce ALI. Some of the WT and KO mice were injected i.p. with melatonin, a rhythm-related protein Rev-erbα agonist. The circadian rhythm in WT mice was disrupted by exposing mice to 24 h of continuous dark or light conditions after intratracheal LPS. Neutrophils were isolated from alveolar lavage fluid of WT and KO mice, and from human peripheral blood. Neutrophils were treated with LPS and melatonin. KEY RESULTS: Disruption of circadian rhythm by either 24-h dark or light conditions exacerbated LPS-induced ALI and decreased expression of Rev-erbα and SerpinB1 protein in lung, whereas melatonin treatment increased SerpinB1 expression and attenuated LPS-induced ALI in WT mice, but not in KO mice. In isolated neutrophils, Rev-erbα was co-localized with SerpinB1 and bound to its promoter to trigger SerpinB1 transcription. Furthermore, LPS stimulation increased formation of neutrophil extracellular traps, which was reversed by melatonin treatment in neutrophils from WT mice, but not from KO mice. CONCLUSION AND IMPLICATIONS: In mice, SerpinB1 is rhythmically regulated by Rev-erbα, and its down-regulation exacerbates LPS-induced ALI by inducing formation of neutrophil extracellular traps.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.